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Identification of the CRB1 gene
and analysis of its role in
autosomal recessive retinal dystrophies
Anneke den Hollander
ISBN:
90-9015558-9
Cover:
Model of the CRB1 protein (11 EGF-like domains). By courtesy of M.
Roeters and Prof. Dr. G. Vriend of the Centre for Molecular and
Biomolecular Informatics (CMBI). Design by M.A. van Driel.
Printed by:
PrintPartners Ipskamp, Enschede, the Netherlands, 2002.
The research presented in this thesis was performed at the Department of Human Genetics,
University Medical Center Nijmegen, Nijmegen, the Netherlands, and was supported by the
Foundation Fighting Blindness USA, Inc. and the British Retinitis Pigmentosa Society.
Identification of the CRB1 gene
and analysis of its role in
autosomal recessive retinal dystrophies
Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen
Proefschrift
ter verkrijging van de graad van doctor
aan de Katholieke Universiteit Nijmegen,
volgens besluit van het College van Decanen
in het openbaar te verdedigen op
vrijdag 8 maart 2002
des namiddags om 1.30 uur precies
door
Antonia Ingrid den Hollander
geboren op 16 oktober 1973
te Sassenheim
Promotor:
Prof. Dr. H.G. Brunner
Co-promotor:
Dr. F.P.M. Cremers
Manuscriptcommissie:
Prof. Dr. F.G.M. Russel (voorzitter)
Prof. Dr. J.A. Schalken
Prof. Dr. M.H. Breuning (UL)
voor mijn ouders
Contents
Abbreviations
8
1
General introduction
9
2
Isolation and mapping of novel candidate genes for retinal disorders using
41
suppression subtractive hybridization
3
Mutations in a human homologue of Drosophila crumbs cause retinitis
63
pigmentosa (RP12)
4
Leber congenital amaurosis and retinitis pigmentosa with Coats-like
77
exudative vasculopathy are associated with mutations in the crumbs
homologue 1 (CRB1) gene
5
CRB1 has a cytoplasmic domain that is functionally conserved between
89
human and Drosophila
6
Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis
107
of its expression pattern in eye and brain
7
General discussion
117
Summary / Samenvatting
133
Acknowledgements / Dankwoord
139
Curriculum vitae
141
List of publications
143
7
Abbreviations
ALPS
AMD
ASO
ATP
cDNA
cGMP
CNS
CRB
CRB1
Crl1
CTL
DIG
ds
EGF
ERG
EST
LCA
LM-PCR
MAGUK
MRI
ORF
PCR
PDE
PDZ
PPRPE
RACE
RP
RPE
RT-PCR
SAC
SH3
SJ
ss
SSCP
SSH
UAS
UTR
ZA
8
agrin, laminin, perlecan, slit
age-related macular degeneration
allele-specific oligonucleotide
adenosine triphosphate
complementary DNA
cyclic guanosine-monophosphate
central nervous system
Crumbs
Crumbs homologue 1
Crumbs-like protein 1
C-type lectin
digoxigenin
double-stranded
epidermal growth factor
electroretinogram
expressed sequence tag
Leber congenital amaurosis
ligation-mediated polymerase chain reaction
membrane-associated guanylate kinase
magnetic resonance imaging
open reading frame
polymerase chain reaction
phosphodiesterase
PSD-95, Discs Large, ZO-1
preserved para-arteriolar retinal pigment epithelium
rapid amplification of cDNA ends
retinitis pigmentosa
retinal pigment epithelium
reverse-transcriptase polymerase chain reaction
subapical complex
Src homology region 3
septate junction
single-stranded
single-strand conformation polymorphism
suppression subtractive hybridization
upstream activator sequence
untranslated region
zonula adherens
Chapter
1
General introduction
General introduction
1.1 The retina
Cell types and layers of the retina
The retina is a highly organized structure dedicated to light perception and signal
transduction. The retina consists of several cell types of which neural cells predominate. The
three principal neural cell types are photoreceptors, bipolar cells, and ganglion cells. The
photoreceptors convert light into a nerve impulse that is transmitted by the bipolar cells to the
ganglion cells. The axons of the ganglion cells leave the eye at the optic disc to form the optic
nerve. Their activity is modulated by other neural cell types such as horizontal cells and
amacrine cells.
Non-neural cell types of the retina include glial cells, vascular endothelium, pericytes, and
microglia. The principal supporting glial cells of the retina are the Müller cells, which may
help to nourish and maintain the retina. Microglia are a highly specialized subpopulation of
the mononuclear phagocyte system, which also reside in the central nervous system. In
humans the retina has a dual blood supply, branches from the central retinal vessels nourish
the inner part, while the choroidal circulation nourishes the outer part. Retinal capillaries are
characterized by complete circumferentially orientated endothelial cells joined by tight
junctions, surrounded by a thick basal lamina and pericytes.
Retinal cells are arranged in a highly organized manner, and in histological sections appear as
ten distinct layers (Figure 1). The most outer layer is the retinal pigment epithelium (RPE),
which is situated between the choroid and the photoreceptors. The second layer, the receptor
layer, is divided into two layers comprising the outer and the inner segments of the
photoreceptor cells. The third layer is the external limiting membrane. This is not a true
membrane, but is composed of closely apposed processes from Müller cells, with cell bodies
10
9
8
7
6
5
4
3
2b
2a
1
Figure 1. Histological section
of a monkey retina. C, choroid;
1, retinal pigment epithelium; 2a,
photoreceptor outer segments;
2b,
photoreceptor
inner
segments; 3, external limiting
membrane; 4, outer nuclear
layer; 5, outer plexiform layer; 6,
inner nuclear layer; 7, inner
plexiform layer; 8, ganglion cell
layer; 9, optic fiber layer; 10,
inner limiting membrane
C
11
Chapter 1
in the sixth layer. The fourth layer is the outer nuclear layer, and it contains the cell bodies of
the photoreceptors. The fifth layer is the outer plexiform layer, which represents a zone of
synaptic interactions between the photoreceptors and the interneurons (the horizontal, bipolar
and amacrine cells). The sixth layer is the inner nuclear layer. It contains the cell bodies of
Müller cells and the interneurons. The seventh layer is the inner plexiform layer, which
represents a zone of synaptic interactions between the interneurons and ganglion cells. The
eighth layer is the ganglion cell layer, and contains the cell bodies of these neurons. The ninth
layer is the optic fiber layer, which contains unmyelinated ganglion axons that course towards
the optic disc. The innermost layer is the inner limiting membrane, which is not a true
membrane, but is formed by apposed processes of the same Müller cells that form the external
limiting membrane.
The photoreceptors
There are two types of photoreceptor cells in the human retina: rods and cones. In humans,
95% of the photoreceptors are rods and 5% are cones. Rods are responsible for sensing
contrast, brightness and motion, while cones subserve fine resolution, spatial resolution and
color vision. The highest concentration of photoreceptors is found in and near the macula, the
central part of the retina, which has a diameter of approximately 4 mm. The center of the
macula is called the fovea, a region with a diameter of 1.5 mm that exclusively contains cone
photoreceptors (Figure 2). In the fovea there is a nearly one-to-one relationship between the
cone photoreceptor, its ganglion cell, and the emerging nerve fiber, which ensures fine spatial
detail. In the peripheral retina many photoreceptors are coupled to the same ganglion cell,
receptor density (104/mm2)
which is called convergent wiring, and a more complex system of relays is necessary.
16
cones
Figure 2. Density of rods
and cones in the retina. Cone
density peaks at the fovea.
rods
12
8
4
0
80o
60o
40o
nasal
20o
0o
optic fovea
disc
12
20o
40o
temporal
60o
General introduction
discs
plasma membrane
outer
segment
cytoplasmic space
Figure 3. Schematic diagram of a rod
photoreceptor cell.
intradiscal space
cilium
mitochondrion
inner
segment
Golgi apparatus
endoplasmatic reticulum
nucleus
synaptic terminal
Convergent wiring is an ideal system for the detection of small amounts of light. The result of
such an arrangement is that the macula is used primarily for central and color (photopic)
vision while the remaining retina, which is populated mostly by rod photoreceptors, is utilized
primarily for peripheral and night (scotopic) vision.
Photoreceptor cells contain an outer segment, an inner segment, a cell body and a synaptic
terminal (Figure 3). Rod photoreceptors are slender, elongated structures; in humans they
have a diameter of 1 µm and a length of 40 µm. The outer segment is characterized by a stack
of about 1000 discs, which are formed by invaginations of the plasma membrane at the base
of the outer segment. The discs are densely packed with the visual pigment rhodopsin, and
provide an extensive surface area for the capture of incoming photons. The discs have a
lifespan of 10 days and are constantly renewed and shedded. A slender immotile cilium joins
the outer segment and the inner segment, which is rich in mitochondria and ribosomes. The
inner segment generates ATP at a very rapid rate and is highly active in synthesizing proteins.
The inner segment is continuous with the nucleus and an axon ending as the synaptic
terminal.
The outer segments of cones are shorter than those of rods, and are generally conical. The
human retina has three types of cones that contain different visual pigments. The three types
are referred to as blue, green, and red, or short, medium and long wavelength sensitive cones.
The tips of the outer segments of the photoreceptors are closely associated with the bordering
RPE. The RPE is a multifunctional and indispensable component of the retina, which is
13
Chapter 1
comprised of a single layer of cuboidal cells. It forms a barrier between the retina and the
choroidal circulation, and exchanges nutrients and waste products. The RPE absorbs stray
light that would otherwise degrade the visual image. Additionally, the RPE phagocytoses the
discs shed by the outer segments of the photoreceptors. Furthermore, the RPE has a crucial
role in the regeneration of visual pigments in the visual cycle1.
Phototransduction
The rod photopigment rhodopsin constitutes 85% of the total amount of protein present in the
rod outer segments. It is composed of an opsin, a seven transmembrane-helix G-proteincoupled receptor, which is bound covalently to the light sensitive chromophore 11-cis-retinal,
a derivative of vitamin A. Light induces isomerization of 11-cis-retinal to all-trans-retinal,
and subsequent conformational changes of the opsin molecule. One of the intermediates,
metarhodopsin II, also called photoexcited rhodopsin, triggers a cascade of enzymatic events2
(Figure 4).
The conformational changes in photoexcited rhodopsin expose G-protein binding sites. This
permits the binding of the photoreceptor G-protein transducin. Each molecule of
photoactivated rhodopsin can activate several hundred transducin molecules, the first step of
an amplification process that continues throughout the enzymatic cascade. The α-subunit of
transducin activates the enzyme cGMP phosphodiesterase (PDE), which then hydrolyses
cGMP. The resulting decrease in cGMP levels leads to closure of cGMP-gated cation
channels in the plasma membrane of the rod outer segments. Influx of sodium and calcium
ions is prevented, and the subsequent hyperpolarization of the entire rod cell plasma
membrane leads to the release of the neurotransmitter glutamate at the synaptic terminal.
Inactivation of the phototransduction cascade occurs via several mechanisms. Rhodopsin is
phosphorylated by rhodopsin kinase, and then binds arrestin. This blocks the interaction of
rhodopsin with transducin, which leads to a decrease in PDE activity. Activation of retinaspecific guanylate cyclases by recoverin increases intracellular cGMP concentrations, which
leads to the opening of the cGMP-gated channels.
Arrestin promotes the dissociation of all-trans-retinal from opsin. All-trans-retinal is
enzymatically reduced to all-trans-retinol, and is transported to the RPE, where it is
isomerized to 11-cis-retinol and oxidated to 11-cis-retinal. 11-cis-retinal is returned to the
outer segment where it binds to opsin and regenerates the visual pigment. This cycle of events
is referred to as the visual cycle.
14
General introduction
R
R*
channel
opening
Tα-GTP
cGMP↓
cGMP↑
GC*
channel
closure
membrane
hyperpolarization
Cai↓
Figure 4. Flow of information in visual excitation and recovery2. Photoisomerization of rhodopsin
triggers a cascade leading to cGMP hydrolysis and the closure of membrane channels, which
generates a nerve signal. Channel closure also induces a drop in the cytosolic calcium level, which
leads to the activation of guanylate cyclase and the reopening of channels. R, rhodopsin; R*,
photoactivated rhodopsin (metarhodopsin II); Tα-GTP, α subunit of transducin (GTP-bound form);
PDE*, activated cGMP phosphodiesterase; GC*, activated guanylate cyclase.
1.2 Inherited retinal dystrophies
Loss or reduction of vision is considered a grave restriction in the quality of human life. The
major causes of visual impairment in the Western world are age-related macular degeneration
(AMD), diabetic retinopathy and glaucoma, together constituting 75% of all cases3. The
development of these diseases is generally multifactorial, with genetic and environmental
components. Monogenic inherited retinal dystrophies constitute only 5% of all causes of
visual impairment, affecting approximately 1 in 2000 individuals. However, many of these
diseases have an earlier onset and a more severe clinical course than the multifactorial
diseases mentioned above, and are generally untreatable. These characteristics, together with
the possibility of using genetic approaches to understand the disease mechanisms, have
focused attention on inherited retinal dystrophies4.
There are a large number of monogenic inherited retinal disorders, which can be classified in
degenerations primarily affecting the peripheral retina, dystrophies primarily affecting the
macula, and color vision defects. Peripheral retina degenerations, for example retinitis
pigmentosa, choroideremia and gyrate atrophy, usually begin with night blindness and
progress to peripheral visual field loss. Macular dystrophies, for example Stargardt disease,
Best disease, and cone or cone-rod dystrophies, are characterized by a degeneration of the
retina, RPE and/or choroidal tissue most severely affecting the macular area. Patients can
develop loss of visual acuity and color vision. A preference for dimmed light when reading
may indicate a macular dystrophy. Color vision defects, for example protanopia, deuteranopia
or tritanopia, are caused by loss of one or more of the cone pigments, or, in rod
monochromatism, by absence of the cone photoreceptors.
15
Chapter 1
The most important retinal dystrophies within the scope of this thesis are retinitis pigmentosa
and Leber congenital amaurosis. This introduction will therefore mainly focus on these
diseases.
Retinitis pigmentosa
Retinitis pigmentosa (RP) is a heterogeneous group of progressive retinal dystrophies, which
affects approximately 1.5 million people worldwide with 100.000 sufferers in Europe5, and
has a prevalence of approximately 1 in 4000 individuals6. Retinitis pigmentosa is a misnomer
since the term retinitis suggests a retinal inflammation, which is not the case in RP. The term
pigmentosa refers to dark pigmentary intraretinal deposits that develop in the midperipheral
retina. These pigmentary changes, known as bone-spicules, result from the release of pigment
by degenerating cells in the RPE.
RP patients first slowly lose the rod photoreceptors (scotopic vision) and then the cone
photoreceptors (photopic vision). The disease is usually first detected in the patient’s
childhood years through the loss of night vision. The diagnosis of RP is then confirmed with
four main tests, an electroretinogram (ERG), measurement of the visual field, the best
corrected visual acuity, and pedigree analysis6,7. Over the course of a few decades, patients
suffer from the gradual degeneration of both peripheral and central vision. Eventually, they
can become legally blind, with central vision of 20/200 or worse. In patients with advanced
RP, the RPE atrophies or depigments, the retinal vessels attenuate, and a pale appearance of
the optic disc become apparent. Other ocular features complicating the disease are myopia
(75%), open-angle glaucoma (3%), posterior subcapsular cataract, keratoconus, posterior
vitreous detachment, and Coats-like exudative vasculopathy8 (1-3%).
RP is a heterogeneous disorder, both clinically and genetically. Several atypical forms of RP
are distinguished, for example RP sine pigmento, a form without the intraretinal
pigmentation, retinitis punctata albescens, characterized by scattered white dots, and RP with
preserved para-arteriolar RPE (PPRPE), characterized by a typical preservation of the RPE
underneath and adjacent to the retinal arterioles. A large variation is seen in age of onset of
symptoms, the nature of visual loss and the speed of progression. The severity is frequently
related to the mode of inheritance. The inheritance of RP can be autosomal dominant (~20%),
autosomal recessive (~30%) and X-linked (~10%)6,9. Autosomal recessive and X-linked
forms are usually severe while autosomal dominant forms are relatively mild. About 40% of
RP cases are isolated, having no family history of the disorder. Although it can be expected
16
General introduction
that most of these represent autosomal recessive cases, it is likely that some represent digenic,
polygenic or multifactorial cases.
Leber congenital amaurosis
Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal
dystrophies, leading to blindness or severe visual impairment from birth. LCA accounts for
more than 5% of all retinal dystrophies10,11, and is one of the main causes of blindness in
children, affecting 18% of all blind children in the Netherlands12. LCA is considered to be a
congenital form of RP, and a classification system to distinguish LCA and RP has been
proposed13. LCA is defined as congenital blindness with extinguished or markedly diminished
photopic and scotopic ERG responses when examined before the age of one, sluggish
pupillary responses to light, and searching nystagmus. Almost all patients remain visually
impaired for life and never achieve a visual acuity better than 20/400. In contrast to RP
patients, LCA patients tend to be hyperopic, with a refractive error of more than 5 diopters.
Patients usually develop the fundus appearances of RP in later years. Inheritance of LCA is
generally autosomal recessive, although also some autosomal dominant cases have been
described6,14.
1.3 Genes involved in retinal disease
In the last decade, the identification of more than 60 genes involved in non-syndromic and
syndromic retinal dystrophies has remarkably improved our understanding of their
pathogenesis4,15-21 (Figure 5; RetNet, for URL see Table 5). The genetic basis of retinal
dystrophies is extremely heterogeneous and the construction of a comprehensive catalogue of
genes involved in retinal dystrophies is a challenging task.
For example, 7 genes involved in LCA have been identified, accounting for 35-65% of the
140
mapped
120
100
80
60
Figure 5. Number of
cloned and mapped
retinal disease genes,
1980-2001 (RetNet).
cloned
40
20
Jan-00
Jan-98
Jan-96
Jan-94
Jan-92
Jan-90
Jan-88
Jan-86
Jan-84
Jan-82
Jan-80
0
17
Chapter 1
cases, and 15 genes involved in autosomal recessive RP have thus far been identified, together
constituting 25-40% of the molecular causes (Table 1). From family and genetic linkage
studies one can deduce that at least 50 autosomal recessive RP genes remain to be identified.
The heterogeneity of retinal disease is emphasized by the observation that mutations in a
single gene can cause more than one phenotype. The most extreme example is RDS;
mutations in this gene can lead to autosomal dominant retinitis pigmentosa, retinitis punctata
albescens, pattern dystrophies, central areolar choroidal dystrophy and various autosomal
dominant macular dystrophies (ref. 50 and references therein). Mutations in the ABCA4 gene
can cause various autosomal recessive retinal dystrophies, ranging from fundus
flavimaculatus, Stargardt disease, cone-rod dystrophy to retinitis pigmentosa22,23,51,52. In
addition, at least two ABCA4 variants have been shown to be a risk factor for age-related
macular degeneration53. A model has been developed in which the severity of retinal disease
correlates inversely with residual ABCA4 activity54.
Table 1. Genes involved in autosomal recessive RP and LCA.
Gene
Number and percentage of cases
Autosomal recessive retinitis pigmentosa:
ABCA4
3 families
CNGA1
4/173
CNGB1
1 family
CRB1
MERTK
3/328*
NR2E3
1 family
PDE6A
6/164
PDE6B
4/92; 1/19; 3/19
RGR
1/182
RHO
1/126
RLBP1
0/50; 1/19
RPE65
3/162
SAG
3/120
TULP1
2/536
USH2A
10/224
Leber congenital amaurosis:
AIPL1
14/202
CRB1
21/233; 7/52
CRX
2/100; 5/176
GUCY2D
6/100; 11/176; 24/118
LRAT
0/38; 3/267**
RPE65
3/100; 12/176; 13/114**; 7/45
RPGRIP1
3/57
* 328 patients with various retinal dystrophies
** early-onset retinal dystrophy
18
References
3.7%
4.3%; 5.3%; 15.8%
0.5%
0.8%
0%; 5.3%
1.9%
2.5%
0.4%
4.5%
22-24
25
26
this thesis
27
28
29
30-32
33
34
35,36
37
38
39
40
6.9%
9.0%; 13.5%
2.0%; 2.8%
6.0%; 6.3%; 20.3%
0%; 1.1%
3.0%; 6.8%; 11.4%; 15.6%
5.3%
41
42, this thesis
43,44
43-45
46,47
37,43,44,48
49
2.3%
2-4%
0.9%
General introduction
Until now, 49 genes have been identified that are involved in non-syndromic retinal disorders.
Expression of 32 (65%) of these genes is restricted to the retina or the RPE, and in some cases
one other tissue (Table 2). Many encode proteins that are part of the phototransduction
cascade, such as rhodopsin (RHO), cone opsins (OPN1LW, OPN1MW, OPN1SW), rhodopsin
kinase (RHOK), transducin (GNAT1), arrestin (SAG), α- and β-subunits of cGMP
phosphodiesterase (PDE6A, PDE6B), subunits of rod and cone cyclic nucleotide gated
channels (CNGA1, CNGA3, CNGB1, CNGB3), guanylate cyclase (GUCY2D), and guanylate
cyclase activating protein (GUCA1A). Other genes encode photoreceptor structural proteins,
including peripherin (RDS) and rod outer segment membrane protein (ROM1), and proteins
involved in the metabolism of retinol, such as cellular retinaldehyde binding protein (RLBP1),
11-cis retinol dehydrogenase (RDH5), RPE65 (RPE65), lecithin retinol acyltransferase
(LRAT) and retina-specific ATP-binding cassette transporter (ABCA4). Also proteins
regulating gene expression are implicated in retinal disease, including the trancription factors
‘cone rod homeobox’ (CRX) and ‘neural retina leucine zipper’ (NRL), and photoreceptorspecific nuclear receptor (NR2E3).
Prior to the onset of molecular genetics, only one gene involved in retinal disease was
identified by characterization of a specific biochemical abnormality. This retinal disease,
gyrate atrophy, is caused by a defective activity of ornithine aminotransferase81. The
remaining 48 retinal disease genes have been identified by two distinct molecular genetic
approaches, the phenotype-orientated approach and the gene-orientated approach123.
The phenotype-orientated approach
In the phenotype-orientated approach, the approximate chromosomal localization of a gene
causing a particular phenotype is either determined by linkage analysis, requiring
(moderately) large families, or physical mapping using deletions or translocations that can be
detected cytogenetically. Genes in the critical region are then isolated by different cloning
strategies. This strategy is referred to as positional cloning, and was successfully used to clone
several retinal disease genes, among which the choroideremia, Norrie disease and RP type 3
genes58,108,109,124,125. With the near completion of the human genome sequence and the
availability of detailed gene maps, candidate genes in the region of interest can be readily
selected. Screening of such candidate genes is referred to as the positional candidate gene
approach.
19
Chapter 1
Table 2. Tissues where non-syndromic retinal disease genes are expressed
Gene
ABCA4
AIPL1
CACNA1F
CHM
CNGA1
CNGA3
CNGB1
CNGB3
CRB1
CRX
EFEMP1
ELOVL4
GNAT1
GUCA1A
GUCY2D
LRAT
MERTK
Tissues
retina
retina, pineal gland
retina
retina, RPE, choroid, various tissues
retina, kidney
retina, testis, kidney, heart
retina
retina
retina, brain
retina, pineal gland
retina, RPE, lung, brain, heart, spleen, kidney
retina, brain
retina
retina
retina
RPE, testis, liver, small intestine, prostate, pancreas, colon, brain
retina, testis, ovary, prostate, lung, kidney, spleen, leukocytes,
placenta, thymus, small intestine, colon, liver
NR2E3
retina
NRL
retina
NYX
retina, kidney, brain, testis, muscle, uterus, mammary, placenta
OAT
nearly all tissues
OPA1
retina, brain, testis, heart, muscle, placenta, lung, liver, kidney
OPN1LW
retina
OPN1MW
retina
OPN1SW
retina
PDE6A
retina
PDE6B
retina, brain
PROML1
retina, fetal liver, bone marrow, kidney, pancreas, placenta
PRPC8
retina, kidney, lung, placenta, heart, liver, brain, spinal cord
RBP4
liver, kidney, lung, spleen, brain, heart, skeletal muscle, eye
RDH5
RPE, retina, liver, kidney, intestine, heart, mammary, testis, brain
RDS
retina
RGR
retina, RPE
RHO
retina
RHOK
retina
RLBP1
RPE, liver, epididymis, lung, kidney, testis, spleen
ROM1
retina
RP1
retina
RP2
retina, heart, brain, spleen, lung, liver, muscle, kidney, testis
RPE65
RPE
RPGR
retina, heart, brain, placenta, lung, liver, muscle, pancreas*
RPGRIP1
retina, testis
RS1
retina
SAG
retina, pineal gland
TIMP3
RPE, placenta, heart, kidney, lung, liver, pancreas, muscle, brain
TULP1
retina
UNC119
retina
USH2A
retina, cochlea, brain, kidney
VMD2
RPE
* RPGR contains a retina-specific exon110
20
References
51
55
56,57
58-60
61,62
63,64
65
66
this thesis
67,68
69
70
71
72
73
74
75,76
77
78
79,80
81
82,83
84
84
84
85
86,87
88,89
90
91
92-94
95
96
97
98
99,100
101
102,103
104
105-107
108-110
111,112
113
114,115
116,117
118,119
118
120
121,122
General introduction
The gene-orientated approach
In the gene-orientated approach the role of a particular gene is investigated in human disease.
This strategy has shown to be particularly useful in the study of diseases that occur in wellstudied, highly specialized tissues such as the retina. Because most retinal disease genes are
expressed predominantly or exclusively in the retina or the RPE (Table 2), any gene that is
expressed only in these tissues is a promising candidate gene for retinal disease.
The gene-orientated approach requires the availability of DNA samples from large patient
groups and efficient methods for large-scale mutation analysis. For most candidate genes the
retinal disease phenotype cannot be predicted, which requires the screening of large groups of
patients with various retinal disorders.
This approach has become increasingly successful in the identification of retinal disease
genes. For example, 10 of the 15 genes involved in autosomal recessive RP have been
identified by the gene-based approach; CNGA125, MERTK27, PDE6A25,126, PDE6B127, RGR33,
RHO34, RPE6537, SAG38, TULP139 and USH2A40.
Due to the extreme genetic heterogeneity of retinal disease, mutations may be identified in
only a small percentage of patients (Table 1). For example, mutation screening of the RGR
gene in 842 patients with various retinal dystrophies, including autosomal dominant RP,
autosomal recessive RP, isolated RP, LCA, retinitis punctata albescens and choroidal
sclerosis, resulted in the identification of disease-causing mutations in only two (0.2%)
patients33.
1.4 Isolation of genes preferentially expressed in the retina
or the RPE
Most retinal disease genes identified thus far are expressed exclusively or predominantly in
the retina or the RPE (Table 2). This makes the gene-orientated approach particularly useful
in the identification of retinal disease genes. Isolation of genes expressed preferentially in
these tissues does not only provide good candidate genes for retinal disorders, but will also
expand our knowledge into the molecular biology of the retina and RPE. Several methods
have been developed for the isolation of tissue-specific genes128,129, and some of them have
been successfully employed to isolate retina- or RPE-specific genes (Table 3).
21
Chapter 1
Table 3. Methods used to isolate human retina- and RPE-specific genes
Method of isolation
Subtractive hybridization:
• retina vs lymphoblastoid cell line
• RPE cell line vs lymphoblastoid cell line
• retina vs fibroblast
• retina vs brain
Differential hybridization:
• human retina cDNA library, probes: bovine
retina vs human fibroblast
• human retina cDNA library, probes: ground
squirrel retina vs human fibroblast
• fovea cDNA library, probes: fovea vs
midperipheral retina
In silico analysis of gene expression:
• sequencing of human retina cDNA library
• BodyMap
• HGI; clusters containing >3 retina ESTs
• HGI; clusters containing retina and pineal gland
ESTs
• UniGene; retina-specific and -enriched clusters
• UniGene; retina-specific and -enriched clusters
Suppression subtractive hybridization:
• retina vs mixture of tissues
• RPE vs mixture of tissues
* not an official gene symbol
Genes isolated
References
GNAS1, NRL
RCV1, ARR3, UNC119
AOC2, MYOC
78,133-135
136,137
118,138,139
140,141
ROM1, CHX10
101,142
USP11, STXBP1, PHR1*
143-146
147,148
FZD5, RRH, PPEF2
CLUL1*, PDE6H
AIPL1
149-151
152,153
154
55,155
C18orf2*, MPP4
156,157
158
CRB1
this thesis
this thesis
Subtractive hybridization
Subtractive hybridization or subtractive cloning is a method used for the isolation of
sequences distinguishing closely related genomes, or mRNAs present in one type but absent
from another type of cells130-132. Denatured double-stranded cDNA from the two samples to
be compared are hybridized so that sequences common to the samples are ‘subtracted’,
leaving a population of cDNAs enriched for sequences preferentially expressed in one of the
samples. The first round of subtraction removes the rapidly hybridizing repetitive classes of
DNA, while the subsequent second and third rounds remove more slowly hybridizing
sequences, leaving only genes unique or massively upregulated in the sample of interest. If
the technique has worked well, less than 5% of the starting cDNA sequences will be left132.
Subtractive hybridization has proven useful in the identification of a number of retina-and
RPE-specific genes, by subtracting retina or RPE cDNA against lymphoblastoid cell line,
fibroblast or brain cDNA134,136,138,140 (Table 3). The technique has also been employed to
isolate genes that are differentially expressed between degenerated and healthy retinas of
22
General introduction
mouse or dog95,159,160, and to isolate genes that are expressed higher in the macula region
compared to the peripheral region of the monkey retina161.
Differential hybridization
Another method to isolate tissue-specific genes is differential hybridization, which involves
the comparison of the intensity of hybridization signals of different cDNA probes hybridized
to cDNA library clones. Differential hybridization of human retina cDNA libraries has been
used to identify retina-specific genes, using retina and fibroblast cDNA probes, or fovea and
midperipheral retina cDNA probes101,144,147 (Table 3). To isolate cone-specific genes,
Swanson and coworkers used a cDNA probe from the retina of the 13-line ground squirrel,
which in contrast to humans consists mainly (95%) of cones144. Differential hybridization has
also been used to isolate bovine and canine retina- and RPE-specific genes96,162,163.
In silico analysis of gene expression
Expressed sequence tags (ESTs) are partial, single-read cDNA sequences of 300-500 bp.
Sequence data and other information on ESTs from a large number of organisms are
contained in dbEST, a division of the GenBank database164. EST sequences are submitted by
individual researchers, or are generated by large-scale EST sequencing projects, such as the
Washu-Merck Human EST Project, an initiative of Washington University sponsored by
Merck, and the Cancer Genome Anatomy Project of the National Cancer Institute (NCI). EST
sequences can be generated from normalized or nonnormalized cDNA libraries. A normalized
cDNA library is one in which each transcript is represented in more or less equal numbers.
The advantage of using normalized cDNA libraries is that redundant sequencing of highly
expressed genes is minimized, and the potential for identification of less abundant transcripts
is maximized165. An advantage of nonnormalized libraries is that the transcript abundance of
the original cell or tissue is accurately reflected in the frequency of clones in the library.
At this moment, more than 27,000 human retina and RPE EST sequences from 17 different
cDNA libraries have been deposited in dbEST134,136,144,148,166,167 (Table 4), representing
approximately 6000 different records. More than half of these ESTs are from two normalized
retina cDNA libraries constructed by Dr. B. Soares165, which were sequenced by the WashuMerck Human EST project. More than 5000 ESTs were submitted by the group of Dr. J.
Nathans. Using their EST sequence information, this group isolated retina-specific genes that
show homology to known proteins149-151 (Table 3).
23
Chapter 1
Table 4. Number of retina and RPE ESTs in dbEST (December 2001)
NCBI
library
number Library name
Retina cDNA libraries:
178
Soares retina N2b5HR
277
Stratagene fetal retina
228
Human retina cDNA randomly primed sublibrary
177
Soares retina N2b4HR
226
Human retina cDNA Tsp509I-cleaved sublibrary
313
Retina II
221
Human fovea cDNA
433
Retina I
165
Subtracted human retina
849
Subtracted retina cDNA
300
Human retina
RPE cDNA libraries:
6359
Human retinal pigment epithelium cDNA
1359
Unizap XR retina pigment epithelium
9
Subtracted human retinal pigment epithelium
606
Human retina cell line ARPE-19
485
Retinal pigment epithelium 0041 cell line
850
Subtracted RPE cDNA
Total:
Number of
ESTs
Submitter
12,124
6,293
3,365
2,298
1,524
1,296
100
40
38
36
16
Washu-Merck
NCI
Nathans
Washu-Merck
Nathans
Kervalage
Bernstein
Kervalage
Swaroop
den Hollander
Swanson
334
76
58
39
22
3
27,662
Swaroop
Paraoan
Swaroop
Stöhr
Kervalage
den Hollander
The first collection of tissue-specific transcriptional profiles, BodyMap, was made available
online in 1991 (ref. 168; for URL see Table 5). BodyMap is a collection of 164,000 3’ ESTs,
or gene signatures, that contain the transcript compositions of 51 human organs and tissues.
The cDNA libraries used to construct BodyMap are nonnormalized, allowing the estimation
of the absolute abundance of transcripts. An initial indication of the tissue distribution of a
gene can be obtained by analysis of the different cDNA libraries in which it is represented.
BodyMap contains 925 retina ESTs, and has proven useful for the isolation of retina-specific
genes153 (Table 3).
The increasing number of cDNA libraries from which human ESTs are obtained paves the
way for in silico analysis of tissue-specific transcription patterns169. ESTs are highly
redundant and, because of their fragmentary nature, rarely represent an entire transcript. This
has prompted several groups to develop gene indexing databases such as the Human Gene
Index (HGI) at the Institute of Genome Research (for URL see Table 5), UniGene at the
National Center for Biotechnology Information (for URL see Table 5), and STACK at the
South African National Bioinformatics Institute, that attempt to group the ESTs encoding the
same gene and thus to reduce the complexity of existing EST databases170. The qualitative and
quantitative analysis of the tissue origins of individual ESTs in the assembled clusters
provides an in silico expression pattern which can be used as an initial indication of its tissue
24
General introduction
distribution169. However, one should keep in mind that most ESTs in these databases are from
normalized cDNA libraries, and therefore do not represent an accurate source for abundance
information165.
The HGI and UniGene gene index databases have been screened for retina154,156 or
retina/pineal-specific EST clusters155, which so far led to the isolation of three retina-specific
genes (Table 3). Because in silico expression profiling only provides a first indication of the
tissue distribution of a gene, Stöhr and coworkers analyzed 180 EST clusters for their in vitro
expression by RT-PCR156.
Recently, a catalog of nearly 5000 UniGene entries containing at least one EST from retina
was constructed, which were ordered according to their level of expression158 (GETProfiles,
for URL see Table 5). Chromosome-specific transcript maps of the human retina were
assembled by ordering approximately 1000 retina-specific UniGene clusters (GETMaps, for
URL see Table 5).
Table 5. Several URLs that are useful for identifying retinal disease genes
Name
URL and description
RetNet
http://www.sph.uth.tmc.edu/RetNet
list of cloned and mapped genes causing inherited retinal degeneration
BodyMap
http://bodymap.ims.u-tokyo.ac.jp
collection of tissue-specific transcriptional profiles; can be searched for genes and
ESTs that are specific for certain library or tissue (e.g. retina)
UniGene
http://www.ncbi.nlm.nih.gov/UniGene/
gene indexing database; can be queried with terms (e.g. retina); has a digital
differential display option
HGI
http://www.tigr.org/tdb/hgi/index.html
gene indexing database; can be searched for entries that are specific for or enriched in
a certain library or tissue (e.g. retina)
GETProfiles http://telethon.bio.unipd.it/GETProfiles/retina/
catalogue of 4974 UniGene entries containing at least one retina EST, ordered
according to levels of expression
GETMaps
http://telethon.bio.unipd.it/GETMaps/retina/
transcription map of 979 retina-specific or -enriched UniGene clusters retina, ordered
by chromosome
TIGEM
http://www.tigem.it/RET
transcription and expression map of retina and RPE ESTs derived from the cDNA
libraries described in this thesis, and retina-specific or -enriched UniGene clusters;
contains semi-quantitative RT-PCR results
25
Chapter 1
1.5 Isolation of low abundant retina- and RPE-specific
genes
The transcriptional profile of the retina is characterized by a small number of highly
expressed genes, accounting for less than 10% of the total number of genes, but for
approximately 50% of the detected transcriptional activity. The percentage of tissue-specific
genes expressed in the retina is estimated to be 10% (ref. 158). The total number of genes
expressed in retina is not known, but has been roughly estimated to be 10,000-30,000 (ref.
153). Early estimates based on reassociation kinetics estimated the mRNA complexity of
typical vertebrate tissues to be 10,000-20,000, and were extrapolated to suggest around
40,000 for the entire genome. Analysis of the nearly finished human genome sequence
estimated the total number of human genes to be approximately 30,000-40,000 (ref. 171),
suggesting that the estimate of 10,000-20,000 genes expressed in a typical vertebrate tissue
may be correct. These estimates suggest that hundreds of retina-specific genes remain to be
identified, most of which are expressed at a low level.
Most techniques used thus far to isolate retina- or RPE-specific genes generally fail to isolate
tissue-specific genes expressed at a low level. Due to the nature of the kinetics of subtractive
hybridization, abundant genes hybridize faster and to a greater level of completion than low
abundance genes. Therefore, this method is less suitable for the isolation of low abundant,
differentially expressed genes132. Differential hybridization with RNA probes is biased to
select for abundantly expressed genes, since a specific cDNA clone will be detected only if its
relative abundance in the probe is 0.2% or greater101. Most ESTs in dbEST are from
normalized cDNA libraries, which have an increased representation of low abundant cDNAs
compared to nonnormalized libraries. However, the use of normalized libraries is not
sufficient to identify rare genes165. Consequently, low abundant genes are underrepresented in
the databases. EST databases now include virtually all of the abundantly expressed human
genes – the easier to reach “low hanging fruit on the tree”. The genes that are still
undiscovered are expressed at low levels and/or are expressed specifically in certain cell
types172.
At the start of the research project described in this thesis a novel technique, suppression
subtractive hybridization, was described to isolate tissue-specific genes that are expressed at a
low level in the tissue of interest.
26
General introduction
Suppression subtractive hybridization
Suppression subtractive hybridization (SSH) is a PCR-based technique that combines
normalization and subtraction173. This method is based on the suppression PCR effect: long
inverted terminal repeats when attached to DNA fragments can selectively suppress
amplification of undesirable sequences in PCR procedures174. SSH overcomes the problem of
differences in mRNA abundance by incorporating a hybridization step that normalizes
sequence abundance during the course of subtraction.
A schematic representation of the SSH method is shown in Figure 6. The differentially
expressed cDNAs are present in the ‘tester’ cDNA but are absent or present at lower levels in
tester cDNA with adaptor1
driver cDNA
tester cDNA with adaptor2
first hybridization
a
b
c
d
second hybridization
a, b, c, d + e
fill in ends
a
b
c
d
e
amplify by PCR
a, d
b
c
e
no amplication
no amplication
linear amplification
exponential amplification
Figure 6. Suppression subtractive hybridization173.
27
Chapter 1
the ‘driver’ cDNA. The tester and driver double-stranded (ds) cDNAs are first digested with
RsaI, a four-base cutting restriction enzyme that yields blunt ends. The tester cDNA
fragments are then divided into two samples (1 and 2) and ligated with two different adaptors
(adaptor 1 and adaptor 2), resulting in two populations of tester, (1) and (2). The ends of the
adaptors are designed without phosphate groups, so that only the longer strand of each adaptor
can be covalently attached to the 5’-ends of the cDNA.
The SSH technique uses two hybridizations. First, an excess of driver is added to each sample
of the tester. The samples are then heat-denatured and allowed to anneal. The single-stranded
(ss) cDNA tester fraction (a) is normalized, i.e. concentrations of high and low abundance
cDNAs become roughly equal. Normalization occurs because the reannealing process
generating homo-hybrid cDNAs (b) is faster for the more abundant molecules, due to the
second order kinetics of hybridization. Furthermore, the ss cDNAs in the tester fraction (a) are
significantly enriched in cDNAs for differentially expressed genes, as ‘common’ nontarget
cDNAs form heterohybrids (c) with the driver.
In the second hybridization, the two samples from the first hybridization are mixed. Only the
remaining normalized and subtracted ss tester cDNAs are able to reassociate and form types
(b), (c), and new hybrids (e). Addition of a second portion of denatured driver at this stage
further enriches fraction (e) for differentially expressed genes. The newly formed (e) hybrids
have an important feature that distinguishes them from hybrids (b) and (c) formed during first
and second hybridizations. This feature is that they have different adaptor sequences at their
5’-ends. One is from sample 1 and the other is from sample 2. The two sequences allow
preferential amplification of the subtracted and normalized fraction (e) using PCR and a pair
of primers, P1 and P2, which correspond to the outer part of adaptor 1 and 2, respectively. To
accomplish this selective amplification, an extension reaction is performed to fill in the sticky
ends of the molecules for primer annealing before the initiation of the PCR procedure.
In all PCR cycles, exponential amplification can only occur with type (e) molecules. Type (b)
molecules contain long inverted repeats on the ends and form stable ‘panhandle-like’
structures after each denaturation-annealing PCR step. The resulting ‘panhandle-like’
structure cannot serve as a template for exponential PCR, because intramolecular annealing of
longer adaptor sequences is both highly favored and more stable than intermolecular
annealing of the much shorter PCR primers. This is the suppression PCR effect. Furthermore,
type (a) and (d) molecules do not contain primer-binding sites, and type (c) molecules can be
amplified only at a linear rate. Only type (e) molecules have different adaptor sequences at
their ends, which allows them to be exponentially amplified during PCR.
28
General introduction
1.6 Aim of this thesis
Genes expressed specifically or preferentially in the retina and the RPE are candidate genes
for retinal disorders. Although a considerable number of retina-specific genes have been
identified, many more await discovery, in particular those expressed at low levels. Until now,
less than ten genes have been identified that are expressed specifically in the RPE. The highly
specialized function of the RPE suggests that many RPE-specific genes remain to be
identified. The main goal of the research described in this thesis is to isolate novel retina- and
RPE-specific genes, in particular those expressed at low levels.
Chapter 2 describes the construction of retina- and RPE-enriched cDNA libraries through
SSH, and the isolation of 33 cDNAs that are expressed preferentially in the retina or the
RPE166. Mapping of these cDNAs in the human genome revealed that one clone represented a
promising positional candidate gene for RP type 12 (RP12). Characterization of the cDNA
sequence and genomic structure of this gene (CRB1) is described in Chapter 3. Mutations
were identified in ten unrelated patients with RP12, a specific and severe form of RP
characterized by a preserved para-arteriolar RPE175. Chapter 4 describes mutation analysis of
CRB1 in a group of LCA patients and in a panel of patients with recessive or sporadic RP.
These studies revealed that CRB1 mutations are an important cause of LCA, and represent an
important risk factor for the development of Coats-like exudative vasculopathy in RP176.
The CRB1 gene is transcribed into two types of mRNA, encoding a 1376- and 1406-aa
protein, respectively. Both proteins contain a signal peptide, 19 EGF-like domains and 3
laminin A G-like domains, and are homologous to Drosophila Crumbs protein. Crumbs is
essential for establishing and maintaining epithelial polarity in Drosophila. Crumbs and the
1406-aa CRB1 protein contain a transmembrane domain and a 37-aa cytoplasmic domain. In
Drosophila, the cytoplasmic domain is crucial for Crumbs function. Overexpression studies in
Crumbs mutant and wild-type Drosophila embryos, described in Chapter 5, revealed that the
cytoplasmic domains of Crumbs and CRB1 are functionally conserved177. These findings
suggest that CRB1 may be involved in maintaining polarity of retinal cells and/or form an
anchor for a retinal protein complex at a specific membrane region.
Isolation of the mouse Crb1 gene and detailed mRNA expression studies in developing and
adult mouse eye and brain are described in Chapter 6. In the adult retina, Crb1 is expressed
in the outer nuclear layer and some nuclei of the inner nuclear layer178. In the developing
neural tube, Crb1 is expressed in a ventral region that is positive for Nkx2.2, which defines
29
Chapter 1
the position of the V3 interneurons. In the adult brain, Crb1 is expressed in regions that
contain differentiating or migrating neural stem cells.
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162. Kuo C-H, Akiyama M and Miki N (1989)
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164. Boguski MS, Lowe TM and Tolstoshev CM
(1993) dbEST--database for "expressed sequence
tags". Nature Genet 4:332-333.
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166. den Hollander AI, van Driel MA, de Kok YJM,
van de Pol DJR, Hoyng CB, Brunner HG,
Deutman AF and Cremers FPM (1999) Isolation
and mapping of novel candidate genes for retinal
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using
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38
167. Paraoan L, Grierson I and Maden BEH (2000)
Analysis of expressed sequence tags of retinal
pigment epithelium: cystatin C is an abundant
transcript. Int J Biochem Cell Biol 32:417-426.
168. Kawamoto S, Yoshii J, Mizuno K, Ito K,
Miyamoto Y, Ohnishi T, Matoba R, Hori N,
Matsumoto Y, Okumura T, Nakao Y, Yoshii H,
Arimoto J, Ohashi H, Nakanishi H, Ohno I,
Hashimoto J, Shimizu K, Maeda K, Kuriyama H,
Nishida K, Shimizu-Matsumoto A, Adachi W,
Ito R, Kawasaki S, Chae KS, Murakawa K,
Yokoyama M, Fukushima A, Hishiki T, Nakaya
A, Sese J, Monma N, Nikaido H, Morishita S,
Matsubara K and Okubo K (2000) BodyMap: a
collection of 3' ESTs for analysis of human gene
expression information. Genome Res 10:18171827.
169. Bortoluzzi S and Danieli GA (1999) Towards an
in silico analysis of transcription patterns. Trends
Genet 15:118-119.
170. Bouck J, Yu W, Gibbs R and Worley K (1999)
Comparison of gene indexing databases. Trends
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171. International Human Genome Sequencing
Consortium (2001) Initial sequencing and
analysis of the human genome. Nature 409:860921.
172. Martin KJ and Pardee AB (2000) Identifying
expressed genes. Proc Natl Acad Sci USA
97:3789-3791.
173. Diatchenko L, Lau Y-FC, Campbell AP,
Chenchik A, Moqadam F, Huang B, Lukyanov
S, Lukyanov K, Gurskaya N, Sverdlov ED and
Siebert PD (1996) Suppression subtractive
hybridization: A method for generating
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probes and libraries. Proc Natl Acad Sci USA
93:6025-6030.
174. Gurskaya NG, Diatchenko L, Chenchik A,
Siebert PD, Khaspekov GL, Lukyanov KA,
Vagner LL, Ermolaeva OD, Lukyanov SA and
Sverdlov ED (1996) Equalizing cDNA
subtraction based on selective suppression of
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transcripts induced by phytohemaglutinin and
phorbol 12-myristate 13-acetate. Anal Biochem
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175. den Hollander AI, ten Brink JB, de Kok YJM,
van Soest S, van den Born LI, van Driel MA, van
de Pol DJR, Payne AM, Bhattacharya SS,
Kellner U, Hoyng CB, Westerveld A, Brunner
HG, Bleeker-Wagemakers EM, Deutman AF,
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AAB (1999) Mutations in a human homologue
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(RP12). Nature Genet 23:217-221.
General introduction
176. den Hollander AI, Heckenlively JR, van den
Born LI, de Kok YJM, van der Velde-Visser SD,
Kellner U, Jurklies B, van Schooneveld MJ,
Blankenagel A, Rohrschneider K, Wissinger B,
Cruysberg JRM, Deutman AF, Brunner HG,
Apfelstedt-Sylla E, Hoyng CB and Cremers FPM
(2001) Leber congenital amaurosis and retinitis
pigmentosa
with
Coats-like
exudative
vasculopathy are associated with mutations in the
crumbs homologue 1 (CRB1) gene. Am J Hum
Genet 69:198-203.
177. den Hollander AI, Johnson K, de Kok Y, Klebes
A, Brunner HG, Knust E and Cremers FPM
(2001) CRB1 has a cytoplasmic domain that is
functionally conserved between human and
Drosophila. Hum Mol Genet 10:2767-2773.
178. den Hollander AI, Ghiani M, de Kok Y,
Wijnholds J, Ballabio A, Cremers FPM and
Broccoli V (2002) Isolation of Crb1, a mouse
homologue of Drosophila crumbs, and analysis
of its expression pattern in eye and brain. Mech
Dev 110:203-207.
39
Chapter
2
Isolation and mapping of novel candidate genes for retinal
disorders using suppression subtractive hybridization
Anneke I. den Hollander1, Marc A. van Driel1, Yvette J.M. de Kok1, Dorien J.R.
van de Pol1, Carel B. Hoyng2, Han G. Brunner1, August F. Deutman2 and Frans
P.M. Cremers1
Departments of 1Human Genetics and 2Ophthalmology, University Medical Center Nijmegen,
Nijmegen, the Netherlands
Genomics 58:240-249 (1999)
Novel candidate genes for retinal disorders
We have constructed human cDNA libraries enriched for retina- and retinal pigment
epithelium
(RPE)/choroid-specific
cDNAs
through
suppression
subtractive
hybridization. The sequence of 314 cDNAs from the retina-enriched library and 126
cDNAs from the RPE/choroid-enriched library was analyzed. Based on the absence of a
database match, 25% of the retina cDNA clones and 16% of the RPE/choroid cDNA
clones are novel cDNAs. The expression profiles of 86 retina and 21 RPE/choroid cDNAs
were determined by a semiquantitative reverse transcription polymerase chain reaction
technique. Thirty-three cDNAs were expressed exclusively or most prominently in retina
or RPE/choroid. These cDNAs were mapped in the human genome by radiation hybrid
mapping. Eleven cDNAs colocalized with loci involved in retinal disorders. One cDNA
mapped in a 1.5-megabase critical region for autosomal recessive retinitis pigmentosa
(RP12). Another cDNA was assigned to the 7.7-cM RP17 linkage interval. Seven cDNAs
colocalized with four loci involved in Bardet-Biedl syndrome.
Introduction
The highly specialized function of the retina requires a large number of specifically expressed
genes. Isolation of such genes has contributed to the understanding of the function of the
retina and the pathogenesis of retinal disease. Over the last few years the molecular causes of
several retinal disorders have been elucidated using positional cloning and candidate gene
approaches. As most nonsyndromic retinal disease genes identified thus far are expressed
exclusively or predominantly in the retina or in the retinal pigment epithelium (RPE), the
candidate gene approach is becoming more and more popular1-3.
Thirteen retinal disease genes encode proteins that are part of the phototransduction cascade,
for example rhodopsin, the α subunit of transducin and the α and β subunits of cyclic
guanosine-monophosphate phosphodiesterase (cGMP-PDE)3-6. Two genes encode proteins
essential for the structure of the photoreceptor disks (RDS/peripherin and ROM-1)5. Other
retina-specific genes involved in retinal disorders include the retina-specific ATP-binding
cassette transporter (ABCR/ABCA4) gene7, a retina-specific transcription factor (CRX)8, the
Tubby-like protein (TULP-1) gene9,10, the X-linked retinoschisis gene11, and a retina-specific
calcium channel (CACNA1F) gene12,13.
Visual processing and survival of the retina depend on a number of highly specialized
functions carried out by the RPE. The molecular mechanisms underlying these processes are
likely to require a broad spectrum of proteins, some of which may be unique and specific to
43
Chapter 2
the RPE. Defects in genes expressed specifically in the RPE may disrupt this supportive
function and can lead to disorders of the retina, as exemplified by RPE65 (refs 14,15), cellular
retinaldehyde-binding protein (CRALBP/RLBP1)16 and bestrophin17. RPE65 and CRALBP
have a role in the transport and metabolism of vitamin A from RPE cells to photoreceptor
cells in the retina18,19. Bestrophin may have a role in the metabolism and transport of
polyunsaturated fatty acids17.
Although a number of genes involved in retinal disorders do not show expression that is
restricted to the eye, e.g. gyrate atrophy (OAT)20, choroideremia (REP1)21, Sorsby’s fundus
dystrophy (TIMP-3)22, RP3 (RPGR)23,24 and RP2 (ref. 25), the isolation of a large number of
novel genes expressed exclusively or predominantly in the eye will contribute greatly to the
understanding of the function of the retina and the pathogenesis of retinal disease. Several
methods have been used to isolate retina- and RPE-specific genes, including differential
hybridization26-28, subtractive hybridization29-35, and large-scale sequencing of retinal cDNA
libraries36,37.
In order to isolate novel retina- and RPE-specific genes, we constructed cDNA libraries
enriched for retina- and RPE/choroid-specific cDNAs using a polymerase chain reaction
(PCR)-based suppression subtractive hybridization technique, which normalizes sequence
abundance and achieves high enrichment for differentially expressed cDNAs by a single
round of subtractive hybridization38. Subtraction was performed against a mixture of cDNAs
from several nonocular tissues. The sequence of 440 cDNAs was analyzed and compared to
sequences deposited in the GenBank and EMBL databases. The expression profiles of 107
(predominantly novel) cDNAs were determined by semiquantitative reverse transcription
polymerase chain reaction (RT-PCR). Thirty-three cDNAs were expressed specifically or at
highest levels in the retina or RPE/choroid compared to other tissues. These were mapped in
the human genome by radiation hybrid mapping. Several cDNAs map near loci involved in
retinal disorders.
Materials and methods
Suppression subtractive hybridization
For suppression subtractive hybridization, poly(A)+ RNA from five human tissues (brain,
kidney, liver, heart, skeletal muscle) and total placental RNA was purchased from Clontech.
Total RNA from retina, RPE/choroid, psoas muscle and stomach (mucosal layer) was isolated
with RNAzol B (Campro Scientific). Retina and RPE/choroid poly(A)+ RNA was isolated
44
Novel candidate genes for retinal disorders
with the Oligotex mRNA Kit (Qiagen). Complementary DNA (cDNA) was synthesized using
the SMART PCR cDNA Synthesis Kit (Clontech). First-strand cDNA synthesis was
performed on 0.4 µg poly(A)+ RNA (brain, kidney, liver, heart, skeletal muscle, retina,
RPE/choroid) or 1 µg total RNA (placenta, stomach, psoas muscle). Single-stranded cDNA
was amplified by long-distance PCR with the Advantage cDNA PCR Kit (Clontech). Prior to
subtraction, cDNA was size fractionated with Chroma Spin-1000 columns (Clontech) and
digested by RsaI. Digested cDNA was purified with the Advantage PCR-Pure Kit (Clontech).
Subtraction of retina and RPE/choroid cDNA was performed with the PCR-Select cDNA
Subtraction Kit (Clontech) according to the method described by Diatchenko et al.38. Tester
cDNA consisted either of retina or RPE/choroid cDNA. Driver cDNA consisted of a mixture
of cDNAs from brain, kidney, liver, heart, skeletal muscle, placenta, stomach (mucosal layer),
and psoas muscle. Tester cDNA was divided into two portions and ligated to two different
adaptors. In a first hybridization step, an excess of denatured driver (450 ng) was added to
each sample of denatured tester (12 ng). In the second hybridization step, the two primary
hybridization samples were mixed without denaturing, and freshly denatured driver cDNA
(300 ng) was added. Differentially expressed cDNAs were amplified by two suppression PCR
amplifications with the Advantage cDNA PCR Kit (Clontech).
Cloning and analysis of subtracted cDNA
Subtracted PCR products were ligated into plasmid vector pCRII using a T/A cloning kit
(Invitrogen). The resulting constructs were transformed to TOP10F’ competent cells
(Invitrogen) and selected by blue/white screening. Approximately 2000 colonies from the
retina-enriched library and 400 colonies from the RPE/choroid-enriched library were picked
manually and grown for 16 hours in 100 µl LB containing ampicillin (50 µg/ml) in 96-wells
tissue culture plates. After the addition of 100 µl 30% (w/v) glycerol, the plates were stored at
-80°C.
Inserts from cDNA clones were recovered by direct PCR amplification of the culture using
the vector primers T7 and M13 Reverse. PCR products were electrophoresed with a
Centipede horizontal electrophoresis system (200 samples/gel; Owl Scientific). Gels were
blotted on Qiabrane Nylon Plus membranes (Qiagen). To determine which cDNA clones had
a poly(A) tail, blots were hybridized with a (T)24A/C/G oligonucleotide. To screen for
abundant cDNA clones, the blots were also hybridized with oligonucleotides designed from
cDNA sequences encoding rhodopsin (RHO; 5'-CTTCTTGCTATTTACAAAGTGC-3'), the β
subunit of cGMP-PDE (PDE6B; 5'-TGATTTCAGAGTTCATGGACC-3'), MEK kinase 1
45
Chapter 2
(MEKK1; 5'-CAGTATTACTTTGTAGATCACC-3') and an abundant retina-specific cDNA
(RET1;
5'-AATCCAAAGGAACAGGGAGG-3').
Oligonucleotides
(20
pmol)
were
radioactively labeled with 2 pmol [γ-32P]ATP using 10 U T4 polynucleotide kinase (New
England Biolabs) and the kinase buffer provided by the manufacturer. Oligonucleotide
hybridizations were performed in 5× SSPE, 0.3% SDS at 50°C for 16 h. Blots were washed
with 5× SSPE, 0.3% SDS at 45°C.
Insert PCR products were isolated from an agarose gel with the Qiaquick Gel Extraction Kit
(Qiagen). DNA sequencing was performed with the Thermo Sequenase Dye Terminator
Cycle Sequencing Pre-mix Kit (Amersham) using an automated DNA sequencer (Applied
Biosystems, Inc, Model 370A). Clones containing a poly(A) tail were sequenced with a
(T)24A/C/G oligonucleotide, the remaining cDNA clones were sequenced with the vector
primers. Nucleic acid homology searches were performed using the FASTA program39
against the GenBank and EMBL databases.
Expression profile analysis by semiquantitative RT-PCR
For semiquantitative RT-PCR, total RNA from eight human tissues (brain, liver, lung, skeletal
muscle, placenta, heart, spleen, kidney) was purchased from Clontech. Total RNA from
retina, RPE/choroid and two RPE cell lines, D407 (ref. 40) and ARPE-19 (ref. 41), was
isolated by RNAzol B (Campro Scientific) and treated with DNase I (Gibco/BRL). Randomly
primed cDNA was synthesized in a volume of 250 µl from 3.1 µg total RNA using 4 pmol
random hexanucleotides [pd(N)6; Pharmacia], 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01%
(w/v) gelatin, 5 mM MgCl2, 1 mM DTT, 95 U RNAguard (Pharmacia) and 2500 U MMLV
reverse transcriptase (Gibco/BRL). Primer sets were chosen with the PRIMER program
(version 0.5; Whitehead Institute for Biomedical Research) from the nucleotide sequence of
107 retina and RPE/choroid cDNA clones, amplifying products of 100-250 bp. The primer
sets were first tested on genomic DNA to test the optimal annealing temperature and MgCl2
concentration of the PCR. Semiquantitative RT-PCRs were performed in a volume of 35 µl
using 87 ng of randomly primed cDNA, 11.2 pmol of each primer, 10 mM Tris-HCl, pH 8.3,
50 mM KCl, 0.01% gelatin, 0.5 mM MgCl2 (for most primer sets), 160 µM of each dNTP and
1.75 units Taq DNA polymerase (Gibco/BRL). Amplification was performed at 94°C for 1
min, 60°C for 1 min (for most primer sets) and 72°C for 1 min in a microtiter PCR machine
(MG Research). After 25 cycles a 10-µl sample was taken from the PCRs, and an additional 5
cycles (for a total of 30 cycles) were performed. Again, a 10-µl sample was taken from the
PCRs, and the PCR was continued for an additional 5 cycles for a total of 35 cycles.
46
Novel candidate genes for retinal disorders
Northern blot analysis
RNA from the tissues used for the RT-PCR was also used for the Northern blot analysis. Ten
micrograms of total RNA of each tissue was loaded on a 1% agarose gel containing 1× MOPS
and 18% formaldehyde. Gels were blotted on GeneScreen Plus membranes (NEN Life
Science Products) with 10× SSC. Hybridizations were performed in 5× SSPE, 50% deionized
formamide, 5× Denhardt’s, 1% SDS at 60°C. Blots were washed in 2× SSPE, 2% SDS at
60°C.
Radiation hybrid mapping
Radiation hybrid mapping was performed with the Stanford G3 Radiation Hybrid Panel
(Research Genetics). PCRs were performed in a volume of 10 µl using 25 ng DNA of each
hamster/human hybrid cell line, 4 pmol of each primer, 10 mM Tris-HCl, pH 8.3, 50 mM
KCl, 1.5 mM MgCl2 (for most primer sets), 5 mM DTT, 200 µM of each dNTP and 0.4 units
Taq DNA polymerase (Gibco/BRL). Amplification was performed for 35 cycles at 94°C for 1
min, 60°C for 1 min (for most primer sets) and 72°C for 1 min in a microtiter PCR machine
(MG Research). The localization of the cDNA fragments in the Stanford G3 Radiation Hybrid
Map was determined at the Stanford Human Genome Center website (http://wwwshgc.stanford.edu).
Results
Subtraction efficiency
An efficient suppression subtractive hybridization decreases the concentration of ubiquitously
expressed (housekeeping) genes, and increases the concentration of tissue-specific genes38.
Equal amounts of subtracted and unsubtracted cDNA from the retina and RPE/choroid were
size-separated and blotted. These blots were hybridized with two housekeeping genes,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB). The retina cDNA
blot was hybridized with three retina-specific genes: rhodopsin (RHO), the β subunit of
cGMP-PDE (PDE6B) and a retina-specific cDNA isolated in this study (RET10), which is
expressed at a low level in retina. The RPE/choroid cDNA blot was hybridized with two RPEspecific genes, i.e. RPE65 and CRALBP. In both libraries the concentration of the two
housekeeping genes was considerably decreased and the concentration of the tissue-specific
genes was increased considerably after subtraction (Figure 1).
47
Chapter 2
retina enriched library
A
ACTB
GAPDH
RHO
PDE6B
B
RPE/choroid
enriched library
A
B
ACTB
GAPDH
RPE65
Figure 1. Southern blot analysis of
retina and RPE/choroid cDNA before
(lane B) and after subtraction (lane
A). The concentrations of two housekeeping genes, GAPDH and ACTB,
are
considerably
lower
after
subtraction. The concentrations of
three retina-specific genes, RHO,
PDE6B, and RET10, and two RPEspecific genes, RPE65 and CRALBP,
are increased considerably after
subtraction.
CRALBP
RET10
Sequence analysis of cDNAs from the retina- and RPE/choroid-enriched
libraries
Prior to sequencing, cDNA clones were analyzed for the presence of a poly(A) sequence by
Southern blot analysis with an (T)24A/C/G oligonucleotide. From the retina-enriched library,
121 of 314 cDNA clones (39%) were positive; from the RPE/choroid-enriched library 65 of
126 (52%) were positive. Positive clones in most cases represent 3'-end cDNA fragments
carrying a poly(A) tail, but may also be cDNA fragments with an internal poly(A) or poly(T)
stretch.
Initially, 118 cDNAs from the retina-enriched library were partially sequenced and compared
to the GenBank and EMBL databases. Four cDNAs were found more than once, including
PDE6B (13/118), RHO (3/118), MEK kinase 1 (MEKK1) (4/118), and RET1 (4/118) (Table
2). The sequencing of additional cDNA clones was preceded by Southern blot analysis of 196
retina cDNAs with radioactively labeled oligonucleotides specific for PDE6B, RHO, MEKK1,
and RET1. Thirty-nine cDNA clones were found to be positive, and were excluded from
sequence analysis. The remaining 157 cDNAs were sequenced. In conclusion, we analyzed a
total of 314 cDNAs from the retina-enriched library, 275 of which were sequenced.
48
Novel candidate genes for retinal disorders
Table 1. Database match of cDNA clones from the retina- and RPE/choroid-enriched cDNA librariesa
Database match
1. Known genes
a. Not retina-specific
b. Retina-specific
2. ESTs/cDNAs
a. Not retina-specific
b. Retina-specific
c. Pineal gland-specific
3. No database match
Total
Retina cDNA library
b
39 (14)
27 (10)b
109 (39)
27 (10)b
4 (2)
69 (25)
275b
RPE/choroid cDNA library
24 (19)
7 (5)
71
2
2
20
126
(56)
(2)
(2)
(16)
a
The number (and percentage) of clones which show more than 95% identity to known genes (1) or
ESTs and cDNAs (2) or that show no database match (3) are given.
b
Thirty-nine repetitively encountered clones were excluded from sequence analysis and were not
included in this table.
From the RPE/choroid-enriched library, 126 cDNA clones were sequenced and compared to
sequences deposited in the GenBank and EMBL databases. Repetitively encountered clones
in this library included insulin-like growth factor binding protein 5 (IGFBP5) (12/126), RHO
(3/126) and MEKK1 (3/126).
Comparison of the cDNA sequences with cDNAs and ESTs deposited in databases resulted in
“expression profiles” based on their representation in other cDNA libraries (Table 1). In the
retina- and RPE/choroid-enriched libraries, 58 (22%) and 11 (9%) of the cDNA clones
respectively show homology to known retina- or pineal gland-specific cDNAs or known
genes; 69 (25%) and 20 (16%) of the cDNA clones respectively represent novel cDNAs; and
148 (53%) and 95 (75%) respectively show homology to cDNAs and known genes expressed
in other tissues as well. However, when the 39 frequently encountered cDNAs from the
retina-enriched library which were not sequenced are also considered, approximately 30% of
the retina-enriched cDNAs show homology to known retina- or pineal gland-specific cDNAs
or genes, 50% show homology to cDNAs expressed in other tissues as well, and 20% are
novel.
Several genes already known to be involved in retinal disorders were isolated from the retinaand RPE/choroid-enriched libraries, including PDE6B, RHO, the α-subunit of cGMP gated
channel (CNGC), myosin VIIA (MYO7A) and tissue inhibitor of metalloproteinases-3 (TIMP3).
The 314 analyzed retina cDNA clones represent 199 different cDNA fragments; 161 were
49
Chapter 2
Table 2. Known genes encountered in the retina-enriched library
Gene
cGMP phosphodiesterase β
MEK kinase 1 (mouse)
Nucleosome assembly protein
Rhodopsin
cGMP gated channel α
Hexokinase II pseudogene
Phospholipase A2
Prothymosin α
α-Tropomyosin
Capping protein α1
Chromosomal protein
Cytochrome c oxidase
Dystrobrevin isoform DTN-2
HS1 protein
Insulin growth factor-binding protein 5
KIAA0168 gene
KIAA0240 gene
Myosin VIIA
Mouse tob family
Na-dependent neurotransmitter transporter (rat)
Protein phosphatase 1 subunit β
scg (mouse)
Stanniocalcin precursor
Transcription factor ETR103
Voltage-dependent Ca2+ channel β-2c
U2 snRNA associated B antigen
X16 gene (mouse)
No. of clones
>20
>11
4
>3
3
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
found only once, 20 were found twice, 6 were found 3 times, and 12 were found 4 times or
more. The 126 analyzed RPE/choroid cDNA clones represent 101 different cDNA fragments;
87 were found only once, 9 were found twice, 3 were found 3 times, and only 2 were found 4
times or more. Seventeen cDNAs were encountered both in the retina and in the RPE/choroidenriched library.
Expression profile analysis of novel retina and RPE/choroid cDNAs
Eighty-six cDNA clones from the retina-enriched library and 21 cDNA clones from the
RPE/choroid-enriched library were selected for expression profile analysis by RT-PCR. Based
on their comparison with DNA sequences in the GenBank and EMBL databases, 60 of these
50
Novel candidate genes for retinal disorders
clones were novel, 11 were identical to cDNAs represented specifically in other retina cDNA
libraries, and 18 were identical to cDNAs represented in retina and/or pineal gland, brain,
neuroepithelium, or cochlea cDNA libraries. Furthermore, 5 were identical to cDNAs listed in
the BodyMap database (http://cookie.imcb.osaka-u.ac.jp/bodymap/) and 13 cDNA clones
were identical to cDNAs represented in other libraries that were of interest because of their
homology to known genes or their high abundance in retina, pineal gland, or brain cDNA
libraries. We performed a semiquantitative RT-PCR (25, 30 and 35 cycles) using RNA from
12 tissues, including liver, lung, skeletal muscle, placenta, heart, brain, spleen, kidney, retina,
two RPE cell lines (ARPE-19 and D407), and RPE/choroid.
To test the usefulness of our semiquantitative RT-PCR, we determined the expression profile
of several known retina- or RPE-specific genes, e.g., ABCR7, RDS42, ROM-1 (ref. 26),
phosducin (PDC)43, and CRALBP44 (Table 3). All genes were expressed considerably higher
in retina or RPE/choroid than in all other tissues, but for some, lower levels of expression
were also detected in other tissues (e.g., Figure 2).
Based on the expression profiles of the 86 cDNA clones selected from the retina-enriched
library, 30 were considered candidate genes for retinal disorders. Of these 30 cDNA clones, 9
were expressed only in retina or RPE/choroid, 12 were expressed considerably higher in
retina or RPE/choroid than in all other tissues tested, and 9 were expressed in retina or
RPE/choroid and in one or two other tissues after the same number of PCR cycles. Of the 21
cDNA clones selected from the RPE/choroid-enriched library, 2 were expressed only in retina
or RPE/choroid, and 1 was expressed considerably higher in the retina and RPE/choroid than
in the other tissues.
Thus, after expression profiling, 33 of 107 cDNAs were considered good candidates for
retinal disorders. Of these 33 cDNA clones, 15 showed no database match; 8 clones were
ABCR
1 2 3 4 5 6 7 8 9 10 11 12
RET1
1 2 3 4 5 6 7 8 9 10 11 12
RET1D2
1 2 3 4 5 6 7 8 9 10 11 12
25
30
35
GAPDH
Figure 2. Expression profile analysis of the ABCR gene and two clones isolated from the
retina-enriched library (RET1 and RET1D2) by semiquantitative RT-PCR (25, 30, and 35
cycles). GAPDH serves as a positive control for the RNA. (1) Liver, (2) lung, (3) skeletal
muscle, (4) placenta, (5) heart, (6) brain, (7) spleen, (8) kidney, (9) retina, (10) ARPE-19, (11)
D407, (12) RPE/choroid.
51
Liver
------+
-----
Lung
------+
-----
Placenta
--+
--+
--+
-----
Heart
----+
--+
-----
Brain
--+
--+
-++
---++
Spleen
----+
-++
-----
Kidney
--+
--+
-++
-----
Retina
-++
+++
+++
--+
+++
ARPE19
-----++
----+
D407
------+
-----
RPE +
choroid
-++
-++
+++
--+++
Chrom.
region
Chrom. (cM)
1
213-216
3
117
117
5
74-79
141
6
13-17
13-17
60-65
Clone
name
RET3C11
RPE63
RPE1H6
RET2E2
RET1A6
RET1F6
RET1F12
RET5
GenBank
Accession
No.
AI444813/14
AI444822
AI444824
AI444808
AI444832
AI444803
AI444804
AI444827
Sk.
ARPELiver Lung muscle Placenta Heart Brain Spleen Kidney Retina
19
D407
--- --------- --------+
------- --------- --------+
------- --------- -------++
------- --------- --+
------+
------- --------- --------+
------- --------- -------++
------+ -++
--+
-++
-++ -++
-++
-++
+++
--+
--+
--+ --+
--+
-++
--- --+
-++
--+
-++
--+
--+
RPE/choroid, or considerably higher in the retina or RPE/choroid than in most other tissues testeda
RPE
+
chor.
----------+
---++
---
Table 4. Chromosomal localization and expression profiles of 33 retina (RET) and RPE/choroid cDNA clones that are expressed specifically in the retina or
RT-PCR products detected in agarose gel electrophoresis after 25, 30 and 35 cycles are indicated by a + at the first, second or third position, respectively, for
each tissue analyzed.
a
Gene
ABCR
RDS
ROM-1
PDC
CRALBP
Skeletal
muscle
------+
-----
Table 3. Expression profile analysis of five known retina- or RPE-specific genes by semiquantitative RT-PCRa
RET1B10b
RET1A4
RET1
RET3b
RET3E4b
RET1B3b
RET2G9
RET1D2
RET2F8b
RPE1G2b
RET3F3b
RET1B8
RET1H7b
RET3B3
RET3E3b
RET1G11
RET29
RET3D11b
RET1F4b
RET2C9b
RET10b
RET1A12b
RET2A6b
RET1D1
RET4B7
AI444837
AI444831
AI444825
AI444826
AI444817/18
AI444835
AI444810
AI444838
AI444809
AI444823
AI444819
AI444836
AI461441
AI444811/12
AI444816
AI444805
AI444830
AI444815
AI444839
AI444807
AI444828/29
AI444833/34
AI444806
AI461440
AI444821
--+
--+
--+
---++
-------------++
----------+
------+
--------+
--+
-++
-++
------+
--+
--+
--------+
-++
---------++
--+
----+
--------+
--+
-++
-++
--+
----+
--------------+
---------++
--+
----+
--------+
--+
-++
-++
--+
-------++
---------++
--------+
--+
--+
----+
----+
--+
--+
---
-++
--+
-++
----+
----+
---------++
---------++
--+
----+
--------+
--+
-++
-++
-++
----+
-++
--+
------+
-++
-++
----+
-----+
-++
--+
---++
-------++
-++
-++
--+
--+
----+
-++
--+
---------++
----------+
------+
----+
----+
-++
-++
--+
--+
--+
-++
--+
--+
---------++
---------++
--+
---++
--------+
--+
+++
+++
+++
--+
-++
-++
-++
+++
--+
-++
-++
+++
--+
-++
--+
-++
+++
-++
+++
-++
--+
-++
-++
-++
+++
--+
--+
--+
--------+
------+
----+
----------+
------+
-------++
-++
--+
--+
--------------------+
----------+
------+
-------++
---
--+
-++
------+
--+
-------++
--+
-++
---------++
---++
------+
----+
-++
Chromosomal localizations were determined by radiation hybrid mapping. RT-PCR products detected in agarose gel electrophoresis after 25, 30 or 35 cycles
are indicated by a + at the first, second or third position, respectively, for each tissue analyzed.
b
Clones that show no database match to other ESTs (<90% homology) are in boldface.
a
?
21
X
17
19
16
12
14
15
8
11
7
60-65
60-66
65-66
73-74
74
82-93
62-65
110
90-96
46-58
22-26
70-71
75-77
75-77
75-77
75-82
50-58
50-58
69-81
44-50
122-150
122-150
122-150
?
?
Chapter 2
identical to cDNAs represented specifically in other retina cDNA libraries; 4 were identical to
cDNAs represented in retina and/or pineal gland, brain, neuroepithelium, or cochlea cDNA
libraries; 3 were identical to cDNAs from the Bodymap database; and 3 were identical to
cDNAs represented in other libraries that were considered interesting by homology to known
genes or high abundance in retina, pineal gland, or brain cDNA libraries. The nucleotide
sequences of the 33 retina- and RPE/choroid-specific cDNAs were submitted to the
GenBank/dbEST databases; GenBank accession numbers are listed in Table 4. Clones that
show no database match to other ESTs (<90% homology) are in boldface.
Radiation hybrid mapping of retina- and RPE/choroid-specific cDNAs
Thirty of the thirty-three selected cDNA clones were mapped in the human genome by
radiation hybrid mapping (Table 4). One cDNA, RET2E2, was mapped by its homology to a
genomic clone of which the complete sequence was deposited in the database. Two cDNAs
(RET1D1 and RET4B7) could not be mapped. The genetic intervals of the mapped cDNA
clones were determined relative to framework markers. These genetic intervals were
compared to linkage intervals of nonsyndromic and syndromic retinal disorders using the
RetNet database (http://www.sph.uth.tmc.edu/Retnet/) and reviews listing retinal disease
loci3,6,45,46. Eleven cDNA clones localized near loci involved in retinal diseases (Table 5).
RET3C11 is a positional candidate for autosomal recessive RP (RP12) on 1q31-q32.1 (ref.
47). The RP12 critical region has been refined to a 1.3-cM interval48. RET3C11 was mapped
on a YAC contig spanning the RP12 critical region48. Recently, a locus for age-related
macular degeneration (ARMD1) was mapped to 1q25-1q31 (ref. 49). The involvement of
RET3C11 in RP and ARMD is currently under investigation.
RET1G11 is a positional candidate for autosomal dominant RP (RP17), the gene defect of
which was localized on 17q22. The RP17 locus was first described in two South African
families50,51. Recently, we found linkage to the RP17 locus in a large Dutch family and
refined the critical region to a 7.7-cM interval between markers D17S1607 and D17S948 (ref.
52). By radiation hybrid mapping, RET1G11 was localized near marker D17S1607 and is
currently being analyzed. RET1G11 may also be a candidate gene for cone-rod dystrophy
(CORD4) on 17q (ref. 53).
Seven cDNA clones are positional candidate genes for Bardet-Biedl syndrome (BBS), which
is characterized by pigmentary retinopathy, mental retardation, obesity, and polydactyly. Five
BBS loci have been identified until now; BBS1 on chromosome 11q13 (ref. 54), BBS2 on
16q21 (ref. 55), BBS3 on 3p13-p12 (ref. 56), BBS4 on 15q22.3-q23 (ref. 57), and BBS5 on
54
Novel candidate genes for retinal disorders
Table 5. Comparison of chromosomal map locations between 11 chorioretinal cDNAs and inherited
retinal disease locia
Chrom.
Disease
region (cM) locus
213-216
RP12
ARMD1
117
BBS3
62-65
BBS1
46-58
ACHM1
70-71
BBS4
75-77
BBS2
75-82
RP17
CORD4
Cytogenetic
location
1q31-q32.1
1q25-q31
3p13-p12
11q13
14
15q22.3-q23
16q21
17q22
17q
Chrom.
Chrom. Clone name
region (cM)
1
RET3C11
213-216
203-216
3
RPE63 / RPE1H6
114-125
11
RET2G9
63-72
14
RPE1G2
?
15
RET1B8
63-71
16
RET1H7/ RET3B3 / RET3E3
72-81
17
RET1G11
76-84
?
19
RET1F4
69-81
OPA3
19q13.2-q13.3 68-69
a
The genetic mapping intervals of these clones were determined relative to framework markers.
2q31 (ref. 58). Positional candidates are RET2G9 for BBS1; RET1H7, RET3B3 and RET3E3
for BBS2; RPE63 and RPE1H6 for BBS3; and RET1B8 for BBS4.
RPE1G2 may be a positional candidate for total colorblindness (ACHM1) on chromosome
1459. RET1F4 maps near a locus for optic atrophy with ataxia and 3-methylglutaconic aciduria
(OPA3) on chromosome 19q13.2-q13.3 (ref. 60).
Some cDNAs mapped in this study were localized in the same chromosomal region, and may
represent different RsaI fragments from the same gene. For example, RPE63 and RPE1H6
map to the same region on chromosome 3, and have similar expression profiles (Table 4).
A cluster of four cDNAs (RET5, RET1B10, RET1A4, RET1) maps to 6p21 (60-66 cM), and
the expression profiles of these cDNAs are very similar (Table 4). We have isolated a cDNA
clone from a full-length retina cDNA library, and demonstrated that RET1 and RET1A4
belong to the same gene. RET1A4 colocalizes with the RDS gene in the radiation hybrid map,
and the expression profile of RDS is similar to that of all four cDNAs (Table 3). This result
suggests that these cDNAs may belong to the RDS gene. Northern blot analysis shows that the
RDS gene is represented by transcripts of approximately 3 and 6.5 kb42. The 3-kb transcript
has been sequenced and deposited in the database. The four cDNAs isolated in this study
could represent RsaI fragments of the 6.5-kb transcript. RET1 was analyzed by Northern blot
analysis and hybridizes to a transcript of approximately 6 kb (Figure 3).
Other cDNAs fragments which may belong to the same gene are RET1F6 and RET1F12 on
chromosome 6 (13-17 cM); RET3 and RET3E4 on chromosome 7 (73-74 cM); RET1H7,
55
Chapter 2
1
2
3
4
5
6
7
8
9
10
11 12
kb
9.49
7.46
4.40
2.37
Figure 3. Northern blot
analysis of RET1. Lanes
contain 10 µg of total RNA
from the following human
tissues: (1) liver, (2) lung, (3)
skeletal muscle, (4) placenta,
(5) heart, (6) brain, (7) spleen,
(8) kidney, (9) retina, (10)
ARPE-19, (11) D407, (12)
RPE/choroid.
RET3B3 and RET3E3 on chromosome 16 (75-77 cM); RET29 and RET3D11 on chromosome
19 (50-58 cM); and RET10, RET1A12, and RET2A6 on the X chromosome (122-150 cM).
Discussion
This study describes the isolation and mapping of 33 retina- and RPE-specific cDNAs.
Libraries enriched for retina- and RPE/choroid-specific cDNAs were constructed through
suppression subtractive hybridization. The sequence of 314 cDNAs from the retina-enriched
library and 126 cDNAs from the RPE/choroid-enriched library was analyzed and compared to
the GenBank and EMBL databases. The expression profiles of 86 retina and 21 RPE/choroid
(predominantly novel) cDNAs were determined by a semiquantitative RT-PCR. Thirty-three
cDNAs were expressed specifically or considerably higher in retina or RPE/choroid than in
most other tissues analyzed, and were mapped in the human genome by radiation hybrid
mapping.
Subtraction efficiency and evaluation of the libraries
The subtraction of the retina and RPE/choroid cDNA was very efficient, as the concentration
of two housekeeping genes (GAPDH and ACTB) is considerably decreased after subtraction,
and the concentration of several tissue-specific genes (RHO, PDE6B, and RET10 for the
retina and RPE65 and CRALBP for the RPE) is considerably increased after subtraction.
Based on homology with retina-specific genes/cDNAs and on expression profiling by RTPCR, approximately 30% of the retina-enriched cDNA library is estimated to be retinaspecific. In an unsubtracted, nonnormalized retina cDNA library, approximately 2% of the
cDNAs is estimated to be retina-specific (S. Bernstein, personal communication), suggesting
a considerable enrichment of retina-specific transcripts in the retina-enriched cDNA library.
The RPE/choroid-enriched library was constructed from RPE that was attached to the choroid.
The attached choroidal tissue hampers the isolation of RPE-specific genes from this library.
56
Novel candidate genes for retinal disorders
Nevertheless, this library is the first human RPE subtracted library reported in the literature.
One subtracted RPE library has been constructed previously from an RPE cell line31. Gene
expression in this cell line differs from that in RPE tissue in vivo, as several of the known
genes isolated from this library are genes involved in cell growth and proliferation31.
In contrast to conventional subtraction methods and differential hybridization techniques
previously used to isolate retina-specific genes, suppression subtractive hybridization allows
the isolation of tissue-specific transcripts expressed at moderate or low levels in the tissue of
interest. Such transcripts are underrepresented in the collection of retina ESTs deposited in the
public databases. Consequently, the suppression subtractive hybridization technique enables
the isolation of novel retinal and RPE/choroidal cDNAs. Twenty-five percent of the retina
cDNA clones and 16% of the RPE/choroid cDNA clones were novel.
Thirty-nine percent of the cDNA clones from the retina-enriched library and 52% of the
cDNA clones from the RPE/choroid-enriched library contain a poly(A) stretch, most of which
represent cDNA fragments containing a poly(A) tail. As most ESTs deposited in the public
databases represent 3' ends of genes, most cDNAs encountered in these libraries that show no
database match are truly novel.
The representation of RsaI fragments for several known genes in the retina-enriched cDNA
library was analyzed (data not shown). Most RsaI fragments encountered are small RsaI
fragments (<500 bp) from the 3' region of the genes. As suppression subtractive hybridization
is a PCR-based technique, small RsaI fragments will preferentially be amplified over large
RsaI fragments. A disadvantage of this method is that genes that lack small RsaI fragments
are underrepresented in the libraries. To bypass this problem, other restriction enzymes might
be used instead of RsaI.
The cDNA used for the subtraction was synthesized by oligo(dT) priming with the SMART
PCR cDNA Synthesis Kit (Clontech). With this kit high yields of cDNA can be amplified
from nanogram amounts of poly(A)+ or total RNA. As the cDNA is amplified by PCR and not
all transcripts will be amplified equally efficiently, the cDNA will not be a complete
representation of the mRNA in vivo. Moreover, we encountered cloning artifacts in two genes
(PDE6B and RHO), which are both probably caused by priming to an internal poly(T) stretch
during the PCR amplification of the cDNA. This may have led to the preferential
amplification of the 3' end of the PDE6B gene during the cDNA synthesis, as one of the RsaI
fragments was encountered 13 times in the first 118 cDNA clones analyzed from the retinaenriched library (Table 2).
57
Chapter 2
Expression profiling
Another important aspect of our strategy in isolating novel retina- and RPE-specific genes is
the expression profiling of a large number of cDNAs from the retina- and RPE/choroidenriched libraries by a semiquantitative RT-PCR method. For most ESTs deposited in the
public databases, no expression data have been determined. The different cDNA libraries in
which an EST is encountered may give an idea of its expression, but will depend largely on
how many cDNAs from each tissue type are deposited in the public databases.
From 314 retina cDNA clones that were analyzed, 86 were selected for expression profile
analysis by a semiquantitative RT-PCR. Thirty cDNAs are expressed specifically in the retina
or considerably higher in the retina than in most other tissues tested. From 126 analyzed
RPE/choroid cDNA clones, 21 were selected for expression profile analysis. Three cDNAs
are expressed specifically in the retina or RPE/choroid. No cDNAs were isolated that are
expressed exclusively in the RPE. This may have two reasons. First, retina and RPE cannot be
separated completely. Therefore, retina RNA will contain approximately 1% RPE RNA and
vice versa. Consequently, RPE-specific cDNAs will also show expression in the retina on RTPCR. Secondly, the RNA used for the semiquantitative RT-PCR was isolated from RPE that
was attached to the choroid. As the amount of RNA from the RPE is only a fraction of the
isolated RNA, truly RPE-specific cDNAs will only be detected after a larger number of
cycles. It is therefore possible that the expression detected in the RPE/choroid by RT-PCR
would actually be higher in pure RPE tissue. To compensate for this problem, we included
two RPE cell lines in the RT-PCR panel. However, the expression of genes in these cell lines
does not represent the in vivo situation.
Mapped retina- and RPE-specific cDNAs
The 33 cDNAs expressed specifically or at a high level in the retina or RPE/choroid represent
good candidate genes for retinal disorders. These cDNAs were mapped in the human genome
by radiation hybrid mapping. Eleven of these cDNAs localized near loci involved in retinal
disorders. Candidate cDNAs were isolated for retinitis pigmentosa, age-related macular
degeneration, Bardet-Biedl syndrome, total colorblindness, cone-rod dystrophy, and optic
atrophy. We are currently investigating two cDNAs (RET3C11 and RET1G11) for their
involvement in two forms of RP (RP12 and RP17).
Several cDNAs mapped in this study localize to the same chromosomal region. These cDNAs
may represent different RsaI fragments of the same gene. A cluster of four cDNAs on 6p21
(60-66 cM) may represent RsaI fragments of a 6.5-kb transcript of the RDS gene.
58
Novel candidate genes for retinal disorders
In the near future we will scale up the analysis of the retina- and RPE/choroid-enriched cDNA
libraries. The expression profiling of cDNAs provides important information that is highly
complementary to the ongoing human genome sequencing project. We will concentrate future
efforts on the structural characterization of retina- or RPE-specific genes which should enable
us to identify several novel genes underlying chorioretinal diseases.
Acknowledgements
We thank Dr. L. Pels of The Netherlands Ophthalmic Research Institute, Amsterdam, The
Netherlands, for the collection of retina and RPE/choroid tissues. We thank Dr. J. Janssen, Dr.
A. Davis and Dr. L. Hjelmeland for kindly providing the RPE cell lines. This work was
supported by The Foundation Fighting Blindness, Inc., USA and The British Retinitis
Pigmentosa Society.
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(RP17) on chromosome 17q22. Hum Genet
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KH, Elbedour K, Parvari R, Naidu Yandava C,
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Bardet-Biedl syndrome to chromosome 16q and
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Rokhlina T, Nishimura D, Duyk GM, Elbedour
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Genet 3:1331-1335.
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57. Carmi R, Rokhlina T, Kwitek-Black AE,
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Sheffield VC (1995) Use of a DNA pooling
strategy to identify a human obesity syndrome
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3' untranslated region of the myotonic dystrophy
protein kinase gene. Hum Mol Genet 6:563-569.
Chapter
3
Mutations in a human homologue of Drosophila crumbs
cause retinitis pigmentosa (RP12)
Anneke I. den Hollander1, Jacoline B. ten Brink2, Yvette J.M. de Kok1, Simone
van Soest2, L. Ingeborgh van den Born2,3, Marc A. van Driel1, Dorien J.R. van
de Pol1, Annette M. Payne4, Shomi S. Bhattacharya4, Ulrich Kellner5, Carel B.
Hoyng6, Andries Westerveld7, Han G. Brunner1, Elisabeth M. BleekerWagemakers2, August F. Deutman6, John R. Heckenlively8, Frans P.M.
Cremers1 and Arthur A.B. Bergen2
1
Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, the
Netherlands; 2The Netherlands Ophthalmic Research Institute, Amsterdam, the Netherlands;
3
The Rotterdam Eye Hospital, Rotterdam, the Netherlands; 4Institute of Ophthalmology,
University College London, London, UK; 5Department of Ophthalmology, University Clinic
Benjamin Franklin, Free University Berlin, Berlin, Germany; 6Department of Ophthalmology,
University Medical Center Nijmegen, Nijmegen, the Netherlands, 7Institute of Human
Genetics, University of Amsterdam, Amsterdam, the Netherlands;
Ophthalmology, University of California, Los Angeles, California, USA.
Nature Genet 23:217-221 (1999)
8
Department of
Mutations in CRB1 cause RP12
Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of
diseases that afflicts approximately 1.5 million people worldwide. Affected individuals
suffer from a progressive degeneration of the photoreceptors, eventually resulting in
severe visual impairment. To isolate candidate genes for chorioretinal diseases, we
cloned cDNAs specifically or preferentially expressed in the human retina and the
retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization
(SSH) method1,2. One of these cDNAs (RET3C11) mapped to chromosome 1q31-q32.1, a
region harboring a gene involved in a severe form of autosomal recessive RP
characterized by a typical preservation of the para-arteriolar RPE (RP12)3. The fulllength cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A Glike domains and a C-type lectin domain. This protein is homologous to the Drosophila
melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten
unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous
AluY insertion disrupting the ORF, five homozygous missense mutations and four
compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for
CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the
retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1
mutations trigger a novel mechanism of photoreceptor degeneration.
To isolate novel retinal disease genes, we constructed cDNA libraries enriched for retina- and
RPE-specific genes using SSH1,2. We sequenced 440 RsaI cDNA fragments and selected 107
cDNAs, 60 of which showed no database match, for expression profile analysis by a
semiquantitative RT-PCR. Thirty-three cDNAs were specifically or preferentially expressed
in the retina or RPE and were mapped in the human genome by radiation hybrid mapping1.
The retina-specific cDNA clones RET3C11 mapped to 1q31-q32.1 near marker SHGC-31318
in the Stanford G3 Radiation Hybrid map and was localized on YACs 772C10 and 755E11
within a 3-cM region harboring the RP12 gene (Figure 1A). RP12 is a clinically distinct and
severe form of autosomal recessive RP. Affected individuals experience night blindness from
early childhood and progressive visual field loss. On ophthalmoscopy and with fluorescein
angiography, typical preservation of the RPE adjacent to and under the retinal arterioles is
noted, whereas there is a general loss of RPE throughout the retina. Due to early macular
involvement, patients experience severe visual impairment before the age of 20 years4,5.
We obtained the full-length cDNA sequence by screening a retina cDNA library with the
RET3C11 subtraction clone and reiterated 5’-RACE. The consensus cDNA sequence consists
65
Chapter 3
A
RP12
33 12
S5 1 S 4
1
D D
D1
S2
6
81
AF
2
M3
18
13 0
3
4
GC S28
H
1
S
D
c1
9v
cen
M2
AF
2
747D11
755E11
772C10
200 kb
1
wb
7
0
B
tel
RET3C11
1
CRB1
2
3
4
5
6
7
8
9
R
10
R
11
5’
3’
ATG
AluY
1 kb
TAA
2978+5G>A
Cys948Tyr
Glu995Stop
Arg764Cys
Met1041Thr
Thr745Met
Leu1071Pro
Ser403Stop
C
Cys250Trp
Ala161Val
H. sapiens CRB1 - 1376 aa
Drosophila crumbs - 2139 aa
signal peptide
EGF-like domain
TM
laminin G-domain
C-type lectin domain
Figure 1. Chromosomal map of the critical chromosomal region for RP12 and the structure of
CRB1 and protein product. (A) Localization of the RP12 gene in a 3-cM region between markers
D1S412 and AFM207wb12 (ref. 3). The RET3C11 subtraction clone was mapped near marker
SHGC-31318 by radiation hybrid mapping, and localized on YACs 772C10 and 755E11. (B) The
intron/exon structure of the CRB1 gene. The size of the introns between exons 1-6 were not
determined. The centromere/telomere orientation of CRB1 is not known. The RET3C11
subtraction clone represents an RsaI (R) fragment of an unspliced RNA transcript. Mutation
analysis in RP patients with preserved para-arteriolar RPE revealed an AluY insertion in exon 7
and a 5’ splice site mutation in intron 8. (C) The CRB1 protein structure and missense/nonsense
mutations found in RP12 patients. The CRB1 protein is 35% identical and 55% similar to
Drosophila CRB. TM, transmembrane region.
of 4,361 bp and encodes a predicted protein of 1,376 amino acids (Figure 1C). A 235-bp
fragment of the cDNA was used as a probe in a northern-blot analysis, which revealed a 5-kb
transcript in neural retina (Figure 2A). Using RT-PCR, expression was detected in neural
retina, brain and fetal brain, but not in RPE/choroid, two RPE cell lines, fetal liver, liver, lung,
skeletal muscle, placenta, heart, spleen and kidney (Figure 2B). The consensus cDNA
sequence contains a small poly(A) tail but lacks an upstream polyadenylation site, suggesting
that the 3’ end of the cDNA remains to be determined.
The predicted protein sequence contains a signal peptide6, 19 EGF-like domains7 contained in
4 clusters, 3 laminin A G-like or ALPS (agrin, laminin, perlecan, slit) domains8,9, and a C66
Mutations in CRB1 cause RP12
type lectin (CTL) domain10 (Figure 1C). The presence of EGF-like domains, a signal peptide
and the absence of a transmembrane region suggests that it is an extracellular protein.
Database searches with the predicted protein sequence revealed highest homology to
Drosophila CRB (35% identity, 55% similarity). The protein was therefore denoted CRB
homologue 1 (CRB1). CRB is larger (2,139 aa), has 30 EGF-like and 4 laminin G-like
domains and is a transmembrane protein11. The typical arrangement of laminin G-like
domains flanked by EGF-like domains, however, is conserved between CRB1 and CRB
(Figure 1C).
On the basis of its preferential expression in the retina and its co-localization with the RP12
locus, we considered CRB1 a candidate gene for RP12. The intron/exon structure of CRB1
was determined by ligation-mediated PCR12 and exon-exon PCR. CRB1 consists of 11 exons
spanning at least 40 kb; the splice junctions follow the AG/GT rule. We designed 25 primer
pairs for mutation analysis of the CRB1 ORF and splice junctions (Table 1).
We diagnosed 15 unrelated RP patients with para-arteriolar preservation of the RPE. Of these,
patient 22147 belongs to a large consanguineous pedigree previously investigated in the
search of the RP12 locus13. The other families were too small for conclusive haplotype
analysis, but patients of five additional families (24868, 25977, 25983, RP112 and RP0136)
showed homozygosity for markers in the RP12 region. We carried out single-strand
conformation polymorphism (SSCP) analysis and nucleotide sequencing using DNA from
B
kb
9.5
7.5
4.4
2.4
l iv
er
lun
g
sk
.m
u
pla scl
ce e
he nta
ar
br t
ain
sp
le e
k id n
ne
re y
tin
AR a
P
D4 E-19
07
RP
E/
fet chor
o id
al
b
fet rain
al
liv
er
re
ti
A
na
these 15 patients.
CRB1
GAPDH
1.4
Figure 2. Expression of CRB1 in human tissues and RPE cell lines. (A) A northern blot
containing poly(A)+ RNA from human retina was incubated with a CRB1 cDNA fragment.
The transcript size is approximately 5 kb. (B) RT-PCR on total RNA from ten human adult
tissues, two fetal tissues and two RPE cell lines (ARPE-19, ref. 29; and D407, ref. 30). CRB1
is expressed specifically in retina, adult brain and fetal brain. GAPDH serves as a control.
67
Chapter 3
Table 1. Primer pairs and PCR conditions for mutation analysis of the ORF and splice junctions of
CRB1
Fragment
exon 1
exon 2 fr 1
exon 2 fr 2
exon 2 fr 3
exon 3 fr 1
exon 3 fr 2
exon 4
exon 5
exon 6 fr 1
exon 6 fr 2
exon 6 fr 3
exon 6 fr 4
exon 6 fr 5
exon 6 fr 6
exon 7 fr 1
exon 7 fr 2
exon 7 fr 3
exon 8
exon 9 fr 1
exon 9 fr 2
exon 9 fr 3
exon 9 fr 4
exon 10
exon 11 fr 1
exon 11 fr 2
Sense primer
(5’→3’)
Antisense primer
(5’→3’)
CAGCAACACACCAGAGGATG
ATAATACGCCAGAACTAAACCAG
GGTTGAGGCAGCACAAAGGTC
CAGGAGTTCTTGCCAACGG
GTACAGTGGGACAATCTGTG
CCAAGTCGCAGTGTCTGCC
GATGGAATTGATGGTTACTCC
TCACCTCTGCCTCTGCCAC
GCTCTGGTAAACAAAGCATTG
GAATCCAGGGGCACAGTCG
GACGAATGTTGGTCCCAGC
CAGAGTGGTAAAATAGTTCATG
GAAACAGTATAAAGATATCTGATC
GCTATAAGCGATATGTGTATTC
CAACGTGATAGATCGATGCC
CACAGCTCTTCCTGCTAATAC
ACAAGTAAATTACGTGAAACTTC
AGTGAGGGATGCATGTTCC
ATTCTCCTGGGCTGTACC
GCTATGTTACAAACTGAGCC
GCGATGGCTTCCTGTGGG
TGCCACTCTCCATCGCTGG
CAGGTCAATAATCAGTCAAAGG
CAAACGAAGGTGTGGATGGC
ACCAGTGGGAATGACCAGC
CTGTGGCAGTCACACTGG
CAACCTTGTCAAAAGCAGAGG
CTCTGAGGCATGGCACTCC
TTCTCCTCCTCCTCTATTTTG
ACACGGATATATTGATAAGTGC
CTCCATGTTTGTCCGAACGC
TCTTGCTTGTCAGGTAGGC
TCAGTCTTCACAAAACCTAGG
ATAAAGTAAAAGTTTAGCATACAG
CAACATTTTTCTATTTAGTTGCC
CTCAAATGTCGCAACTTAACTG
AATGATCATTACTATTAATAACGG
GTGCCATCATTCACTGACTG
GTGGCAACAGCTTTTATATGC
CATGAACATTTTCAAAGTAAGAG
ATATAAAGGGCCTGCAAGGG
GCTGCAACTCTGTCAGAGC
GAACTCAACATCGATGAATGC
CAGTGATGCAGAGTATAGCTTC
CTTGAATGAGATGAACAAGATG
GAGGAGAAGATGAACTTTGAG
ACCAATGTATTCAACAGGGACC
TTACGTCCACCTCGCAGC
TCTGTGCCAGGACTTACTC
CAGAGATCTAAAATGAATCAAG
Size
(bp)
158
375
254
229
200
242
275
383
281
212
235
279
252
140
196
303
308
276
346
303
325
282
261
183
279
Tann
(°C)
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
58
60
60
62
60
56
MgCl2
(mM)
1.5
1.5
1.5
1.5
1.5
1.5
3.0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
3.0
1.5
1.5
1.5
3.0
1.5
1.5
3.0
3.0
1.5
3.0
Mutations are summarized (Table 2) and indicated in CRB1 (Figure 1B) or the predicted
protein (Figure 1C). In patient 25977, a homozygous insertion of an Alu repeat DNA element
was found in exon 7 at nt 2,320 (Figure 3A). The Alu element belongs to the AluY
subfamily14, is orientated in the antisense direction, contains a more than 70-nt poly(A) tail
and is flanked by a 12-bp direct repeat consisting of CRB1 nt 2,309-2,320. This insertion is
heterozygously present in the unrelated parents and two siblings of 25977 (Figure 3B), but not
in 185 healthy controls. The Alu sequence shows a 5’ truncation that was also observed in a
pathologic Alu element inserted in F9 (ref. 15). The Alu insertion disrupts the ORF, and most
likely results in the inactivation of CRB1.
We identified a homozygous non-conservative Met1041Thr missense mutation in patient
22147 (Figure 4A). As expected, the mutation co-segregates with the RP12 phenotype (Figure
4B). The same mutation was found heterozygously in 1 of 100 healthy control individuals
68
Mutations in CRB1 cause RP12
living in the same region as patient 22147. This is not unexpected, assuming an allele
frequency for this mutation of 1 in 200 and considering the small size of the population in this
region (~1.5 million).
Mutations in other patients include two stop mutations, a splice-site mutation and several
missense mutations (Table 2 and Figure 1). The 2977+5G→A 5’ splice-site mutation of exon
8 found in patient 25710 lowers the splice potential score16 from 76.4 to 62.0, suggesting that
the splice site is inactivated. The same patient and patient 25540 carry a 3’ splice-site
mutation (2978G→A) in exon 9, which has a mild effect on the splice potential score (82.2
compared with the normal 85.6). This alteration introduces a Cys→Tyr change in the
fourteenth EGF-like domain of CRB1. In the EGF-like motif, Cys948 is a conserved residue
and is probably used in protein folding by the formation of a disulfide bridge with Cys933
(ref. 17). Similarly, a conserved Cys in the sixth EGF-like domain is substituted
homozygously by a Trp in a British Asian patient (RP112). Analysis of this mutation in the
family of this patient revealed that two affected siblings are also homozygous for the
mutation; the parents and three unaffected siblings are heterozygous (data not shown). The
mutation was not found in 100 racially matched controls. The Thr745Met mutation found in
patients 24868 and 25540 alters a threonine residue that is conserved in the last three laminin
A G-like domains of Drosophila CRB and in all three G-like domains of CRB1. Of the
remaining CRB1 missense mutations, Arg764Cys is a non-conservative substitution, whereas
Ala161Val and Leu1071Pro are conservative substitutions.
On SSCP or sequence analysis of the entire CRB1 ORF, we identified 20 mutant alleles in 10
patients of 15 patients. SSCP analysis in 25 control individuals did not reveal any
polymorphisms in the CRB1 ORF. Except for the Met1041Thr mutation, none of the
mutations were found in 100 healthy individuals, providing convincing evidence that CRB1 is
Table 2. CRB1 mutations in RP patients with preserved para-arteriolar RPE
Patient
25983
RP112
24228
25977
24868
25540
26023
25710
22147
RP0136
Allele 1
Mutation
Effect
617C→T
Ala161Val
885T→G
Cys250Trp
1343C→G
Ser403Stop
2320insAlu
truncation
2369C→T
Thr745Met
2369C→T
Thr745Met
2425C→T
Arg764Cys
2977+5G→A
splice defect
3257T→C
Met1041Thr
3347T→C
Leu1071Pro
Allele 2
Mutation
617C→T
885T→G
2425C→T
2320insAlu
2369C→T
2978G→A
3118G→T
2978G→A
3257T→C
3347T→C
Effect
Ala161Val
Cys250Trp
Arg764Cys
truncation
Thr745Met
Cys948Tyr
Glu995Stop
Cys948Tyr
Met1041Thr
Leu1071Pro
69
Chapter 3
A
nt. 2309
nt. 2320
G T T AT G T CAT C T T T A CT C T TG A TG A
1
co
ntr
ol
bl a
nk
normal
B
1
I
2
2
3
II
AluY
insertion
patient 25977
normal
direct repeat
AluY insertion
G T T A T G T C A T C T T T A C T C T T T T T T ..
AluY insertion
direct repeat
..T C C C A A T C A T C T T T A C T C T T G A T G A
Figure 3.
3. Homozygous
AluAlu
Figure
Homozygous insertion
insertionofofanan
repetitiveelement
elementin in
exon
of CRB1
in
repetitive
exon
7 of7 CRB1
in patient
patient
25977.
(A)
DNA
sequence
analysis
25977. (A) DNA sequence analysis of a part of
of a 7part
7 of
a normal(top)
individual
exon
of of
a exon
normal
individual
and the
(top)
and
the
boundaries
of
the 25977
Alu
boundaries of the Alu insertion in patient
insertion
in
patient
25977
(bottom).
The
(bottom). The insertion is flanked by a 12-bp
insertion
is flanked
by a 12-bp (B)
direct
repeat of
direct
repeat
of nt 2,309-2,320.
Analysis
of ntfragment
2,309-2,320.
(B) Analysis
PCR of
PCR
1 of exon
7 in the offamily
fragment
1
of
exon
7
in
the
family
of
patient 25977. The insertion is present
patient
25977.
The
insertion
is
present
heterozygously in the parents and two siblings
in the parents and two
ofheterozygously
patient 25977 (II-1).
siblings of patient 25977 (II-1).
involved in RP12. No mutations have been detected so far in five patients. Sequence analysis
of the CRB1 promotor and of larger parts of the introns could identify additional mutations. It
is also possible that these patients have been misdiagnosed or that this specific form of RP is
genetically heterogeneous.
To our knowledge, we are the first to identify a human disease gene using SSH. In contrast to
conventional subtractive hybridization techniques previously used to isolate retina-specific
genes, SSH allows the isolation of tissue-specific transcripts expressed at moderate or low
levels in the tissue of interest by combining normalization and subtraction1,2. Screening of an
amplified retina cDNA library with RET3C11 revealed 16 cDNA clones of 1.2 million,
suggesting that CRB1 expression is relatively low (<0.01%). Comparison of the RET3C11
subtraction clone with the CRB1 cDNA and gene suggests that RET3C11 represents an RsaI
fragment of an incompletely spliced or unspliced CRB1 transcript. Most retinal SSH clones
identified in the same procedure correspond to properly processed mRNA molecules (ref. 1,
and A.I.d.H. and F.P.M.C., unpublished data). Most RsaI fragments encountered in the SSH
libraries are small RsaI fragments (<500 bp) from the 3’ region of genes1. As SSH is a PCRbased technique, small RsaI fragments will preferentially be amplified over large RsaI
fragments1. CRB1 cDNA contains only 4 RsaI sites at positions 439, 1,365, 1,470 and 3,429.
70
Mutations in CRB1 cause RP12
The scarcity of RsaI sites in CRB1 cDNA has probably resulted in the enrichment of an
incompletely spliced RNA carrying suitable RsaI sites at its 3’ end.
The biochemical function of CRB1 and its role in RP pathogenesis remain to be elucidated.
Drosophila CRB is involved in the organization of ectodermally derived epithelia and the
establishment of polarity of epithelial cells, but mutations in crb also lead to abnormalities in
the development of neural tissues11,18. CRB co-localizes with the zonula adherens, suggesting
that it is involved in cell-cell interactions19. EGF-like domains, laminin A G-like domains and
CTL domains function in protein-protein interactions. Many proteins essential for neuronal
development, such as reelin, agrin, tenascin and proteins of the notch family, contain multiple
EGF-like domains20. Besides CRB and CRB1, G-like domains are also associated with EGF-
A
normal
patient 22147
Ser Met Thr
Ser Thr Thr
T T T C C A T G A C A G A T T T C C AC GA C A G A
B
X
5
6
8
9
11 12
C/C T/T C/C
C
12
T/C
T/C T/C
XI
9
13
T/C
16
18 19
20 21
T/C C/C C/C T/C C/C
38 39 40
C/C T/C C/C
Figure 4. Homozygous
Met1041Thr
missense
mutation found in patient
22147. (A) DNA sequence
analysis of part of exon 9 of
a normal individual (left
panel) and of patient 22147
carrying a homozygous
3257T→C mutation (right).
(B) Co-segregation of the
mutation with the RP12
phenotype in a large
consanguineous
pedigree
from the Netherlands. Only a
small part of the 11generation pedigree5,13 is
drawn. The arrow indicates
patient 22147. All patients
are homozygous for the
mutant cytosine at nt 3,257,
whereas all parents are
heterozygous. (C) Fundus
photograph of the right eye
of patient 22147, 41 years of
age, showing preservation of
the RPE adjacent to the
retinal arterioles (arrow).
71
Chapter 3
like domains in several other proteins, including neurexins21, heparan sulfate proteoglycan
core protein22, and Drosophila perlecan, slit, agrin and fat proteins8,11,23,24. The combination
of EGF-like domains and G domain-like repeats in CRB1 and its preferential expression in
the retina suggest that CRB1 may be involved in neuronal development of the retina,
presumably through protein-protein interactions. Its highest similarity to CRB suggests that it
may have a role in the organization or polarity of retinal cells.
We have identified CRB1 and demonstrated its involvement in RP12, a clinically distinct
form of autosomal recessive RP. The processes underlying RP12 pathogenesis are distinct
from those operating in other forms of autosomal recessive RP, for which most defects have
been found in components of the phototransduction cascade or retinol metabolism25,26,
suggesting that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.
The (sub)cellular localization of CRB1 and the identification of interacting proteins may shed
light on the pathogenesis of RP12 and possibly on the etiology of other forms of retinal
degeneration.
Methods
cDNA cloning and sequencing
For cDNA library screening, we amplified a 155-bp PCR product from the RET3C11
subtraction clone and labeled it with α-32P-dCTP by random primer extension. We used the
probe to screen 1.2×106 plaques of a human retina 5’-STRETCH cDNA library (Clontech) in
λgt10 at 80,000 plaques/150-mm dish by standard protocols using Hybond-N+ membranes
(Amersham). Hybridizations were performed at 65°C in 7% (w/v) SDS, PO4 (0.5 M) and
EDTA (1 mM). We purified 5 of 16 positive clones by secondary and tertiary screening using
nitrocellulose filters and NC hybridization buffer (6×SSC, 5×Denhardt’s, 0.2% (w/v) SDS,
10% (w/v) dextrane-sulphate). Phage DNA was isolated with the Lambda Miniprep Kit
(Qiagen). Inserts were cloned in pBluescript (Stratagene) and sequenced with the Thermo
Sequenase Dye Terminator Cycle Sequencing Pre-mix kit (Amersham) using an ABI 370A
automated sequencer. To identify the 5’ end of the gene, we performed reiterated 5’-RACE
using human fetal brain Marathon-Ready cDNA (Clontech). The first 5’-RACE was
performed by a primary PCR using primer AP1 and a gene-specific primer at position 2,9362,956, and a nested PCR using primer AP2 and a primer at position 2,896-2,918. We cloned
the resulting PCR products, ranging from 400 to 1,500 bp, in a T/A vector (Invitrogen) and
sequenced them. A second 5’-RACE was performed using primer AP1 and a primer at
72
Mutations in CRB1 cause RP12
position 1,689-1,712, and in a nested PCR primer AP2 and a primer at position 1,648-1,671.
The resulting PCR products ranged from 300 to 1,700 bp. We confirmed the 5’ end of the
cDNA by a third 5’-RACE using primer AP1 and a primer at position 382-404, and in a
nested PCR primer AP2 and a primer at position 351-373. We carried out nucleic acid and
protein database searches using the FASTA, TFASTA and BLAST programs27,28 against the
GenBank, EMBL and SWISS-PROT databases.
Northern-blot and RT-PCR analysis
For northern-blot analysis, we isolated total RNA from human retina by RNAzol B (Campro
Scientific). We isolated retina poly(A)+ RNA from total RNA with an mRNA purification kit
(Pharmacia Biotech). Retina poly(A)+ RNA (5 µg) was separated on a 1% agarose gel
containing 1×MOPS and 18% (w/v) formaldehyde and transferred to a Hybond-N+ membrane
(Amersham). A cDNA fragment containing CRB1 nt 1,604-1,838 was labeled with α-32PdCTP by random primer extension and hybridized to a multiple-tissue northern blot
(Clontech) and the retina northern blot in ExpressHyb hybridization solution (Clontech) at
65°C. Blots were washed twice in 0.1×SSC, 0.1% SDS at 65°C for 20 min. We performed
RT-PCR for 35 cycles on RNA from 10 human adult tissues, 2 fetal tissues and 2 RPE cell
lines (ARPE-19, ref. 29; D407, ref. 30) as described1 with primers amplifying CRB1 nt 2,9413,233.
Intron/exon structure
The intron/exon boundaries of exons 1-5 were determined by ligation-mediated (LM) PCR12.
Genomic or YAC DNA was digested with AluI, BalI, DraI, EcoRV, HaeIII, PvuII, RsaI, ScaI
or StuI before ligation of adaptors. We performed LM-PCR with an exon-specific primer and
an adaptor primer. To determine the intron/exon boundaries of exons 6-11, we designed
primers from the cDNA sequence and performed exon-exon PCR reactions on genomic DNA
or on DNA from YAC 772C10 or 755E11 (ref. 3) using Taq DNA polymerase (BRL) or ExTaq (TaKaRa). LM-PCR products and exon-exon PCR products were separated on agarose
gel, purified with the Qiaquick Gel Extraction kit (Qiagen), and (partially) sequenced as
described above.
RP12 patients
The 15 unrelated RP patients with preserved para-arteriolar RPE were identified by L.I.v.d.B.
(patients 22147 (ref. 5), 24228, 24868 and 25710), J.R.H. (patients 25976, 25977, 25978,
73
Chapter 3
25979, 25980, 25982 and 25983 (ref. 4)), U.K. (patient 25540), M. van Schooneveld (patient
26023) or D. Bessant (patient RP112). We isolated genomic DNA of these patients from
blood samples by standard procedures.
Mutation analysis
For mutation analysis of the CRB1 ORF and splice junctions, we designed 25 primer pairs
from exonic and intronic sequences that amplified genomic DNA fragments of 150-350 bp.
Primer sequences and PCR conditions are available (Table 1). We performed mutation
analysis for 15 unrelated RP patients by SSCP analysis and sequencing of PCR products.
Exons 1, 2.1, 4 and 5 were screened by SSCP analysis and the remaining amplicons by
sequence analysis. PCR reactions for SSCP were performed with γ-32P end-labeled primers or
in the presence of α-32P-dCTP. PCR products were denatured and electrophoresed on 5%
polyacrylamide gels. Sequencing was performed on PCR products as above or using a
SequiTherm cycle sequencing kit (Epicentre) and α-32P-dCTP.
GenBank accession number
cDNA sequence of CRB1, AF154671.
Acknowledgements
We thank R. Roepman and H. Yntema for their help with northern-blot analysis and cDNA
library screening; M. van Schooneveld and D. Bessant for ascertaining patients with RP12;
and P. de Jong for continuous interest in this project. This work was supported by The
Foundation Fighting Blindness, Inc., USA and The British Retinitis Pigmentosa Society.
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den Hollander AI, van Driel MA, de Kok YJM,
van de Pol TJR, Hoyng CB, Brunner HG,
Deutman AF and Cremers FPM (1999) Isolation
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Heckenlively JR (1982) Preserved para-arteriole
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van den Born LI, van Soest S, van Schooneveld
MJ, Riemslag FCC, de Jong PTVM and BleekerWagemakers EM (1994) Autosomal recessive
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Doolittle RF, Feng DF and Johnson MS (1984)
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12. Kere J, Nagaraja R, Mumm S, Ciccodicola A,
D'Urso M and Schlessinger D (1992) Mapping
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Bleeker-Wagemakers LM, Westerveld A,
Humphries P, Sandkuijl LA and Bergen AAB
(1994) Assignment of a gene for autosomal
recessive retinitis pigmentosa (RP12) to
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genetically heterogeneous disease population.
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14. Batzer MA, Deininger PL, Hellmann-Blumberg
U, Jurka J, Labuda D, Rubin CM, Schmid CW,
Zietkiewicz E and Zuckerkandl E (1996)
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15. Vidaud D, Vidaud M, Bahnak BR, Siguret V,
Gispert Sanchez S, Laurian Y, Meyer D,
Goossens M and Lavergne JM (1993)
Haemophilia B due to a de novo insertion of a
human-specific Alu subfamily member within the
coding region of the factor IX gene. Eur J Hum
Genet 1:30-36.
16. Shapiro MB and Senapathy P (1987) RNA splice
junctions of different classes of eukaryotes:
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17. Appella E, Weber IT and Blasi F (1988) Structure
and function of epidermal growth factor-like
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18. Knust E, Dietrich U, Tepass U, Bremer KA,
Weigel D, Vässin H and Campos-Ortega JA
(1987) EGF homologous sequences encoded in
the genome of Drosophila melanogaster, and
their relation to neurogenic genes. EMBO J
6:761-766.
19. Wodarz A, Hinz U, Engelbert M and Knust E
(1995) Expression of crumbs confers apical
character on plasma membrane domains of
ectodermal epithelia of Drosophila. Cell 82:6776.
20. Nakayama M, Nakajima D, Nagase T, Nomura
N, Seki N and Ohara O (1998) Identification of
high-molecular weight proteins with multiple
EGF-like motifs by motif-trap screening.
Genomics 51:27-34.
21. Ushkaryov YA, Petrenko AG, Geppert M and
Südhof TC (1992) Neurexins: synaptic cell
surface proteins related to the alpha-latrotoxin
receptor and laminin. Science 257:50-56.
22. Kallunki P and Tryggvason K (1992) Human
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membrane
heparan
sulfate
proteoglycan core protein: a 467-kD protein
containing multiple domains resembling elements
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neural cell adhesion molecules, and epidermal
growth factor. J Cell Biol 116:559-571.
23. Joseph DR and Baker ME (1992) Sex hormonebinding globulin, androgen-binding protein, and
vitamin K-dependent protein S are homologous to
laminin A, merosin, and Drosophila crumbs
protein. FASEB J 6:2477-2481.
24. Patthy L (1992) A family of laminin-related
proteins controlling ectodermal differentiation in
Drosophila. FEBS Lett 298:182-184.
25. Dryja TP and Li T (1995) Molecular genetics of
retinitis pigmentosa. Hum Mol Genet 4:17391743.
26. van Soest S, Westerveld A, de Jong PTVM,
Bleeker-Wagemakers EM and Bergen AAB
(1999) Retinitis pigmentosa: defined from a
molecular point of view. Surv Ophthalmol
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27. Pearson WR and Lipman DJ (1988) Improved
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Natl Acad Sci USA 85:2444-2448.
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28. Altschul SF, Madden TL, Schäffer AA, Zhang J,
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29. Dunn KC, Aotaki-Keen AE, Putkey FR and
Hjelmeland LM (1996) ARPE-19, a human
retinal pigment epithelial cell line with
differentiated properties. Exp Eye Res 62:155169.
76
30. Davis AA, Bernstein PS, Bok D, Turner J,
Nachtigal M and Hunt RC (1995) A human
retinal pigment epithelial cell line that retains
epithelial characteristics after prolonged culture.
Invest Ophthalmol Vis Sci 36:955-964.
Chapter
4
Leber congenital amaurosis and retinitis pigmentosa with
Coats-like exudative vasculopathy are associated with
mutations in the crumbs homologue 1 (CRB1) gene
Anneke I. den Hollander1, John R. Heckenlively3, L. Ingeborgh van den Born4,
Yvette J.M. de Kok1, Saskia D. van der Velde-Visser1, Ulrich Kellner5,
Bernhard Jurklies6, Mary J. van Schooneveld7, Anita Blankenagel8, Klaus
Rohrschneider8, Bernd Wissinger9, Johan R.M. Cruysberg2, August F.
Deutman2, Han G. Brunner1, Eckart Apfelstedt-Sylla9, Carel B. Hoyng2 and
Frans P.M. Cremers1
Departments of 1Human Genetics and 2Ophthalmology, University Medical Center Nijmegen,
Nijmegen, the Netherlands; 3Department of Ophthalmology, University of California, Los
Angeles;
4
Rotterdam Eye Hospital, Rotterdam, the Netherlands;
5
Department of
Ophthalmology, University Clinic Benjamin Franklin, Free University of Berlin, Berlin,
6
Universitäts-Augenklinik, Essen, Germany, 7Department of Ophthalmology, University
Medical Center Utrecht, Utrecht, the Netherlands; 8Universitäts-Augenklinik, Ruprecht-KarlsUniversität, Heidelberg; 9Universitäts-Augenklinik, Tübingen, Germany
Am J Hum Genet 69:198-203 (2001)
Mutations in CRB1 cause LCA and RP with Coats-like exudates
Mutations in the crumbs homologue 1 (CRB1) gene cause a specific form of retinitis
pigmentosa (RP) that is designated “RP12” and is characterized by a preserved paraarteriolar retinal pigment epithelium (PPRPE) and by severe loss of vision at age <20
years. Because of the early onset of disease in patients who have RP with PPRPE, we
considered CRB1 to be a good candidate gene for Leber congenital amaurosis (LCA).
Mutations were detected in 7 (13%) of 52 LCA patients from the Netherlands, Germany
and the United States. In addition, CRB1 mutations were detected in five of nine patients
who had RP with Coats-like exudative vasculopathy, a relatively rare complication of
RP that may progress to partial or total retinal detachment. Given that four of five
patients had developed the complication in one eye and that not all siblings with RP
have the complication, CRB1 mutations should be considered an important risk factor
for the Coats-like reaction; its development probably requires additional genetic or
environmental factors. Although no clear-cut genotype-phenotype correlation could be
established, patients with LCA, which is the most severe retinal dystrophy, carry null
alleles more frequently than do patients with RP. Our findings suggest that CRB1
mutations are a frequent cause of LCA and are strongly associated with the
development of Coats-like exudative vasculopathy in patients with RP.
We have described mutations in the CRB1 gene (MIM 604210) in a severe, autosomal
recessive form of retinitis pigmentosa (RP) that is designated “RP12” (MIM 600105)1. The
gene consists of 12 exons and exhibits alternative splicing at its 3’ end (A.I. den Hollander
and F.P.M. Cremers, unpublished data). The CRB1 protein contains 19 epidermal growth
factor (EGF)-like domains, 3 laminin A globular-like domains, a transmembrane domain, and
a 37-amino acid cytoplasmic tail; in addition, it is homologous to Drosophila crumbs protein.
RP12 is a specific form of RP characterized by a preserved para-arteriolar retinal pigment
epithelium (PPRPE) in the early-to-middle stages of disease. Patients experience night
blindness and develop a progressive loss of their visual field at <10 years of age. Because of
early macular involvement, patients have severe visual impairment at <20 years of age. Other
features of this type of RP are high hyperopia, nystagmus, optic-nerve-head drusen, vascular
sheathing, and maculopathy2,3. Mutations have now been identified in 15 patients who have
isolated or autosomal recessive RP with PPRPE (ref. 1; U. Kellner, A.I. den Hollander,
Y.J.M. de Kok, L.I. van den Born, F.P.M. Cremers, J.R. Heckenlively, unpublished data).
Leber congenital amaurosis (LCA) is considered the earliest and most severe form of retinal
dystrophy, causing blindness or severe visual impairment at birth or during the first months of
79
Chapter 4
life. Mutations that lead to LCA have been detected in GUCY2D (MIM 600179)4, RPE65
(MIM 180069)5-7, CRX (MIM 602225)8-10, and AIPL1 genes (MIM 604392)11. Mutations in
these four genes account for 15%-30% of LCA cases12-15, an indication that more LCA genes
await discovery. Because of the early onset of symptoms in patients who have RP with
PPRPE - and the observation that mutations in RPE65 and CRX can lead to both LCA and
RP5,7,9,16 - we considered CRB1 to be a good candidate gene for LCA.
Fifty-two unrelated patients with LCA were ascertained by ophthalmologists from six centers
in the Netherlands, Germany, and the United States. The diagnosis of LCA is made when
patients are nonseeing or visually inattentive in infancy and have a nonrecordable
electroretinogram when investigated at <1 year of age17. In early stages, the fundus is,
typically, blond; however, in three patients (13067, 16507, and 16690), there was a
preservation of the retinal pigment epithelium (RPE) that is characteristic for RP with PPRPE.
We used 25 primer sets to screen exons 1-11 of the CRB1 gene by single-strand conformation
analysis, as described elsewhere1, but we replaced the primer set for exon 5 by primers 5’TAATTCAACACCTTTGACTTAGC-3’
and
5’-TGCCATAAAATACCAGAAAGTC-3’.
Primers used to amplify exon 12 were 5’-CCTGAGTAGTTCCATTGTCC-3’ and 5’ATTCACAGTGTGTGGATCCC-3’. Products that migrated differently through the gel were
analyzed by sequencing. When only one allele was identified in a patient, the patient’s sample
was also subjected to sequence analysis of all 26 amplicons and of the promoter region of
CRB1, which contains several putative photoreceptor-gene regulatory sites (A.I. den
Hollander
and
F.P.M.
Cremers,
unpublished
data),
with
primers
5’-
GTAAAAATCAGCTATAGAAATTGC-3’ and 5’-TTTTCTGTTCATAAATTATATTCCC3’ (-800 to -345), and primers 5’-TAAGTTTTCTTCTGTCTTGGCC-3’ and 5’CTGAGGTAGAAGATGAGAAGG-3’ (-421 to +179).
In eight patients with LCA, nine distinct sequence changes in the coding region or splice sites
of CRB1 were found (Table 1; homozygous Thr821Met polymorphism in proband 12864 not
depicted – see below). Two of the changes (Lys801stop and Cys948Tyr) had been identified
by us previously (ref. 1; U. Kellner, A.I. den Hollander, Y.J.M. de Kok, L.I. van den Born,
F.P.M. Cremers, J.R. Heckenlively, unpublished data) in patients who have RP with PPRPE,
but seven of them (748-754del, Thr821Met, Ile1100Arg, Glu1111stop, 4013+1G→T,
Arg1331His, and Glu1333stop) had not been identified previously and were not found on
180 control chromosomes. For two LCA patients (12859 and 12864), DNA samples of
family members were available for cosegregation analysis (for patient 12859, see Figure 1A).
Proband 12864 and one of her unaffected sisters were homozygous for Thr821Met, which
80
Mutations in CRB1 cause LCA and RP with Coats-like exudates
Table 1. CRB1 mutations in patients who have LCA or RP with Coats-like exudates
Allele 1
Disease and
patient
Inheritance Mutation
Effect
number
LCA:
12831
Isolated
2978G→A
Cys948Tyr
12859
Recessive
3434T→G
Ile1100Arg
12862
Isolated
2536A→T
Lys801stop
12872
Recessive
4127G→T
Arg1331His
13067
Isolated
3466G→T
Glu1111stop
16507
Isolated
2978G→A
Cys948Tyr
16690
Isolated
748-754del
Frameshift
RP with Coats-like exudates:
9439
Recessive
1343C→G,
Ser403stop,
1433A→G
Tyr433Cys
16894
Recessive
2816A→G
Asn894Ser
16937
Recessive
2644G→C,
Asp837His,
4195G→A
Ala1354Thr
16968
Isolated
2978G→A
Cys948Tyr
17658
Recessive
Lys801stop
2536A→T
Allele 2
Mutation
Effect
2978G→A
4132G→T
2536A→T
?
4013+1G→T
2978G→A
2536A→T
Cys948Tyr
Glu1333stop
Lys801stop
2425C→T
Arg764Cys
?
2978G→A
Cys948Tyr
2977+5G→A
3676T→C
Splice defect
Cys1181Arg
Splice defect
Cys948Tyr
Lys801stop
suggested that this sequence change is not pathogenic. In patient 12872, only one allele was
identified. No sequence changes were detected in the 800 bp that precede the transcriptionstart site, which suggested that the second allele may be present in other, unidentified
regulatory elements or splice variants of the gene or may involve a large deletion that was not
detected by PCR analysis.
Of 13 alleles identified in patients with LCA, 7 were nonsense, frameshift, or splice-site
mutations, which is a larger proportion than is found on CRB1 alleles of patients who have RP
with PPRPE (4 of 30 alleles; ref. 1; U. Kellner, A.I. den Hollander, Y.J.M. de Kok, L.I. van
den Born, F.P.M. Cremers, J.R. Heckenlively, unpublished data). In three patients we
identified null mutations on both CRB1 alleles, which suggests that LCA is the most severe
phenotype that can be associated with mutations in CRB1. Two patients with LCA were
homozygous for the mutation (Cys948Tyr) that is most frequently found in patients who have
RP with PPRPE. In four patients who had RP with PPRPE, this mutation was found in
combination with another missense mutation; and in one patient who had RP with PPRPE, it
is found together with a splice site mutation (2977+5G→A) that does not necessarily render
the mutant splice site completely inactive1. These findings suggest that Cys948Tyr is a severe
mutation that leads to a severe phenotype when it is present homozygously. Cys948Tyr
changes the 4th conserved cysteine residue of the 14th EGF-like domain of CRB1, which is
involved in the formation of disulfide bridges and thus the correct folding of the EGF-like
domain.
81
Chapter 4
Pathogenic CRB1 mutations were identified in 7 (13%) of 52 unrelated patients with LCA
from the Netherlands, Germany, and the United States. Mutations in GUCY2D, RPE65,
AIPL1, and CRX, account for 6%-20%, 3%-16%, 7%, and 2%-3% of LCA cases,
respectively7,13,14,16,18,19, which suggests that CRB1 mutations contribute significantly to the
etiology of LCA.
To determine whether mutations in the CRB1 gene are a common cause of RP in the Dutch
population, we screened 97 unrelated patients who had isolated or autosomal recessive RP for
the presence of Cys948Tyr and Arg764Cys. These mutations had been previously identified
in 5 and 3 alleles, respectively, of a total of 30 CRB1 alleles of unrelated patients who had RP
with PPRPE (ref. 1; U. Kellner, A.I. den Hollander, Y.J.M. de Kok, L.I. van den Born, F.P.M.
Cremers, J.R. Heckenlively, unpublished data). The presence of the nucleotide alteration
2425C→T
(Arg764Cys)
was
analyzed
by
allele-specific
oligonucleotide
(ASO)
hybridization20, using wild-type primer 5’-AATATATCCGTGTCTGG-3’ and mutant primer
12859
A
1
I
2
1
II
2
I1100R/
E1333X
I1100R/
E1333X
B
9439
1
I
Figure 1. Cosegregation analysis of
CRB1 mutations in (A) one family
with
LCA1.andCosegregation
(B) five families
with of
Figure
analysis
RP
Coats-like
CRB1and
mutations
in (A)exudates.
one familyAwith
question
mark
denotes
the and
LCA and (B)
five (?)
families
with RP
unidentified
second
allele;
an
asterisk
Coats-like exudates. A question mark (?)
(*)
denotes
patients with
RP allele;
who an
denotes
the unidentified
second
have
the Coats-like
asterisk developed
(*) denotes patients
with RP who
complication.
Arrows
have
developed
the indicate
Coats-like
probands.
IVS8
denotes
the splicecomplication.
Arrows
indicate
probands.
site
mutation
of
exon
8, of
mutation
IVS8 denotes
the splice-site
2977+5G→A.
exon 8, 2977+5G→A.
2
R764C/+
1
II
2
3
I
16894
1
II
*
16968
2
2
3
N894S/?
1
I
16937
1
I
*
N894S/?
5
S403X, S403X,
Y433C/ Y433C/
R764C R764C
+/+
1
*
4
1
II
2
IVS8/+
+/+
3
*
17658
I
2
D837H, A1354T/+
II
*
1
2
*
D837H, A1354T/
C948Y
82
II
1
2
*
K801X/
*
K801X/
C1181R
C1181R
4
C948Y/
IVS8
IVS8/+
1
2
2
3
Mutations in CRB1 cause LCA and RP with Coats-like exudates
5’-AATATATCTGTGTCTGG-3’. The presence of 2978G→A (Cys948Tyr), was analyzed
with
the
amplification-refractory
ATTATCACCTTCTCTCATTAGG-3’
ATTATCACCTTCTCTCATTAGA-3’
mutation
system21
with
(wild-type
(mutant
allele)
sense
allele)
and
antisense
primers
5’-
or
5’-
primer
5’-
GTGCCATCATTCACTGACTG-3’. One of the 97 patients (patient 9439) carried the
Arg764Cys mutation on one allele. Sequencing of the 12 exons of CRB1 revealed two more
sequence changes (Ser403stop and Tyr433Cys), both of which are located on the second
allele, as determined by allele-specific PCR (data not shown) and segregation analysis (Figure
1).
As a complication of RP, proband 9439 (individual II-4) (Figure 2) and his affected brother
(II-5) had developed a Coats-like exudative vasculopathy, which caused additional loss of
vision. Coats-like exudative vasculopathy, a relatively rare complication of RP, can develop
in later stages of the disease and is characterized by vascular abnormalities (aneurysmal
dilations and telangiectatic retinal veins), yellow extravascular lipid depositions, and retinal
detachment. Patients with RP who develop Coats-like changes show a wide spectrum of
disorders, ranging from mild visual difficulties or nyctalopia, as observed in classical RP, to
the other extreme, in which a proliferative vasculopathy develops. If untreated, this
proliferative vasculopathy may result in a painful blind eye due to rubeosis, retinal
neovascularization, or serous retinal detachment. It has been suggested that genetic factors
may be involved in RP with Coats-like exudative vasculopathy22. We therefore hypothesized
that CRB1 mutations may be associated with the development of this complication of RP, and
we ascertained eight additional isolated or autosomal recessive patients who had RP with
Coats-like exudative vasculopathy. These patients were tested for mutations in CRB1 by
sequence analysis of all 26 amplicons. Five of eight patients had sequence changes, and
clinical descriptions of these patients are summarized in Table 2.
In one patient, we identified three sequence changes; in two patients, we found compound
heterozygous mutations; and in one patient, we identified one allele (Table 1). Screening of
the promoter region of CRB1 in the patient with one allele revealed no sequence changes.
Cosegregation analysis in family members of all five proband confirmed autosomal recessive
inheritance of CRB1 mutations (Figure 1). Of the 10 different sequence changes identified in
five patients who had RP with Coats-like exudates, five mutations (Ser403stop, Arg764Cys,
Lys801stop, Cys948Tyr, and 2977+5G→A) have been identified previously in patients who
had RP with PPRPE. The other five changes (Tyr433Cys, Asp837His, Asn894Ser,
Cys1181Arg, and Ala1354Thr) had not previously been identified in patients who had RP
83
Chapter 4
with PPRPE or in patients with LCA, and the changes were not found on 180 control
chromosomes.
Coats-like exudative vasculopathy occurs in only 1.2%-3.6% of patients with RP22. Among
patients who had both RP and PPRPE and were described by Van den Born et al.3, 2 (8.3%)
of 24 had Coats-like changes, and CRB1 mutations were found in these patients1. At least one
patient described in the present study (patient II-4 in family 9439) had RP with Coats-like
changes and PPRPE. However, the disorders of two patients (patient II-2 in family 16894 and
patient II-2 in family 16937) were clearly distinct from RP with PPRPE. In both patients, the
onset of RP occurred when patients were >10 years old. Neither patient showed a preservation
of the RPE surrounding the arterioles, and one patient was highly myopic.
Our findings show that CRB1 mutations are associated with Coats-like exudative
vasculopathy in patients who have RP with and without the PPRPE phenotype. These findings
demonstrate that patients with PPRPE should be checked regularly for the Coats-like
complication. Furthermore, the routine screening of patients with autosomal recessive or
isolated RP may be important because of its ability to reveal those patients who are at
increased risk of developing exudative retinal detachment. If the process is detected before it
becomes proliferative, cryotherapy can be used to prevent further progression.
Not all affected siblings of patients who have RP with Coats-like exudative vasculopathy
develop the Coats-like complication (e.g., families 16894 and 17658 [Figure 1]). This finding,
together with the observation that most patients with CRB1 mutations had developed
unilateral Coats-like exudates, strengthens the idea that CRB1 mutations are an important risk
Figure 2. Fundus photograph of the
inferior part of the left eye of a patient
who had RP with unilateral Coats-like
exudative vasculopathy (proband 9439;
individual II-4). Note the widespread
subretinal white deposits between the
neural retina and RPE in the lower part.
This region of the fundus is out of focus
as a result of the elevation of tissue
caused by subretinal accumulation of
fluid. Triangles (▲) indicate retinal
vessels with white sheathing that is
indicative of vasculitis. The RPE shows
some preservation near retinal arterioles
as seen in patients who had RP with
PPRPE (arrow).
84
Mutations in CRB1 cause LCA and RP with Coats-like exudates
Table 2. Clinical features of patients with RP and Coats-like exudates
Patient and age at
onset of RP
9439, <10 years
16894, 28 years
16937, >10 years
16968, <10 years
17658, <10 years
Note. ? = unknown.
a
B = bilateral; U = unilateral.
b
Spherical equivalent.
Coats-reaction
and age at onseta
U, 20 years
U, 30 years
U, 27 years
U, 20 years
B, 20 years
PPRPE
Refractionb
yes
no
no
?
no
+5.25
-6.00
+4.50
?
+1.00
factor for the development of the Coats-like reaction and that other genetic or environmental
factors may be involved. Interestingly, no CRB1 mutations were identified in 13 (45%) of 29
patients who had RP with PPRPE (U. Kellner, A.I. den Hollander, Y.J.M. de Kok, L.I. van
den Born, F.P.M. Cremers, J.R. Heckenlively, unpublished data), and in the present study we
did not detect CRB1 mutations in four of nine patients who had RP with Coats-like exudates,
which suggests that another gene may be involved in these two specific forms of RP.
We found no obvious genotype-phenotype correlation when we compared mutations in
patients who had both RP and PPRPE with those in patients who had LCA or RP with Coatslike exudates. However, the absence of clear-cut null mutations on both CRB1 alleles of 15
patients who had RP with PPRPE and of 5 patients who had RP with Coats-like exudates,
together with the presence of null mutations on both CRB1 alleles in 3 patients with LCA,
suggests that LCA may be associated with complete loss of function of CRB1. In contrast,
patients who have RP with PPRPE and patients who have RP with Coats-like exudates may
have residual CRB1 function.
RP with PPRPE, RP with Coats-like exudative vasculopathy, and LCA represent different
(but partly overlapping) clinical entities, as evidenced by the presence of the PPRPE
characteristics in some patients with LCA and the higher-than-average incidence of Coats-like
changes in patients who had RP with PPRPE. Because our genotype-phenotype comparison
did not reveal conclusive evidence for qualitative or quantitative differences in CRB1
function in these patient groups, functional studies of CRB1 are necessary to shed light on this
intriguing issue. We suggest that other genetic - and possibly environmental - factors
influence the expression of CRB1 mutations, thereby contributing to the wide spectrum of
features that have been described in the present study.
85
Chapter 4
Acknowledgements
We thank Carolien Vink and Bellinda van den Helm for their technical assistance. A.I.d.H.
and Y.J.M.d.K. were supported by the Foundation Fighting Blindness. J.R.H. was supported
by a Center grant from the Foundation Fighting Blindness and a Clinician-Scientist Award
from Research to Prevent Blindness.
Electronic-Database Information
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim (for
CRB1 [MIM 604210], RP12 [MIM 600105], GUCY2D [MIM 600179], RPE65 [MIM
180069], CRX [602225], and AIPL1 [MIM 604392])
References
1.
den Hollander AI, ten Brink JB, de Kok YJM,
van Soest S, van den Born LI, van Driel MA, van
de Pol TJR, Payne AM, Bhattacharya SS, Kellner
U, Hoyng CB, Westerveld A, Brunner HG,
Bleeker-Wagemakers
EM,
Deutman
AF,
Heckenlively JR, Cremers FPM and Bergen AAB
(1999) Mutations in a human homologue of
Drosophila crumbs cause retinitis pigmentosa
(RP12). Nature Genet 23:217-221.
2.
Heckenlively JR (1982) Preserved para-arteriole
retinal pigment epithelium (PPRPE) in retinitis
pigmentosa. Brit J Ophthalmol 66:26-30.
3.
van den Born LI, van Soest S, van Schooneveld
MJ, Riemslag FCC, de Jong PTVM and BleekerWagemakers EM (1994) Autosomal recessive
retinitis pigmentosa with preserved para-arteriolar
retinal pigment epithelium. Am J Ophthalmol
118:430-439.
4.
Perrault I, Rozet J-M, Calvas P, Gerber S,
Camuzat A, Dollfus H, Châtelin S, Souied E,
Ghazi I, Leowski C, Bonnemaison M, Le Paslier
D, Frézal J, Dufier J-L, Pittler S, Munnich A and
Kaplan J (1996) Retinal-specific guanylate
cyclase gene mutations in Leber's congenital
amaurosis. Nature Genet 14:461-464.
5.
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Gu S, Thompson DA, Srikumari CRS, Lorenz B,
Finckh U, Nicoletti A, Murthy KR, Rathmann M,
Kumaramanickavel G, Denton MJ and Gal A
(1997) Mutations in RPE65 cause autosomal
recessive
childhood-onset
severe
retinal
dystrophy. Nature Genet 17:194-197.
6.
Marlhens F, Bareil C, Griffoin J-M, Zrenner E,
Amalric P, Eliaou C, Liu S-Y, Harris E, Redmond
TM, Arnaud B, Claustres M and Hamel CP
(1997) Mutations in RPE65 cause Leber's
congenital amaurosis. Nature Genet 17:139-141.
7.
Morimura H, Fishman GA, Grover SA, Fulton
AB, Berson EL and Dryja TP (1998) Mutations in
the RPE65 gene in patients with autosomal
recessive retinitis pigmentosa or Leber congenital
amaurosis. Proc Natl Acad Sci USA 95:30883093.
8.
Freund CL, Wang Q-L, Chen S, Muskat BL,
Wiles CD, Sheffield VC, Jacobson SG, McInnes
RR, Zack DJ and Stone EM (1998) De novo
mutations in the CRX homeobox gene associated
with Leber congenital amaurosis. Nature Genet
18:311-312.
9.
Sohocki MM, Sullivan LS, Mintz-Hittner HA,
Birch D, Heckenlively JR, Freund CL, McInnes
RR and Daiger SP (1998) A range of clinical
phenotypes associated with mutations in CRX, a
photoreceptor transcription-factor gene. Am J
Hum Genet 63:1307-1315.
10. Swaroop A, Wang Q-L, Wu W, Cook J, Coats C,
Xu S, Chen S, Zack DJ and Sieving PA (1999)
Leber congenital amaurosis caused by a
homozygous
mutation
(R90W)
in
the
homeodomain of the retinal transcription factor
CRX: direct evidence for the involvement of
CRX in the development of photoreceptor
function. Hum Mol Genet 8:299-305.
Mutations in CRB1 cause LCA and RP with Coats-like exudates
11. Sohocki MM, Bowne SJ, Sullivan LS, Blackshaw
S, Cepko CL, Payne AM, Bhattacharya SS,
Khaliq S, Mehdi SQ., Birch DG, Harrison WR,
Elder FFB, Heckenlively JR and Daiger SP
(2000) Mutations in a new photoreceptor-pineal
gene on 17p cause Leber congenital amaurosis.
Nature Genet 24:79-83.
12. Perrault I, Rozet J-M, Gerber S, Ghazi I, Leowski
C, Ducroq D, Souied E, Dufier J-L, Munnich A
and Kaplan J (1999) Leber congenital amaurosis.
Mol Genet Metab 68:200-208.
13. Dharmaraj SR, Silva ER, Pina AL, Li YY, Yang
J-M, Carter RC, Loyer M, El-Hilali H, Traboulsi
E, Sundin O, Zhu D, Koenekoop RK and
Maumenee IH (2000) Mutational analysis and
clinical correlation in Leber congenital amaurosis.
Ophthalmic Genet 21:135-150.
14. Lotery AJ, Namperumalsamy P, Jacobson SG,
Weleber RG, Fishman GA, Musarella MA, Hoyt
CS, Héon E, Levin A, Jan J, Lam B, Carr RE,
Franklin A, Radha S, Andorf JL, Sheffield VC
and Stone EM (2000) Mutation analysis of 3
genes in patients with Leber congenital
amaurosis. Arch Ophthalmol 118:538-543.
15. Sohocki MM, Daiger SP, Bowne SJ, Rodriquez
JA, Northrup H, Heckenlively JR, Birch DG,
Mintz-Hittner H, Ruiz RS, Lewis RA, Saperstein
DA and Sullivan LS (2001) Prevalence of
mutations causing retinitis pigmentosa and other
inherited retinopathies. Hum Mut 17:42-51.
17. Foxman SG, Heckenlively JR, Bateman JB and
Wirtschafter JD (1985) Classification of
congenital and early onset retinitis pigmentosa.
Arch Ophthalmol 103:1502-1506.
18. Perrault I, Rozet J-M, Gerber S, Ghazi I, Ducroq
D, Souied E, Leowski C, Bonnemaison M, Dufier
J-L, Munnich A and Kaplan J (2000) Spectrum of
retGC1 mutations in Leber's congenital
amaurosis. Eur J Hum Genet 8:578-582.
19. Sohocki MM, Perrault I, Leroy BP, Payne AM,
Dharmaraj S, Bhattacharya SS, Kaplan J,
Maumenee IH, Koenekoop R, Meire FM, Birch
DG, Heckenlively JR and Daiger SP (2000)
Prevalence of AIPL1 mutations in inherited
retinal degenerative disease. Mol Genet Metab
70:142-150.
20. Shuber AP, Skoletsky J, Stern R and Handelin
BL (1993) Efficient 12-mutation testing in the
CFTR gene: a general model for complex
mutation analysis. Hum Mol Genet 2:153-158.
21. Newton CR, Graham A, Heptinstall LE, Powell
SJ, Summers C, Kalsheker N, Smith JC and
Markham AF (1989) Analysis of any point
mutation in DNA: the amplification refractory
mutation system (ARMS). Nucleic Acids Res
17:2503-2516.
22. Khan JA, Ide CH and Strickland MP (1988)
Coats'-type retinitis pigmentosa. Surv Ophthalmol
32:317-332.
16. Thompson DA, Gyürüs P, Fleischer LL,
Bingham EL, McHenry CL, Apfelstedt-Sylla E,
Zrenner E, Lorenz B, Richards JE, Jacobson SG,
Sieving PA and Gal A (2000) Genetics and
phenotypes of RPE65 mutations in inherited
retinal degeneration. Invest Ophthalmol Vis Sci
41:4293-4299.
87
Chapter
5
CRB1 has a cytoplasmic domain that is functionally
conserved between human and Drosophila
Anneke I. den Hollander*1, Kevin Johnson*2, Yvette J.M. de Kok1, Ansgar
Klebes2, Han. G. Brunner1, Elisabeth Knust2 and Frans P.M. Cremers1
1
Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, the
Netherlands; 2Institüt für Genetik, Heinrich-Heine Universität, Düsseldorf, Germany
* The authors wish it to be known that, in their opinion, the first two authors should be regarded as
joint First Authors
Hum Mol Genet 10:2767-2773 (2001)
CRB1 has a functionally conserved cytoplasmic domain
Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal
dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The
CRB1 gene is expressed specifically in human retina and brain and encodes a protein
homologous to Drosophila Crumbs protein. In crumbs mutant embryos apico-basal
polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small
cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines
the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be
partially rescued by expression of just the membrane-bound cytoplasmic domain, and
overexpression of this domain in a wild-type background results in a multilayered
epidermis. A striking difference between CRB1 and Crumbs was that the latter contains
a transmembrane region and a 37-amino acid cytoplasmic domain. Here we describe an
alternative splice variant of human CRB1 that encodes a cytoplasmic domain 72%
similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary
for function in Drosophila are absolutely conserved. Rescuing and overexpression
studies in Drosophila show that the cytoplasmic domains are functionally related
between these distant species. This suggests that CRB1 organizes an intracellular
protein scaffold in the human retina. Human homologues of proteins binding to Crumbs
may be part of this complex and represent candidate genes for retinal dystrophies.
Introduction
Mutations in the Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging
from retinitis pigmentosa (RP) to Leber congenital amaurosis (LCA)1-3. Two specific types of
RP are caused by mutations in the CRB1 gene, RP with preserved para-arteriolar retinal
pigment epithelium (PPRPE) and RP with Coats-like exudative vasculopathy1,2. RP with
PPRPE is a clinically distinct and severe form of RP, which is characterized by a
morphological preservation of the RPE adjacent to and under the retinal arterioles, whereas
there is a general loss of the RPE throughout the retina4. Coats-like exudative vasculopathy is
a relatively rare complication of RP, characterized by vascular abnormalities, yellow
extravascular lipid depositions and retinal detachment. CRB1 mutations have been detected in
59% (17/29) of unrelated, isolated and autosomal recessive cases of RP with PPRPE (ref. 1;
unpublished data), in 56% (5/9) of RP patients who had developed Coats-like exudative
vasculopathy2, and in 9-13% of patients with LCA, the most severe retinal dystrophy leading
to blindness or severe visual impairment from birth2,3. In our work, three patients had null
91
Chapter 5
mutations on both alleles, suggesting that LCA is the most severe phenotype that can be
associated with mutations in the CRB1 gene2.
The CRB1 gene is expressed in human retina, brain and fetal brain. The protein encoded by
CRB1 (GenBank accession no. AF154671) was predicted as an extracellular protein with a
signal peptide, 19 EGF-like domains and three laminin A G-like domains. It is likely that
these domains interact with other extracellular or transmembrane proteins. CRB1 is
homologous to Drosophila Crumbs protein1. However, Crumbs is larger, has 30 EGF-like
domains, 4 laminin A G-like domains and is a transmembrane protein with a small, 37 amino
acid cytoplasmic domain5. The largest part of the Crumbs protein (98%) is localized
extracellularly.
During embryonic development, Drosophila Crumbs is expressed mainly in the epithelia
derived from the ectoderm5,6. Epithelial cells normally show pronounced apico-basal polarity,
demonstrated by a polarized cytoskeleton, an asymmetric distribution of organelles and
proteins and the separation of the plasma membrane into distinct apical and basolateral
domains. Crumbs is expressed on the apical face of ectodermally-derived epithelia and is
particularly concentrated in a narrow region just apical to the zonula adherens (ZA), a beltlike structure encircling the apex of epithelial cells5,7. The ZA connects apico-laterally
localized actin belts of adjacent cells with each other, and thereby participates in cell-cell
adhesion and intercellular communication.
Mutations in crumbs are characterized by a severe disruption of ectodermally derived
epithelia and extensive cell death in the epidermal primordium and some other epithelia of
ectodermal origin5. Consequently, crumbs mutant embryos fail to develop a continuous
cuticle, a secretion product of the epidermis, and only develop ‘crumbs’ of cuticle6. Loss of
epithelial polarity in crumbs mutant embryos is associated with a misdistribution of DEcadherin and Armadillo8 and consequently the failure to assemble a ZA. The mutant
phenotype can be caused by mutations abolishing transcription of crumbs as well as a
mutation deleting only the last 23 amino acids of the cytoplasmic domain, emphasizing the
importance of this domain9.
The crumbs mutant phenotype can be partially rescued by GAL4/upstream activator sequence
(UAS)-mediated overexpression of full-length Crumbs protein. Major parts of the cuticle are
restored, visualized by a contiguous cuticular shield with well-developed denticle belts,
characteristic cuticular structures indicative of normal epidermal development10. A similar
degree of rescue was observed when overexpressing only the membrane-bound cytoplasmic
domain of Crumbs, tagged with a Myc epitope (crbintra-myc)10. Furthermore, embryos rescued
92
CRB1 has a functionally conserved cytoplasmic domain
by overexpression of crbintra-myc show continuous patches of epidermis, in which the ZA
appears to be properly organized11.
Moderate overexpression of either full-length Crumbs or strong overexpression of the
membrane-bound cytoplasmic domain (crbintra-myc) in wild-type embryos leads to an abnormal,
multilayered epidermis10,11. The outermost cells still exhibit aspects of polarity, such as
polarized distribution of apical membrane marker Stranded at Second (SAS) and basolateral
membrane marker Fasciclin III (FASIII), whereas the cells in the inner layer show only
basolateral characteristics11. The cells lose their columnar shape, DE-cadherin localization is
affected and a proper ZA fails to form11. As a consequence, morphogenetic movements of the
epidermis such as `dorsal closure` fail, resulting in embryonic lethality. Interestingly,
overexpression of crbextra, a construct containing the extracellular domain of Crumbs but
lacking the transmembrane and cytoplasmic domain, does not have any obvious consequences
for epithelial development10.
The fundamental role of the cytoplasmic domain of Drosophila Crumbs prompted us to reexamine the human CRB1 locus for additional transcripts and protein isoforms. This analysis
identified an alternative 3’ exon of CRB1 that predicts a transmembrane domain and a 37
amino acid cytoplasmic domain, which is 72% similar to Crumbs. Rescuing experiments in
Drosophila crumbs mutant embryos and overexpression studies in wild-type Drosophila show
that the cytoplasmic domains are functionally related between these distant species.
Results
Alternative splicing of CRB1
The previously published CRB1 cDNA sequence consists of 4361 bp and encodes a predicted
protein of 1376 amino acids (GenBank accession no. AF154671)1. Screening of EST
databases identified 13 human ESTs that showed overlap with the CRB1 cDNA sequence.
Interestingly, comparison of the EST sequences revealed that they diverge, suggesting that the
CRB1 gene may exhibit alternative splicing at its 3’ end. EST sequences all diverged at the
same position, which would result in different protein products that differ at their C-termini
after amino acid 1335.
Four classes of mRNAs predicted to encode four different proteins can be distinguished
(Figure 1A). Class I molecules contain exons 11a and 11b and encode a 1376 amino acid
protein, which was originally described as CRB1 (GenBank accession no. AF154671)1. The
cDNA sequence has a 94 bp 3’-UTR that contains a small poly(A) tail but lacks an upstream
93
Chapter 5
A
10
11a 11b
*
*
I
II
III
IV
10
12
11a
12
*
10
11a
12a
AAA
12
*
10
B
AAA
11a
1 2 3 4 5 6 7 8 9 10 11 12
AAA
13
*
13 14 15
I
II
GAPDH
C
I
1336
1349
1362
1375
II
1336
1349
1362
1375
1388
1401
GTAAGCAGCCTCTCCTTTTATGTCTCTCTCTTATTCTGG
V S S L S F Y V S L L F W
CAGAATCTTTTTCAGCTTCTTTCTTACCTCATTTTGCGT
Q N L F Q L L S Y L I L R
ATGAATGACGAGCCAGTTGTTGAGTGGGGTGAACAGGAA
M N D E P V V E W G E Q E
GATTATTAA
D Y *
TTGGCAGATGACTTGATCTCCGACATTTTCACCACTATT
L A D D L I S D I F T T I
GGCTCAGTGACTGTCGCCTTGTTACTGATCCTCTTGCTG
G S V T V A L L L I L L L
GCCATTGTTGCTTCTGTTGTCACCTCCAACAAAAGGGCA
A I V A S V V T S N K R A
ACTCAGGGAACCTACAGCCCCAGCCGTCAGGAGAAGGAG
T Q G T Y S P S R Q E K E
GGCTCCCGAGTGGAAATGTGGAACTTGATGCCACCCCCT
G S R V E M W N L M P P P
GCAATGGAGAGACTGATTTAG
A M E R L I *
D
Hs CRB1 SNKRATQGTYSPSRQEKEGSRVEMWNLMPPPAMERLI*
Dm crb RNKRATRGTYSPSAQEYCNPRLEMDNVLKPPPEERLI*
Ce CRB1 RGNNAMHGHYSPSSHEFTQNRMAMPTVIKLPPQERLI*
Ce CRL1 RQSRKLHGKYNPAREEHNLSAAYAMPMSHIAKEERLI*
Figure 1. Alternative splicing at the 3’ end
of the CRB1 gene. (A) Isoform I contains
exons 1-10, 11a and 11b; one cDNA was
encountered in the database that also contains
exon 12. Isoform II contains exons 1-10, 11a
and 12; isoform III contains exons 1-10, 11a,
12a and 12; and isoform IV contains exons 110, 11a and 13. Asterisk, the site of the
stopcodon. AAA, a poly(A) tail, preceded by
a polyadenylation signal. Triangles, primers
used for RT-PCR. (B) RT-PCR analysis of
isoforms I (464 bp) and II (557 bp) on a
panel of tissues, performed with a forward
primer in exon 10 and the reverse primer
behind the stop codon. Lane 1, liver; lane 2,
kidney; lane 3, lung; lane 4, retina; lane 5,
skeletal muscle; lane 6, RPE cell line ARPE19; lane 7, placenta; lane 8, RPE/choroid;
lane 9, heart; lane 10, fetal eye; lane 11,
brain; lane 12, fetal cochlea; lane 13, testis;
lane 14, fetal liver; lane 15, fetal brain.
GAPDH serves as a control. (C) Nucleotide
sequence of the coding regions of exon 11b
(isoform I) and exon 12 (isoform II), and
predicted protein sequence of the C-terminal
amino acids of the two alternative splice
forms of CRB1. Isoform I (GenBank
accession nos AF154671 and AY043324)
encodes a 1376 amino acid protein with a
possible transmembrane region (boxed) and a
20 amino acid cytoplasmic domain. Isoform
II (GenBank accession no. AY043325)
encodes a 1406 amino acid protein with a
transmembrane region (boxed) and a 37
amino
acid
cytoplasmic
domain.
Transmembrane regions were predicted by
TopPred2. (D) Alignment of cytoplasmic
domains of Homo sapiens (Hs) CRB1
(GenBank accession no. AY043325),
Drosophila melanogaster Crumbs (Dm Crb)
(GenBank accession no. M33753), C.
elegans (Ce) Crumbs homologue 1 (CRB1;
GenBank accession no. U42839) and
Crumbs-like protein 1 (CRL1; GenBank
accession no. AL008869). Boxed amino
acids are conserved in all homologues.
Asterisks denote the C-termini.
polyadenylation signal, which suggests that this sequence does not contain the 3’ end of the
transcript1. 3’-RACE experiments using a forward primer in exon 11b resulted in a product
with a 704 bp 3’-UTR that contains a poly(A) tail, which is not preceded by a polyadenylation
signal. In the database we encountered one cDNA clone from the Soares fetal liver/spleen
94
CRB1 has a functionally conserved cytoplasmic domain
1NFLS cDNA library that contains exons 11a, 11b and 12, and has a poly(A) tail that is
preceded by a polyadenylation signal (GenBank accession nos R09831 and AY043322). Class
II molecules (GenBank accession no. AY043325) contain exons 11a and 12 and are
represented by an EST from the Soares fetal liver/spleen 1NFLS cDNA library (GenBank
accession nos N59646 and N78199) and an EST from an anaplastic oligodendroglioma cDNA
library (GenBank accession no. BF347919). The stop codon is present in exon 12, leading to a
1406 amino acid protein. Class III molecules contain exons 11a, 12a and 12, and are
represented by two cDNA clones from the Soares fetal liver/spleen 1NFLS cDNA library
(GenBank accession nos AA033939, AY043323, T87786 and T87787). Exon 12a starts 116
bp upstream of the splice acceptor site of exon 12. The stop codon is present in exon 12a, and
the cDNA is predicted to encode a 1336 amino acid protein. Class IV molecules contain
exons 11a and exon 13, a novel exon located 3 kb downstream of exon 12. This isoform is
represented by two ESTs derived from a cDNA library made from a mixture of cDNA from
fetal lung, testis and B-cells (GenBank accession nos AI805296 and AA909366), and by one
EST from a testis cDNA library (GenBank accession no. AI026624). The stop codon is
present in exon 13 and this isoform is predicted to encode a 1364 amino acid protein.
To determine whether isoforms I, II, III or IV are transcribed, a reverse transcriptionpolymerase chain reaction (RT-PCR) was performed on RNA from 10 adult tissues, four fetal
tissues and a RPE cell line. RT-PCR was performed with a sense primer in exon 10 and
antisense primers chosen behind the stop codons of the different splice forms (Figure 1A).
Isoforms III and IV could not be amplified from any of these tissues (data not shown).
Isoforms I and II were successfully amplified from adult retina, adult brain, fetal eye and fetal
brain, but not from any other tissues (Figure 1B). The primersets for isoform I amplified a
weak product on RPE/choroid RNA, which is likely to be caused by a contamination with
retinal tissue. By RNA in situ hybridization we detected expression of the gene in the outer
and inner nuclear layers of the retina but not in the RPE12.
The predicted protein sequences encoded by the class I and class II isoforms of CRB1 were
subjected to four different transmembrane prediction programs (PRED-TMR, DAS, TMpred
and TopPred2). For both isoforms putative transmembrane regions were predicted near the Cterminus. However, the prediction of the transmembrane region in isoform II is much stronger
than that in isoform I, as can be exemplified by the prediction scores of the DAS program (5.8
versus 2.9), the TMpred program (2457 versus 1307) and the TopPred2 program (2.346
versus 1.086). The protein encoded by isoform II is predicted to contain a 22 amino acid
transmembrane region and a 37 amino acid cytoplasmic domain (Figure 1C). Although
95
Chapter 5
isoform I was previously described as an extracellular protein1, detailed analysis with these
transmembrane prediction programs suggests that it may contain a transmembrane region and
a small, 20 amino acid cytoplasmic domain (Figure 1C).
Conservation of residues in the cytoplasmic domains of Crumbs homologues
supports the presence of two functional domains
The cytoplasmic domain of isoform II is highly (72%) similar to that of Drosophila Crumbs
protein. Twenty-two out of 37 (59%) amino acids are identical between human CRB1 and
Drosophila Crumbs cytoplasmic domains (Figure 1D). Eight amino acids are identical
between CRB1, Drosophila Crumbs, a Crumbs homologue (C.e. CRB1; GenBank accession
no. U42839) and a Crumbs-like protein (C.e. CRL1; GenBank accession no. AL008869 and
EAT-20; GenBank accession no. AB032748) both recently identified in Caenorhabditis
elegans (Figure 1D)11,13,14. The conserved residues are clustered in two regions of the
cytoplasmic domains, the C-terminal amino acids ERLI and an N-terminal region including
residues at positions 8, 10, 12 and 16 of the cytoplasmic domains (G8, Y10, P12 and E16
respectively).
These two conserved regions coincide with two functional subdomains recently identified by
site-directed mutational analysis in the cytoplasmic domain of Drosophila Crumbs11. The Cterminal amino acids EERLI, as well as residues Y10 and E16 in the N-terminal region, are
necessary to rescue the crumbs mutant phenotype. However, if mutated constructs are
overexpressed in a wild-type background, only constructs including an intact C-terminus (the
EERLI motif) produce a multilayered epidermis irrespective of replacement of additional
conserved residues. The C-terminal EERLI motif binds to the PDZ-domain containing protein
Discs Lost in vitro15 and recruits in into an apically localized protein complex11,15, whereas
the region circumspanning the amino acids Y10 and E16 may be involved in other protein
interactions11. The stretch of proline residues preceding the ERLI motif in human CRB1,
Drosophila Crumbs and C. elegans CRB1 may serve to break any secondary structures,
allowing free movement of the C-terminal ERLI-motif.
Overexpression of CRB1intra-myc rescues the crumbs mutant phenotype
The crumbs mutant phenotype can be partially rescued by GAL4/UAS-mediated
overexpression of either the full-length Crumbs protein or of only the membrane-bound
cytoplasmic domain of Crumbs, tagged with a Myc epitope (crbintra-myc)10. To determine
whether the cytoplasmic domain of human CRB1 can substitute the cytoplasmic domain of
96
CRB1 has a functionally conserved cytoplasmic domain
Drosophila Crumbs in rescuing the crumbs phenotype, GAL4/UAS-mediated expression of
the membrane-bound cytoplasmic domain of CRB1 tagged with a Myc epitope (CRB1intra-myc)
was induced in crumbs mutant embryos. Major parts of the cuticle were restored to a
contiguous cuticular shield with belts of denticles, which is a sign of normal patterning and
Figure 2. Rescue of the crumbs phenotype by overexpression of CRB1intra-myc. (A, B) Cuticle
preparations of (A) crumbs mutant embryos and (B) crumbs mutant embryos overexpressing
CRB1intra-myc. Rescued embryos show a contiguous shield of cuticle and normal denticle belts [inset
in (B)] as a sign of normal patterning and differentiation. (C-E) Confocal optical sections through
the ventral epidermis of embryos during germ band retraction (st12) showing the distribution of
pY-epitopes in red and nuclei (Yoyo-1) in green. In wild-type (C), anti-pY staining marks the ZA
at the apex of epithelial cells. Crumbs mutant embryos (D) do not develop a ZA thus pY-epitopes
never localize. Crumbs mutant embryos overexpressing CRB1intra-myc (E) show a partial restoration
of the ZA and a return to a more columnar shape of the epithelium. (F, G) TUNEL-labelling of
cells dying through apoptosis. Crumbs mutant embryos (F) show extensive, ectopic cell death in
the epidermis in addition to endogenously occurring apoptosis. Crumbs mutant embryos
overexpressing CRB1intra-myc (G) in the epidermis show much reduced epidermal cell death whereas
apoptosis in the CNS proceeds as normal. Anterior always to the left. (B) View onto the ventral
epidermis. (C-E) Apical is up. (F and G) Confocal sections of whole embryos focussing on the
head and abdominal epidermis. In red unspecific yolk staining. Scale bar 5µm. Genotypes: (A, D,
F) crb11A22/crb 11A22; (B, E, G) CRB1intra-myc 9.1/+; GAL4 385.3/+; crb11A22/crb 11A22.
97
Chapter 5
differentiation (compare Figure 2A and B, for comparison to a wild-type cuticle see Figure
3A). The phenotypic rescue reached by the strongest expression line (CRB1intra-myc 9.1) is
similar compared to embryos rescued by full-length Crumbs or the membrane-bound
cytoplasmic domain of Crumbs10.
The basis for the generation of contiguous cuticle is normal epithelial polarity (not shown)
and restoration of the ZA, analyzed by the expression pattern of phosphotyrosine (pY)containing epitopes that mark the ZA (compare Figure 2C-E). Crumbs mutant embryos do not
develop a ZA, while crumbs mutant embryos overexpressing CRB1intra-myc show a partial
restoration of the ZA and a more columnar shape of the epithelial cells.
Crumbs mutant embryos show extensive, ectopic cell death in the epidermis in addition to the
endogenously occurring apoptosis. Crumbs mutant embryos overexpressing CRB1intra-myc in
the epidermis show much reduced epidermal cell death whereas outside the expression
domain of the GAL4 activator, e.g. the central nervous system (CNS), endogenous apoptosis
proceeds as normal (compare Figure 2F and 2G).
Overexpression of CRB1intra-myc transforms the single-layered epidermis into a
multilayered tissue
Moderate overexpression of either full-length Crumbs or strong overexpression of the
membrane-bound cytoplasmic domain (crbintra-myc) in wild-type embryos leads to an abnormal,
multilayered epidermis10,11. To determine whether the cytoplasmic domain of CRB1 has a
similar effect on the development of epithelia as the cytoplasmic domain of Drosophila
Crumbs, GAL4/UAS-mediated expression of CRB1intra-myc was induced in a wild-type
background. Overexpression of CRB1intra-myc in wild-type embryos leads to defects
comparable to those caused by overexpression of crbintra-myc (refs. 10,11).
Cuticle preparations of embryos overexpressing CRB1intra-myc show normal patterning,
although the embryo appears smaller, not extending the complete length of the inner eggshell.
Embryonic lethality is in 98% accompanied by the incomplete morphogenetic movement of
dorsal closure, resulting in large dorsal holes (compare Figure 3A and B, arrowheads). In
embryos overexpressing CRB1intra-myc, the single-layered epidermis has lost its columnar
shape and has become multilayered. The outer layer of cells still exhibit aspects of polarity,
such as polarized distribution of apical membrane marker SAS and basolateral membrane
marker FASIII, whereas the cells in the inner layer show only basolateral characteristics
(compare Figure 3C and D).
98
CRB1 has a functionally conserved cytoplasmic domain
Discussion
Previously, we described a human homologue of Drosophila Crumbs, CRB1, which exhibits
striking structural and sequence similarity to Crumbs1. The most striking difference between
CRB1 and Crumbs was that the latter contains a transmembrane and 37 amino acid
cytoplasmic domain, whereas CRB1 was predicted to encode an extracellular protein1.
Analysis of splice variants of CRB1 presented here revealed that the gene exhibits alternative
splicing at its 3’ end, and transcription of two splice forms was confirmed by RT-PCR. The
transcripts are predicted to encode a 1376 amino acid extracellular or transmembrane protein,
and a 1406 amino acid membrane-bound protein, respectively. It is not clear which 3’-UTR is
used by the first isoform (Figure 1A). The cDNA sequence that was described by den
Hollander et al.1 contains a 94 bp 3’-UTR with a small poly(A) tail but no polyadenylation
signal. By 3’-RACE the 3’-UTR was extended to 704 bp, but the poly(A) tail of this product
was not preceded by a polyadenylation signal either. One EST was identified in the database
that contains exon 12 and has a poly(A) tail preceded by a polyadenylation signal. However, it
is not clear whether this is the 3’-UTR used in vivo, since in that case an RT-PCR with
primers in exon 10 and exon 12 would amplify two products; 557 bp for isoform II and 799
bp for isoform I (Figure 1A and B). Extensive 3’-RACE studies using a forward primer in
exon 11b only revealed the 704 bp 3’-UTR indicating that the isoform I EST containing exon
12 represents a rare transcript or is an artifact.
The presence of two other 3’ end splice variants, represented by ESTs in the database, could
not be confirmed by RT-PCR. Possibly, these splice variants are expressed at a very low level
not detectable by RT-PCR. They also could be expressed during particular developmental
stages or just in a small subset of cells of the retina or brain, which were not tested sufficiently
in our experiments. Another possibility is that the ESTs representing these transcripts are
artifacts caused by aberrant splicing. However, one of these splice forms was found on two
independent EST clones from two different cDNA libraries, suggesting that this explanation
is less likely. Alternative splicing has not been detected for Drosophila crumbs, although
northern blot analysis shows two different mRNA transcripts5. In embryos and pupae only a
7.5 kb transcript was detected, whereas in larvae and adult flies an additional 7.7 kb transcript
was present.
The predicted 1406 amino acid protein encoded by CRB1 isoform II contains a
transmembrane domain and a 37 amino acid cytoplasmic domain 72% similar to that of
Crumbs. The identification of this cytoplasmic domain of CRB1 suggests that it is a true
99
Chapter 5
Figure 3. Overexpression of CRB1intra-myc in wild-type embryos leads to a multilayered phenotype.
(A, B) Cuticle preparations of wild-type embryos (A) and CRB1intra-myc overexpressing embryos
(B) showing the dorsal closure defect which results in a dorsal hole (arrowheads) at the end of
development. The cuticle patterning is normal though the embryo appears smaller, possibly due to
the number of epidermal cells found in the second epidermal layer. (C, D) Confocal optical section
through the ventral epidermis of embryos at the end of germ band retraction (st12/13). The wildtype embryo (C) shows apical localization of SAS (green) and basolateral localization of FASIII
(red) in a single-layered epidermis. In a CRB1intra-myc overexpressing embryo (D) the epidermis has
lost its columnar shape and has become multilayered (asterisks). The outer layer of cells maintains
normal membrane polarity whereas the inner cells lose the expression of apical SAS, instead
FASIII is localized all around the membrane.
homologue of Drosophila Crumbs. At first sight, mutations in human CRB1 and Drosophila
Crumbs seem to cause different phenotypes. However, diseases associated with mutations in
CRB1 and phenotypes resulting from crumbs loss of function both lead to progressive
degeneration of particular polarized cell types. The different phenotypes are a reflection of the
expression domains of CRB1 and crumbs. CRB1, which is expressed in the retina and the
brain, causes severe retinal dystrophies ranging from RP to LCA. These diseases are
characterized by a progressive degeneration of the retina, a highly organised and polarized
tissue. Crumbs is expressed in ectodermally derived epithelia of the embryo. Crumbs mutant
embryos fail to maintain cell polarity in epithelia that are derived from the blastoderm,
namely the amnioserosa, the epidermis, the fore- and hindgut, the trachea, the Malpighian
tubules and the salivary glands. Depending on the epithelium considered, the defects range
from slight disturbances in epithelial organization, disruption of epithelial integrity to
widespread cell death. In late larval stages Crumbs is expressed in all imaginal disc cells but
100
CRB1 has a functionally conserved cytoplasmic domain
enriched in the developing photoreceptor clusters and the inner and outer optic anlagen of the
brain (ref. 6; K. Johnson and E. Knust, unpublished data). So far, no phenotype has been
described for crumbs mutant clones in adult eyes but preliminary results suggest
morphological abnormalities and inducible degeneration of the photoreceptor cells (K.
Johnson and E. Knust, in preparation).
The function of Drosophila Crumbs has been studied extensively, and in particular it was
shown that the cytoplasmic domain is of crucial importance for the function of Crumbs.
Deletion of the 23 C-terminal amino acids of the cytoplasmic domain completely abolishes
Crumbs function9. Furthermore, the membrane-bound cytoplasmic domain can partially
rescue the crumbs phenotype, and overexpression in a wild-type background induces a multilayered epidermis10. In this work we show that the cytoplasmic domain of CRB1 can
substitute the function of Drosophila Crumbs in rescuing and overexpression studies.
Complete rescue could not be obtained, neither using the Drosophila nor the human
cytoplasmic domain, because the GAL4 expression of the activator line was not sufficiently
similar to the expression of wild-type Crumbs10.
The cytoplasmic domain of Crumbs has been shown to contain two functional subdomains,
the C-terminal EERLI motif and a N-terminal region containing residues Y10 and E16, which
are both highly conserved between Crumbs homologues (Figure 1D). The EERLI motif binds
the multi-PDZ-domain protein Discs Lost in vitro15 and is essential for its recruitment into an
apical protein complex in epithelial cells11. Crumbs, discs lost and stardust may constitute a
pathway to organize the apical cortical scaffold that influences the assembly of a continuous
ZA. Stardust encodes a scaffolding protein of the MAGUK family and also interacts with the
C-terminal EERLI motif in the yeast two-hybrid system16.
Similarly, the C-terminus of CRB1 may interact with a human homologue of Discs Lost
and/or Stardust to organize a protein scaffold in the human retina. It has been speculated that
CRB1 may play a role in localizing the phototransduction complex to the apical membrane of
the photoreceptors17. The subsequent lack of coordinated phototransduction activity in the
photoreceptors may lead to their progressive decay and indirectly affect the supporting retinal
pigment epithelium, resulting in LCA or RP phenotypes.
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Chapter 5
Materials and methods
Analysis of EST clones
To identify splice variants at the 3’ end of the CRB1 gene, a BLAST search was performed
with the CRB1 cDNA sequence against the dbEST database. Thirteen human cDNA
sequences were identified that showed overlap with the CRB1 cDNA sequence. These ESTs
were derived from retina (GenBank accession no. AI444814), brain (GenBank accession nos
BF347919 and R35113), testis (GenBank accession no. AI026624), fetal liver/spleen
(GenBank accession nos N78199, N59646, AA033939, R09831, T87786 and T87787) and
mixed tissue (fetal lung, testis, B-cell; GenBank accession nos AI805296, AA909366 and
AA906086) cDNA libraries. ESTs with GenBank accession nos N78199 and N59646
represent the same IMAGE clone (IMAGE clone no. yv74f07) sequenced from both sides, as
well as ESTs with GenBank accession nos T87786 and T87787 (IMAGE clone no. yd93e09).
The two cDNA clones represented by GenBank accession nos AA033939 (IMAGE clone no.
zi06c01) and R09831 (IMAGE clone no. yf30a11), were ordered from the Resource Center/
Primary Database (RZPD) of the German Human Genome Project, Berlin, Germany. Plasmid
DNA was prepared using a miniprep plasmid DNA isolation kit (Qiagen) and analyzed by
cycle sequencing. The complete sequences of the inserts of these clones were deposited in the
GenBank database under accession nos AY043322 and AY043323.
RT-PCR analysis of 3’ end splice variants
For RT-PCR analysis, total RNA was isolated from adult human retina, ARPE-19,
RPE/choroid and testis by RNAzol B (Campro Scientific) and from fetal eye and fetal cochlea
by CsCl purification. Total RNA samples from human liver, kidney, lung, skeletal muscle,
placenta, heart, brain, fetal liver and fetal brain were purchased from Clontech. Randomly
primed cDNA was synthesized from DNaseI-treated RNA using random hexanucleotides as
described by den Hollander et al.18. RT-PCR reactions were performed for four putative
CRB1 3’ end splice variants, using a forward primer in exon 10 (2529; 5’TGCAGACAGAGCAGATTACC-3’) and reverse primers downstream of the predicted stop
codons.
Primers
were
located
in
CAGAGATCTAAAATGAATCAAG-3’),
TCAGGTATGTCAGAGATACC-3’),
ACAATGGAACTACTCAGG-3’),
102
in
and
exon
in
exon
exon
in
11b
exon
12
12a
13
for
for
for
for
isoform
I
(2530;
5’-
II
(3123;
5’-
isoform
III
(3850;
5’-
isoform
IV
(3851;
5’-
isoform
CRB1 has a functionally conserved cytoplasmic domain
TTGCATCCAGCAGGCACGG-3’). PCR was performed as described by den Hollander et
al.18 for 35 cycles.
Transmembrane prediction
Transmembrane predictions of CRB1 protein sequences were performed with four different
transmembrane prediction programs. Prediction with PRED-TMR was performed at the
University of Athens, Biophysics Laboratory (http://o2.db.uoa.gr), with DAS at the
Stockholm Bioinformatics Center (http://www.sbc.su.se), with TopPred2 at the Institut
Pasteur (bioweb.pasteur.fr) and with TMpred at the European Molecular Biology Network
(http://www.ch.embnet.org).
Generation of a CRB1/UAS construct and germline transformation
Directed gene expression in Drosophila was performed as described by Brand and
Perrimon19. A GAL4-dependent target gene is constructed by subcloning it behind a tandem
repeat of GAL4-binding sites, known as the UAS. To activate the target gene, flies carrying
the target are crossed to flies expressing GAL4. Depending on the genomic enhancer driving
GAL4 expression, GAL4 and subsequently the target gene can be expressed in a cell- or
tissue-specific pattern. The UAS vector (pUAST)19 was previously used to construct crbintramyc
, which expresses a transgene containing the N-terminal region of Crumbs including the
signal peptide and the C-terminal region including the transmembrane and cytoplasmic
domains, separated by a Myc epitope10. An UAS construct expressing the cytoplasmic domain
of human CRB1 (CRB1intra-myc) was generated by replacing the Drosophila cytoplasmic
domain in crbintra-myc with the cytoplasmic domain of CRB1. The cytoplasmic domain of
CRB1 was amplified in a RT-PCR reaction on human fetal brain cDNA using the primer
combination
5’-GATCTGGCCAGGAACAAAAGGGCAACTCAGGG-3’
and
5’-
GATCTCTAGACTAAATCAGTCTCTCCATTGCA-3’. The primers introduced MscI and
XbaI sites (underlined), respectively. The EcoRI-XbaI insert of crbintra-myc was cloned in
pBluescript. The cytoplasmic domain of crumbs was excised by MscI-XbaI digestion and the
PCR product containing the cytoplasmic domain of CRB1 was cloned into the MscI-XbaI
sites. The EcoRI-XbaI fragment containing the signal peptide of Crumbs, the Myc epitope, the
transmembrane domain of Crumbs and the cytoplasmic domain of CRB1 was excised from
pBluescript and ligated into EcoRI-XbaI-digested pUAST. The pUAST P-element vector
construct carrying CRB1intra-myc was stably transformed into the germline of flies according to
Spradling20. More than 20 lines were established, 10 of which were compared in more detail.
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Chapter 5
They showed different degrees of the same phenotype depending on their genomic
localization and expressivity.
Fly stocks and overexpression
Effector lines shown are CRB1intra-myc 9.1 (first chromosome) and CRB1intra-myc 18.1 (second
chromosome). In a wild-type background, overexpression was mediated by GAL4daG32 in a
uniform, daughterless expression pattern. Rescue experiments were carried out with
GAL4385.3, crb11A22/TM3 which results in a broad, segmentally reiterated pattern of expression
in the epidermis11.
Histology, cuticle preparations and immunocytochemistry
Cuticle preparations were prepared according to Wieschaus and Nüsslein-Volhard21. Standard
fixation protocols (4% formaldehyde) were applied. Antibody dilutions were as follows:
mouse anti-FASIII (7G10, 1:3, ref. 22), rabbit anti-SAS (1:500, E. Organ and D. Cavener,
unpublished data), mouse anti-phosphotyrosine (PY20, 1:300, Tranduction Labs). Jackson
Immunoresearch supplied Cy2- and Cy3-conjugated secondary antibodies. The DNA dye
Yoyo-1 (Molecular probes) was used at 1:8000. For TUNEL stainings (LaRoche), 4%
paraformaldehyde-fixed embryos were devitellinized, washed in PBS, 0.3% Triton X-100,
incubated in 100 mM Na-citrate, 0.1% Triton X-100 at 65°C for 30 min, washed again in
PBT, then TUNEL dilution buffer, and subsequently incubated in 40 µl TUNEL reaction mix
at 37°C for 3h according to the manufacturer’s recommendations.
Stainings were analyzed with a Leica TCS NT confocal microscope and images were
processed and arranged using Photoshop 5.5 (Adobe) and Canvas 6 (Deneba) on an Apple
Macintosh.
Acknowledgements
We thank M. Luijendijk for providing fetal eye and cochlea RNA samples. A.I.d.H. and
Y.J.M.d.K. are supported by the Foundation Fighting Blindness USA, Inc. K.J. and E.K. are
grateful for the support of the German Research Foundation, DFG grant Kn250/15-2 and the
German “Fonds der Chemischen Industrie”.
104
CRB1 has a functionally conserved cytoplasmic domain
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amaurosis. Arch Ophthalmol 119:415-420.
4.
Heckenlively JR (1982) Preserved para-arteriole
retinal pigment epithelium (PPRPE) in retinitis
pigmentosa. Brit J Ophthalmol 66:26-30.
5.
Tepass U, Theres C and Knust E (1990) crumbs
encodes an EGF-like protein expressed on apical
membranes of Drosophila epithelial cells and
required for organization of epithelia. Cell
61:787-799.
6.
Tepass U and Knust E (1990) Phenotypic and
developmental analysis of mutations at the
crumbs locus, a gene required for the
development of epithelia in Drosophila
melanogaster. Roux's Arch Dev Biol 199:189206.
7.
Tepass U (1996) Crumbs, a component of the
apical membrane, is required for zonula adherens
formation in primary epithelia of Drosophila. Dev
Biol 177:217-225.
8.
Grawe F, Wodarz A, Lee B, Knust E and Skaer H
(1996) The Drosophila genes crumbs and
stardust are involved in the biogenesis of
adherens junctions. Development 122:951-959.
9.
Wodarz A, Grawe F and Knust E (1993)
CRUMBS is involved in the control of apical
protein targeting during Drosophila epithelial
development. Mech Dev 44:175-187.
10. Wodarz A, Hinz U, Engelbert M and Knust E
(1995) Expression of crumbs confers apical
character on plasma membrane domains of
ectodermal epithelia of Drosophila. Cell 82:6776.
11. Klebes A and Knust E (2000) A conserved motif
in Crumbs is required for E-cadherin localisation
and zonula adherens formation in Drosophila.
Curr Biol 10:76-85.
12. den Hollander AI, Ghiani M, de Kok Y,
Wijnholds J, Ballabio A, Cremers FPM and
Broccoli V (2002) Isolation of Crb1, a mouse
homologue of Drosophila crumbs, and analysis of
its expression pattern in eye and brain. Mech Dev
110:203-207.
13. Shibata Y, Fujii T, Dent JA, Fujisawa H and
Takagi S (2000) EAT-20, a novel transmembrane
protein with EGF motifs, is required for efficient
feeding in Caenorhabditis elegans. Genetics
154:635-646.
14. Bossinger O, Klebes A, Segbert C, Theres C and
Knust E (2001) Zonula adherens formation in
Caenorhabditis elegans requires dlg-1, the
homologue of the Drosophila gene discs large.
Dev Biol 230:29-42.
15. Bhat MA, Izaddoost S, Lu Y, Cho K-O, Choi KW and Bellen HJ (1999) Discs lost, a novel multiPDZ domain protein, establishes and maintains
epithelial polarity. Cell 96:833-845.
16. Bachmann A, Schneider M, Theilenberg E,
Grawe F and Knust E (2001) Drosophila Stardust
is a partner of Crumbs in the control of epithelial
cell polarity Nature 414:638-643.
17. Rashbass P and Skaer H (2000) Cell polarity:
Nailing Crumbs to the scaffold. Curr Biol
10:R234-R236.
18. den Hollander AI, van Driel MA, de Kok YJM,
van de Pol TJR, Hoyng CB, Brunner HG,
Deutman AF and Cremers FPM (1999) Isolation
and mapping of novel candidate genes for retinal
disorders
using
suppression
subtractive
hybridization. Genomics 58:240-249.
19. Brand AH, Perrimon N (1993) Targeted gene
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20. Spradling AC (1986) P-element mediated
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22. Patel NH, Snow PM and Goodman CS (1987)
Characterization and cloning of fasciclin III: a
glycoprotein expressed on a subset of neurons
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106
Chapter
6
Isolation of Crb1, a mouse homologue of Drosophila
crumbs, and analysis of its expression pattern
in eye and brain
Anneke I. den Hollander1, Michela Ghiani2,3, Yvette J.M. de Kok1, Jan
Wijnholds4, Andrea Ballabio2,5, Frans P.M. Cremers1 and Vania Broccoli2,3
1
Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, the
Netherlands; 2Téléthon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 3Stem
Cell Research Institute (SCRI), Hospital San Raffaele, Milan, Italy; The Netherlands
Ophthalmic Research Institute, Royal Netherlands Academy of Arts and Sciences (KNAW),
Amsterdam, the Netherlands; Faculty of Medicine, Second University of Naples, Naples, Italy
Mech Dev 110:203-207 (2002)
Expression pattern of Crb1
Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal
dystrophies. CRB1 is homologous to Drosophila crumbs, a protein essential for
establishing and maintaining epithelial polarity. We have isolated the mouse orthologue,
Crb1, and analyzed its expression pattern in embryonic and post-natal stages. Crb1 is
expressed exclusively in the eye, and the central nervous system. In the developing eye,
expression of Crb1 is detected in the retinal progenitors, and later on becomes restricted
to the differentiated photoreceptor cells where it remains active up to the adult stage. In
the developing neural tube, expression of Crb1 is restricted to its most ventral
structures, coinciding with the expression domain of Nkx2.2. In the adult brain, Crb1
expression is defined to areas where the production and migration of neurons occurs in
adulthood.
Introduction
In humans, mutations in the Crumbs homologue 1 (CRB1) gene cause Leber congenital
amaurosis and retinitis pigmentosa, progressive degenerative diseases of the retina causing
severe visual impairment from birth or from childhood, respectively1-3. CRB1 is homologous
to Drosophila Crumbs, a protein that is essential for establishing and maintaining apico-basal
polarity in epithelia derived from the ectoderm4. Crumbs forms a transmembrane anchor for
an intracellular protein scaffold that defines the localization of the zonula adherens, a belt-like
structure encircling the apex of epithelial cells5,6.
Although human CRB1 and Drosophila Crumbs are homologous, their expression patterns
are completely different. By reverse transcriptase-polymerase chain reaction (RT-PCR), we
showed that the expression of CRB1 is restricted to the retina and brain1, while Drosophila
crumbs is expressed in all epithelia derived from the ectoderm, including the epidermis, the
foregut and hindgut, the frontal sac, the tracheal system, the salivary glands, the malpighian
tubules and the optic lobe4,7. We isolated the mouse Crb1 gene and analyzed its expression
pattern in detail during embryonic development and in post-natal stages.
Results and discussion
Identification of the mouse Crb1 gene
The mouse Crb1 gene was identified by RT-PCR and rapid amplification of cDNA ends
(RACE). The deduced amino acid sequence is 76% identical to human CRB1. Both proteins
consist of 19 epidermal growth factor-like domains, 3 laminin A G-like domains, a
109
Chapter 6
A
m
h
1 MKLKRTAYLLFLYLSSSLLICIKNSFCNKNNTRCLSGPCQNNSTCEHFPQDNNC-CLDTANNLDKDCEDLKDPCFSSPCQGIATCVKIPGEGNFLCQCPP
1 MALKNINYLLIFYLSFSLLIYIKNSFCNKNNTRCLSNSCQNNSTCKDFSKDNDCSCSDTANNLDKDCDNMKDPCFSNPCQGSATCVNTPGERSFLCKCPP
SP
EGF1→
EGF2→
m
h
100 GYSGLNCETATNSCGGNLCQHGGTCRKDPEHPVCICPPGYAGRFCETDHNECASSPCLNGAMCQDGINGYSCFCVPGYQGRHCDLEVDECVSDPCKNEAV
101 GYSGTICETTIGSCGKNSCQHGGICHQDPIYPVCICPAGYAGRFCEIDHDECASSPCQNGAVCQDGIDGYSCFCVPGYQGRHCDLEVDECASDPCKNEAT
EGF3→
EGF4→
EGF5→
m
h
200 CLNEIGRYTCVCPQEFSGVNCELEIDECRSQPCLHGATCQDAPGGYSCDCAPGFLGEHCELSVNECESQPCLHGGLCVDGRNSYHCDCTGSGFTGMHCES
201 CLNEIGRYTCICPHNYSGVNCELEIDECWSQPCLNGATCQDALGAYFCDCAPGFLGDHCELNTDECASQPCLHGGLCVDGENRYSCNCTGSGFTGTHCET
EGF6→
EGF7→
m
h
300 LIPLCWSKPCHNDATCEDTVDSYICHCRPGYTGALCETDINECSSNPCQFWGECVELSSEGLYGNTAGLPSSFSYVGASGYVCICQPGFTGIHCEEDVDE
301 LMPLCWSKPCHNNATCEDSVDNYTCHCWPGYTGAQCEIDLNECNSNPCQSNGECVELSSEKQYGRITGLPSSFSYHEASGYVCICQPGFTGIHCEEDVNE
EGF8→
EGF9→
EGF
m
h
400 CLLHPCLNGGTCESLPGNYACHCPFDDTSRTFYGGENCSEILLGCTHHQCLNNGKCIPHFQNGQHGFTCQCLSGYAGPLCETVTTLSFGSNGFLWVTSGS
401 CSSNPCQNGGTCENLPGNYTCHCPFDNLSRTFYGGRDCSDILLGCTHQQCLNNGTCIPHFQDGQHGFSCLCPSGYTGSLCEIATTLSFEGDGFLWVKSGS
10→
EGF11→
G1→
m
h
500 HTGIGPECNISLRFHTVQPNALLLIRGNKDVSMKLELLNGCVHLSIEVWNQLKVLLSISHNTSDGEWHFVEVTIAETLTLALVGGSCKEKCTTKSSVPVE
501 VTTKGSVCNIALRFQTVQPMALLLFRSNRDVFVKLELLSGYIHLSIQVNNQSKVLLFISHNTSDGEWHFVEVIFAEAVTLTLIDDSCKEKCIAKAPTPLE
m
h
600 NHQSICALQDSFLGGLPMGTANNSVSVLNIYNVPSTPSFVGCLQDIRFDLNHITLENVSSGLSSNVKAGCLGKDWCESQPCQNRGRCINLWQGYQCECDR
601 SDQSICAFQNSFLGGLPVGMTSNGVALLNFYNMPSTPSFVGCLQDIKIDWNHITLENISSGSSLNVKAGCVRKDWCESQPCQSRGRCINLWLSYQCDCHR
EGF12→
m
h
700 PYTGSNCLKEYVAGRFGQDDSTGYAAFSVNDNYGQNFSLSMFVRTRQPLGLLLALENSTYQYVSVWLEHGSLALQTPGSPKFMVNFFLSDGNVHLISLRI
701 PYEGPNCLREYVAGRFGQDDSTGYVIFTLDESYGDTISLSMFVRTLQPSGLLLALENSTYQYIRVWLERGRLAMLTPNSPKLVVKFVLNDGNVHLISLKI
G2→
m
h
800 KPNEIELYQSSQNLGFISVPTWTIRRGDVIFIGGLPDREKTEVYGGFFKGCVQDVRLNSQTLEFFPNSTNNAYDDPILVNVTQGCPGDNTCKSNPCHNGG
801 KPYKIELYQSSQNLGFISASTWKIEKGDVIYIGGLPDKQETELNGGFFKGCIQDVRLNNQNLEFFPNPTNNASLNPVLVNVTQGCAGDNSCKSNPCHNGG
EGF13→
m
h
900 VCHSLWDDFSCSCPTNTAGRACEQVQWCQLSPCPPTAECQLLPQGFECIANAVFSGLSREILFRSNGNITRELTNITFAFRTHDTNVMILHAEKEPEFLN
901 VCHSRWDDFSCSCPALTSGKACEEVQWCGFSPCPHGAQCQPVLQGFECIANAVFNGQSGQILFRSNGNITRELTNITFGFRTRDANVIILHAEKEPEFLN
EGF14→
G3→
m 1000 ISIQDARLFFQLRSGNSFYTLHLMGSQLVNDGTWHQVTFSMIDPVAQTSRWQMEVNDQTPFVISEVATGSLNFLKDNTDIYVGDQSVDNPKGLQGCLSTI
h 1001 ISIQDSRLFFQLQSGNSFYMLSLTSLQSVNDGTWHEVTLSMTDPLSQTSRWQMEVDNETPFVTSTIATGSLNFLKDNTDIYVGDRAIDNIKGLQGCLSTI
m 1100 EIGGIYLSYFENLHGFPGKPQEEQFLKVSTNMVLTGCLPSNACHSSPCLHGGNCEDSYSSYRCACLSGWSGTHCEINIDECFSSPCIHGNCSDGVAAYHC
h 1101 EIGGIYLSYFENVHGFINKPQEEQFLKISTNSVVTGCLQLNVCNSNPCLHGGNCEDIYSSYHCSCPLGWSGKHCELNIDECFSNPCIHGNCSDRVAAYHC
EGF15→
EGF16→
m 1200 RCEPGYTGVNCEVDVDNCKSHQCANGATCVPEAHGYSCLCFGNFTGRFCRHSRLPSTVCGNEKRNFTCYNGGSCSMFQEDWQCMCWPGFTGEWCEEDINE
h 1201 TCEPGYTGVNCEVDIDNCQSHQCANGATCISHTNGYSCLCFGNFTGKFCRQSRLPSTVCGNEKTNLTCYNGGNCTEFQTELKCMCRPGFTGEWCEKDIDE
EGF17→
EGF18→
EGF
m 1300 CASDPCINGGLCRDLVNRFLCICDVAFAGERCELDLADDRLLGIFTAVGSGTLALFFILLLAGVASLIASNKRATQGTYSPSGQEKAGPRVEMWIRMPPP
h 1301 CASDPCVNGGLCQDLLNKFQCLCDVAFAGERCEVDLADDLISDIFTTIGSVTVALLLILLLAIVASVVTSNKRATQGTYSPSRQEKEGSRVEMWNLMPPP
19→
TM
m 1400 ALERLI
h 1401 AMERLI
B
M.m.
H.s.
D.m.
C.e.
C.e.
Crb1
CRB1
Crb
Crb1
Crl1
SNKRATQGTYSPSGQEKAGPRVEMWIRMPPPALERLI*
SNKRATQGTYSPSRQEKEGSRVEMWNLMPPPAMERLI*
RNKRATRGTYSPSAQEYCNPRLEMDNVLKPPPEERLI*
RGNNAMHGHYSPSSHEFTQNRMAMPTVIKLPPQERLI*
RQSRKLHGKYNPAREEHNLSAAYAMPMSHIAKEERLI*
Figure 1. (A) Alignment of the mouse (m) Crb1 and human (h) CRB1 protein sequences. The mouse
and human sequences are 82% similar and 76% identical. Identical and similar amino acids are
marked by gray boxes. Protein domains are indicated at the first amino acid position of the domain.
Hydrophobic regions representing the signal peptide and the transmembrane region are underlined. SP,
signal peptide; EGF1-19, epidermal growth factor-like domains; G1-3, laminin A G-like domains;
TM, transmembrane region. (B) Alignment of the cytoplasmic domains of mouse Crb1 (M.m. Crb1;
GenBank accession number AF406641), human CRB1 (H.s. CRB1; GenBank accession number
AY043325), Drosophila Crumbs (D.m. crb; GenBank accession number M33753), and C. elegans
Crumbs homologue 1 (C.e. Crb1; GenBank accession number U42839) and Crumbs-like protein (C.e.
Crl1; GenBank accession number AL008869). Residues conserved between all homologues are
marked by gray boxes. Asterisks denote the C-termini.
110
Expression pattern of Crb1
transmembrane region and a 37-amino acid cytoplasmic domain (Figure 1A). The
cytoplasmic domain of Drosophila Crumbs has two functional domains, the C-terminal amino
acids ERLI and an N-terminal region encompassing a tyrosine residue at the tenth position
and a glutamic acid at the 16th position of the cytoplasmic domain6. These residues are
completely conserved in the cytoplasmic domains of mouse Crb1, human CRB1, Drosophila
Crumbs and a Crumbs homologue (Crb1) and Crumbs-like protein (Crl1) recently identified
in Caenorhabditis elegans8,9 (Figure 1B).
Expression of the mouse Crb1 gene during embryonic and post-natal stages
To determine the temporal and spatial expression of Crb1 during mouse development, we
performed detailed in situ hybridization experiments on histological sections at several
developmental and post-natal stages. Crb1 is expressed exclusively in the eye and the brain,
which is in agreement with the expression of CRB1 in human tissues determined previously
by RT-PCR1.
In the developing eye, Crb1 expression was first detected at stage E11.5 in the retina anlage
(not shown). Crb1 expression is enhanced at the following embryonic stages E12.5, E14.5
and E16.5 (Figure 2A-C), labeling the proliferative retinoblasts. In early post-natal stages
(Figure 2D-F) and in the adult eye (Figure 2I and J) Crb1 expression is strongly detected in
the photoreceptor cells, which are densely packed in the deepest part of the retina.
Furthermore, some cells in the inner nuclear layer express Crb1. Based on their location and
morphology, they may represent bipolar neurons10. Finally, a strong Crb1 hybridization signal
labels the iris up to adult stage (Figure 2G and H).
Outside of the eye, Crb1 transcripts were only detected in the central nervous system, starting
from stage E10.5. At this stage, Crb1 is expressed predominantly in the ventral part of the
neural tube including the ventral spinal cord, the ventral part of the mesencephalon
(tegmentum), the mammillary and the hypothalamic regions and the optic area (Figure 3A).
Interestingly, Crb1 expression is detected as a distinct spot in the zona limitans intrathalamica
(arrow in Figure 3A), an early embryonic landmark that separates the prospective dorsal and
ventral thalami11. No expression was detected in the telencephalon, including the basal
ganglia and the cerebral cortex.
To examine Crb1 expression in the ventral neural tube in more detail, we compared it with the
expression domains of Sonic hedgehog (Shh) and transcription factor Nkx2.2, genes that are
expressed in ventral domains along the rostrocaudal length of the neural tube and in the
forebrain (Figure 3A-C). Shh and Nkx2.2 are molecular markers for the floor plate and the
111
Chapter 6
Figure 2. Crb1 expression in developing and adult mouse eye. (A-C) Sagittal sections of
embryonic eyes at (A) 12.5, (B) 14.5 and (C) 16.5 days post coitum. Crb1 is expressed in the
retinal progenitor cells. (D-F) Sagittal sections of a 5 days post-natal eye, when the retina
differentiation process is almost completed. Crb1 mRNA is detected in the outer nuclear layer and
in some cells of the inner nuclear layer. (G, H) High magnification of Crb1 expression in the adult
iris. (I, J) Sagittal sections of adult mouse retina. Crb1 is expressed in the outer nuclear layer, the
photoreceptor layer and in some cells of the inner nuclear layer. (F, H, J) are bright fields of
hematoxylin-eosin stainings. CB, ciliary body; CO, cornea; GNL, ganglion cell layer; INL, inner
nuclear layer; IPL, inner plexiform layer; IR, iris; LE, lens; ONL, outer nuclear layer; OSD; outer
segment discs of the photoreceptor layer; RE, retina; RPE, retinal pigment epithelium.
most ventral (V3) interneurons, respectively12,13. The Crb1 positive domain lies near the floor
plate region positive for Shh, in an area that is positive for Nkx2.2 (Figure 3D-F). Crb1
expression almost completely overlaps with the expression domain of Nkx2.2, although the
expression of Nkx2.2 seems to be broader. Thus, Crb1 is expressed by the V3 interneurons
placed between the floor plate and the motorneurons all along the spinal cord axis.
In late embryogenesis, Crb1 is expressed mainly in ventral neural structures of the
developing brain, including the mammillary and tuberalis regions of the hypothalamus
(Figure 4A-D, H, I) and the preoptic area (Figure 4F and G). However, starting from E12.5, a
112
Expression pattern of Crb1
strong hybridization signal was detected in the neural area that gives rise to the dorsal
thalamus (Figure 4A-D, F, G). Finally, we investigated Crb1 expression in the adult brain by
in situ hybridization with both 35S- (not shown) and digoxigenin (DIG)-labeled probes (Figure
4J-M). In both procedures, we detected Crb1 transcripts in the granular layer of the
cerebellum (Figure 4J), the hippocampal dentate gyrus (Figure 4K), the olfactory bulbs
(Figure 4L), the subventricular region lining the telencephalic ventricles and the rostral
migratory stream (Figure 4M). Interestingly, these regions of the brain are sites where the
production or migration of neurons occurs in adulthood, and from which adult neural stem
cells can be retrieved14,15.
Figure 3. Crb1 expression during early neural tube development. (A-C) Medial sagittal sections
of the anterior neural tube at 10.5 days post coitum. Comparison of expression domains of (A)
Crb1, (B) Shh (B) and (C) Nkx2.2 in adjacent sections. (D-F) Transversal sections through the
neural tube at 10.5 days post coitum. Comparison of expression domains of (D) Crb1, (E) Shh and
(F) Nkx2.2. Crb1 is expressed in the most ventral (V3) population of interneurons, which are
positive for Nkx2.2 expression and negative for Shh. A schematic representation is shown of genes
defining the different neuronal populations in the neural tube. ap, alar plate; ba, branchial arches;
bp, basal plate; die, diencephalon; drg; dorsal root ganglia; F, floor plate; hyp, hypothalamus; mes,
mesencephalon; mge, medial ganglionic eminence; MN, motorneurons; N, notochord; rh,
rhomboencephalon; SP, spinal cord; tel, telencephalon; tg, tegmentum; zli, zona limitans
intrathalamica.
113
Chapter 6
Figure 4. (A-I) Crb1 expression in late embryonic stages of mouse brain. (A-D) Sagittal sections
of mouse brain at stages (A, B) E12.5 and (C, D) E14.5. (F-I) Transversal sections at stage E16.5;
the levels of transverse sections are indicated by black lines in (E). Crb1 transcripts are present in
the hypothalamic mammillary and tuberalis regions, the preoptic area and the dorsal thalamus. (A,
C, F, H) are bright fields of hematoxylin-eosin stainings. (J-M) Crb1 expression in adult mouse
brain. (J) Medial-lateral sagittal section of a 2-month-old brain, and high magnifications in (K) the
hippocampal area, (L) the olfactory bulb and (M) the telencephalic ventricle. Crb1 expression was
detected by DIG-staining in the granular layer of the cerebellum, the dentate gyrus, the glomerular
layer of the olfactory bulb, the subventricular layer of the telencephalon and the rostral migratory
stream. cb; cerebellum; cx, cortex; dg, dentate gyrus; dt, dorsal thalamus; et, epithalamus; hip,
hippocampus; ma, hypothalamic mammillary area; mes, mesencephalon; ob, olfactory bulb; pop,
preoptic area; rh, rhomboencephalon; rms, rostral migratory stream; ser, subventricular layer; tu,
hypothalamic tuberalis region.
Experimental procedures
Isolation of mouse Crb1 and sequence analysis
Total RNA was isolated from mouse eyes with RNAzol B (Campro Scientific). Primer pairs
deduced from the human CRB1 gene were used for RT-PCR reactions on mouse eye RNA at
an annealing temperature of 48°C. Successfully amplified cDNA fragments were sequenced.
The Crb1 cDNA sequence was completed by RT-PCR on mouse eye RNA, 5’ and 3’ RACE
experiments using mouse brain cDNA (Clontech) and subsequent sequencing of PCR
products. The sequence of the mouse Crb1 gene was deposited in the GenBank database
(accession number AF406641).
Expression studies
A 509 bp fragment (position 200-708) and a 1002 bp fragment (position 1719-2720) of mouse
Crb1 were cloned in pBluescript II KS+. Antisense and sense Crb1 RNA probes were
114
Expression pattern of Crb1
synthesized by linearization with BamHI or HindIII, and transcription with T3 or T7 RNA
polymerase, respectively. In situ hybridizations on embryonic, post-natal and adult stages
were performed with the 509 bp or 1002 bp probe, labeled with
35
S-uridine triphosphate
(UTP) as described previously16. In situ hybridizations on adult brain sections were also
performed with the 1002 bp probe, labeled with DIG. A
35
S-labeled RNA probe for Nkx2.2
was derived from IMAGE cDNA clone mj56c03 (GenBank accession number AI323063). A
35
S-labeled Shh RNA probe was synthesized from a cDNA clone kindly provided by Dr. W.
Wurst. In situ hybridization experiments were performed as described previously16, with
minor modifications.
Acknowledgements
We thank Dr. S. Banfi and Dr. A. Bulfone for helpful discussions, Dr. W. Wurst for the Shh
probe, and C.A.A.M. Neefjes-Mol for excellent technical assistance. A.I.d.H. and Y.J.M.d.K.
were supported by The Foundation Fighting Blindness, Inc., USA.
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14. Vescovi AL and Snyder EY (1999)
Establishment and properties of neural stem cell
clones: plasticity in vitro and in vivo. Brain
Pathol 9:569-598.
15. Weissman IL (2000) Stem cells: units of
development, units of regeneration, and units in
evolution. Cell 100:157-168.
16. Bulfone A, Puelles L, Porteus MH, Frohman
MA, Martin GR and Rubenstein JLR (1993)
Spatially restricted expression of Dlx-1, Dlx-2
(Tes-1), Gbx-2, and Wnt-3 in the embryonic day
12.5 mouse forebrain defines potential transverse
and longitudinal segmental boundaries. J
Neurosci 13:3155-3172.
Chapter
7
General discussion
General discussion
7.1 Isolation of retina- and RPE-specific genes through SSH
At the start of the research project described in this thesis a novel technique, suppression
subtractive hybridization (SSH), was published to isolate tissue-specific genes that are
expressed at a low level in the tissue of interest1. This technique was successfully employed to
construct cDNA libraries enriched for retina- and RPE-specific genes2. Because complete
removal of cDNAs common to both driver and tester pools is not possible, we performed
expression studies by a semi-quantitative RT-PCR method using RNA from 12 different
tissues. We identified 33 cDNAs that are expressed specifically or preferentially in the retina
or the RPE. These cDNAs were mapped in the human genome by radiation hybrid mapping.
Two clones (RET1D1 and RET4B7) that initially could not be mapped by radiation hybrid
mapping2, have now been localized with the nearly finished human genome sequence3, and
map to 22q12 and 2q33.3 respectively.
Our method was instrumental for the identification of CRB1, a gene expressed exclusively in
retina and brain4. The power of the SSH technique to isolate novel, tissue-specific genes that
are expressed at low abundance is strengthened by the observation that CRB1 was not
represented by any retina ESTs in the public databases. The success of our method to isolate
candidate genes for retinal disease is demonstrated by the fact that two of the 33 novel genes
isolated in this initial screen are implicated in retinal disease. Besides the CRB1 gene (clone
RET3C11) that was analyzed in our group, NR2E3 (clone RET1B8) was characterized by two
independent groups, and was shown to be involved in enhanced S-cone syndrome5 and in
RP6. Recently, SSH was used by another group to isolate bovine retina-specific genes7, using
bovine retina cDNA as tester and bovine frontal cortex cDNA as driver. This led to the
successful isolation of a photoreceptor-specific retinal dehydrogenase.
The success of our method to isolate novel retina-specific genes prompted us to analyze more
clones from the retina-enriched library. In collaboration with Dr. Sandro Banfi of the Téléthon
Institute of Genetics and Medicine (TIGEM) and Dr. Alfredo Ciccodicola of the International
Institute for Genetics and Biophysics (IIGB) in Naples, Italy, we have now sequenced
approximately 1.000 additional retina clones. Due to the near completion of the human
genome sequence3, nearly all clones can be localized in the genome by in silico mapping.
Until now, expression profiles have been determined for 195 retina clones by semiquantitative
RT-PCR, which enabled us to isolate 56 cDNAs that are expressed preferentially in the eye.
All sequence, mapping and expression information of the analyzed clones is available to the
entire research community through an online database (http://www.tigem.it/RET/). Selected
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Chapter 7
genes are currently being characterized and will be analyzed for mutations in patients with
retinal disease.
Until now, less than ten genes have been identified that are expressed specifically in the RPE.
This is due to an underrepresentation of RPE ESTs in the public databases; at this moment
only 532 RPE ESTs from 6 different libraries have been deposited in dbEST (Chapter 1). Five
of these libraries were constructed from RPE cell lines or cultured RPE cells8,9 (Chapter 1).
Although the RPE tissue used to construct our RPE cDNA library was attached to the
underlying choroidal tissue, it is the only cDNA library described in literature that has been
constructed from human RPE tissue. Large-scale identification of cDNA clones from this
library will therefore be of great value for the identification of novel RPE-specific genes,
which is currently being performed in a parallel project (Van den Hurk et al., unpublished
data).
Parallel to the construction of the retina- and RPE-enriched libraries, a fetal cochlea-enriched
cDNA library was constructed to isolate candidate genes for inherited sensorineural deafness.
Large-scale analysis of clones from this library is currently being performed in another project
(Luijendijk et al., unpublished data).
7.2 Gene structure of CRB1
The intron-exon structure of the CRB1 gene was determined by PCR techniques4, and consists
of 12 exons. The near completion of the human genome sequence3 now enables us to examine
the genomic structure and sequence of the gene. In the draft version of April 1, 2001, the gene
spans more than 300 kb of genomic sequence at 1q31.3, and is orientated from 5’ to 3’ in a
centromeric to telomeric direction. Transcribed sequences represent approximately 1% of the
entire genomic sequence of CRB1. It is located 500 kb downstream of F13B, encoding the B
subunit of coagulation factor XIII, and 1 Mb upstream of PTPRC, encoding protein tyrosine
phosphatase receptor type C.
The region upstream of the transcription start site contains several putative photoreceptor
gene regulatory sites (Figure 1). Three regulatory elements (Ret-1/PCE-1, OTX and NRE)
and three transcription factors (RX, CRX and NRL) have been experimentally determined to
be involved in photoreceptor-specific transcriptional activation. The Ret-1 or PCE-1
photoreceptor conserved element serves as a binding site for transcription factor RX, which is
essential for normal eye development and is found upstream of all known photoreceptor cellspecific genes10,11. A Ret-1 or PCE-1 motif (CAATTAG) is found 714 bp upstream of the
120
General discussion
-800
PCE-1/Ret-1
gtaaaaatcagctatagaaattgcattttaaagtgtaacagtgaacagttttaacaatggaaaaaggctctgtactagcactaacaccaattagaaaata
-700
aagcattctggttttaattaatggatgttttcatggtgtctaataaaaatacaattttaaggacttgaacaatttaatctgagaaaatattgtgtattta
-600
aagcaagtgtattttttaaataagcaaacatgtttacattcatcaattatagtaaaacaaaagctcaagaaatcccagagcatataatttcatgaaccga
-500
gaataaaatgagctctggacttacgacaaaatctgcccctgaatgatcttcagaactctgttcattttatgcagtagtttaagttttcttctgtcttggc
-400
ccaacttacaaacagcagaaatctgagttgtgggaatataatttatgaacagaaaagattacttgtctgtgaattattttctgaagatgaaagtaaatat
CRX
acaggaacatacggtattcctttaaaagttgccagatcataattgtgtggcaaggcagttatcagaaattaatccctctattgagagcaattgaagacac
-300
-200
-100
tattctaatgtaggcccttttgaggaggcagcatgaacagaagaaaactcgcagcaaaggcttgaggggggaatgaatccaatccagcctgaaaaaatct
Nrl
gcaccaggtttgaaaaatcaccccatcctcccgtgtaagtgatgctaagaagcacaaactgcattttgaatctaagtccctgtattttctgtgaaggagc
Figure 1. Sequence of the promoter region of the CRB1 gene. Nucleotide positions are relative to the
transcription start site (position +1). Putative PCE-1/Ret-1, CRX and Nrl binding sites are underlined.
CRB1
transcription
start
site.
CRX
binds
to
the
OTX
regulatory
sequence
[(C/T)TAATC(C/A)], which has been found upstream of several photoreceptor cell-specific
genes12,13. At position –232 of the CRB1 promoter region, a binding site for CRX is present.
CRX acts synergistically with transcription factor Nrl in photoreceptor gene activation12. Nrl
binds to a motif [GC(N)6-7GCA]14. This motif is located 58 bp upstream of the CRB1
transcription start site.
7.3 The role of CRB1 in recessive retinal dystrophies
We have shown that CRB1 mutations cause retinitis pigmentosa (RP) with preserved paraarteriolar retinal pigment epithelium (PPRPE)4, RP with Coats-like exudative vasculopathy15
and Leber congenital amaurosis (LCA)15. RP with PPRPE (RP12) is a relatively rare form of
autosomal recessive RP, characterized by an early onset of symptoms, a relative preservation
of the RPE surrounding the arterioles and hyperopia. This form of RP was first described in
1982 (ref. 16), and has since then been reported in several countries17-22. The frequency has
been estimated to be less than 1% of RP cases16,17,23, although it may be responsible for a
significant proportion of autosomal recessive RP in the Pakistani population22. Coats-like
exudative vasculopathy is a rare complication of RP, which has an estimated frequency of 13% (ref. 24). This suggests that CRB1 mutations are the cause of 2-4% of autosomal recessive
RP, which is comparable to the frequencies found for other autosomal recessive RP genes
(Chapter 1). We and others identified mutations in the CRB1 gene in 13% and 9% of patients
with LCA, respectively15,25, rendering it one of the most frequent known causes of LCA
(Chapter 1).
RP with PPRPE, RP with Coats-like exudative vasculopathy and LCA represent different but
partly overlapping clinical entities as evidenced by the fact that LCA patients can show the
121
Chapter 7
PPRPE characteristics15,25 and that RP patients with PPRPE reveal a higher than average
incidence of Coats-like changes19. In the families of five probands with RP and Coats-like
exudative vasculopathy, ten patients were affected with RP, and two of them had not
developed the Coats-like complication15. This finding, together with the observation that all
patients but one had developed the complication unilaterally, strengthens the idea that CRB1
mutations are an important risk factor for the development of this severe complication and
that other genetic or environmental factors may be involved.
The disorders of two probands with RP and Coats-like exudative vasculopathy were clearly
distinct from RP12. They experienced a later onset of their RP, showed no preservation of the
RPE surrounding the arterioles, and one patient was highly myopic. These findings show that
CRB1 mutations can cause RP12, characterized by an early onset, PPRPE and hyperopia, as
well as ‘classic’ RP, characterized by a later onset, a higher occurrence of myopia and
absence of PPRPE.
CRB1 mutation spectrum
We have now identified CRB1 mutations in 17 patients with RP and PPRPE, in 6 patients
with RP and Coats-like exudative vasculopathy, and in 7 patients with LCA4,15 (den Hollander
et al., unpublished data; Figure 2). In total, we found 57 sequence changes in 60 CRB1 alleles.
Four mutations were found in more than two individuals, i.e. C948Y (11/57), T745M (5/57)
K801X (5/57), and R764C (4/57), which together constitute 44% of all mutations. The most
frequent mutation found by Lotery and coworkers also was C948Y, which was found on 7/27
alleles25.
In total, 44 different CRB1 mutations have now been identified, including 8 different deletions
or insertions, 6 different nonsense mutations, 2 splice site mutations and 28 different missense
mutations4,15,25 (den Hollander et al., unpublished data). Sixteen missense mutations are
located in the EGF-like domains, and 8 of them affect the conserved cysteine residues. Ten
missense mutations are located in the second and third laminin A G-like domains, while no
missense mutations have been found in the first laminin A G-like domain. One mutation has
been found in the transmembrane region and one in the cytoplasmic domain.
Interestingly, no CRB1 mutations were identified in 12 (41%) of 29 patients who have RP
with PPRPE (den Hollander et al., unpublished data) and in four of ten patients who have RP
with Coats-like exudates15, which suggests that one or more additional genes may be involved
in these two specific forms of RP. Currently no linkage data are available to support or
repudiate this conjecture.
122
General discussion
‘classic’ RP
16894#
16937#
N894S
D837H
C948Y
A1354T
RP12
25983*
A161V
17679*
RP112*
9439*,#
C195F
C250W
S403X -Y433C
24228*
25977*
S403X
R764C
R764C
2320insAlu
16968#
IVS8+5 C948Y
IVS8+5 C948Y
25710
24868*
12723*
14489*
25540*
13066*
26023*
T745M
T745M
T745M
T745M
R764C K801X
R764C
15278*
C948Y
C948Y
C948Y
E995X
G850S
17659#
C948Y
G959S
M1041T
L1071P
22147*
RP0136*
I1100T
15850*
17658#
C1181R
K801X
R1383H
15849*
1
2
3
4
5
6
7
9
8
10 11 12
LCA
16690*
748-754del
12862
K801X
K801X
13067*
E1111X
12831
C948Y
16507*
C948Y
12859
12872
I1100R
IVS10+1
E1333X
R1331H
Figure 2. Mutation spectrum of the CRB1 gene (refs. 4, 15; den Hollander et al, unpublished
data). Exons are drawn to scale; introns are not. Homozygous mutations are underlined; null
alleles are in boldface. Clinical features: *PPRPE; #Coats-like exudative vasculopathy.
Genotype-phenotype correlation
Looking at the complete mutation spectrum of CRB1, we can draw preliminary conclusions
on a genotype-phenotype correlation (Figure 2). Most importantly, there seems to be a
correlation between mutation and phenotype severity. In 3 LCA patients we identified null
mutations on both CRB1 alleles, which suggests that LCA is the most severe phenotype that
can be associated with mutations in CRB1. Two LCA patients are homozygous for C948Y,
which presumably is a severe mutation.
123
Chapter 7
In RP patients, we did not identify clear-cut null mutations on both alleles. Nonsense
mutations and the severe C948Y mutation were found in combination with another missense
mutation, or with a splice site mutation (IVS8+5G>A) that does not necessarily inactivate the
mutant splice site completely4. The consequence of the homozygous Alu insertion found in
one RP patient (25977) is not clear. Possibly it does not completely inactivate CRB1 function.
One explanation could be that a cryptic 3’ splice site in exon 7 at position 2384 of the cDNA
is used, which has a splice potential score that is lower than the normally used 3’ splice site of
exon 7 (84 vs 91; ref. 26), but is comparable with the average splice potential score of all
CRB1 3’ splice sites. The use of this splice site would lead to an in frame deletion of amino
acid residues 710-749, deleting the N-terminal region of the second laminin A G-like domain.
The absence of clear-cut null mutations on both CRB1 alleles of RP patients, and the presence
of null mutations on both CRB1 alleles in at least three LCA patients suggest that LCA may
be associated with complete loss-of-function of CRB1, whereas RP patients may have
residual CRB1 function. The inverse relationship between residual protein activity and the
severity of the retinal dystrophy has also been proposed for the ABCA4 gene27-29. According
to this model, the combination of two severe ABCA4 mutations causes RP, the combination of
a severe and a moderately severe ABCA4 mutation causes cone-rod dystrophy, and the
combination of a severe and mild ABCA4 mutation causes Stargardt disease. Analogous to the
ABCA4 model, we propose that a combination of two severe CRB1 mutations leads to LCA,
the combination of a severe with a moderately severe CRB1 mutation results in RP12, and
combinations of severe and mild mutation, or two moderately severe mutations may be
associated with ‘classic’ RP30 (Figure 3). Additional mutation analysis and functional assays
of individual mutations are required to test this hypothesis.
CRB1 activity
Phenotype
arRP#
RP12#,*
LCA*
CRB1 allele 1
severe
severe
severe
CRB1 allele 2
mild
moderate
severe
Figure 3. Genotype-phenotype correlation model for CRB1 based on our findings in patients
with ‘classic’ RP, RP12 and LCA. We hypothesize that combinations of severe and mild, or two
moderately severe CRB1 mutations are associated with ‘classic’ RP. #Patients with Coats-like
exudative vasculopathy; *patients with preserved para-arteriolar retinal pigment epithelium.
124
General discussion
Mechanisms of photoreceptor death and pathogenesis of CRB1 mutations
In all forms of retinal degeneration, photoreceptors eventually die via apoptosis. Four
mechanisms by which mutations can lead to photoreceptor cell death have been proposed31;
faulty outer segment disc formation (RHO, RDS, TULP1, CRX, RPGR), metabolic overload
(PDE6B), RPE dysfunction (ABCA4, RPE65, MERTK), and continuous activation of
phototransduction (SAG, RHOK, PDE6A, GUCY2D). The distinct features observed in RP
patients with PPRPE and Coats-like exudates suggest that another pathogenic pathway may
be induced by mutations in CRB1.
The PPRPE phenotype and Coats-like exudates observed in patients with mutations in CRB1
suggest that the integrity of the retinal blood vessels may be affected. Although the
development of PPRPE remains unclear, it has been suggested that the preservation of the
RPE is caused by leakage of a soluble factor from the retinal arterioles, which may
temporarily retard the degeneration of the photoreceptors and underlying RPE near the retinal
arteriole16.
7.4 Biochemical function of CRB1 and putative interactors
The CRB1 gene encodes a protein that is homologous to Drosophila Crumbs4, a
transmembrane protein with 30 EGF-like domains, 4 laminin A G-like domains and a 37-aa
cytoplasmic domain32. The CRB1 gene exhibits alternative splicing at its 3’ end, and encodes
two CRB1 isoforms33. One isoform represents a putative extracellular protein, while the
second encodes a transmembrane protein with a 37-aa cytoplasmic domain that is functionally
conserved between human and Drosophila. The biochemical function of CRB1 remains to be
elucidated. However, our knowledge on Drosophila Crumbs allows us to speculate on its
function and on putative interactors of CRB1.
Epithelial polarity in Drosophila
Many eukaryotic cells exhibit a polarized phenotype, which is manifested in their shape, the
asymmetric distribution of organelles and proteins and the polarized orientation of the
cytoskeleton. The typical cell of a simple monolayered epithelium is polarized along an
apical-basal axis34,35. The plasma membrane is separated into two functionally distinct
membrane domains: an apical and a basolateral domain. The basolateral membrane domain
communicates to neighboring cells and the extracellular matrix via membrane proteins and
cell junctions. Invertebrate epithelia exhibit two types of epithelial junctions, the zonula
125
Chapter 7
adherens (ZA) and the septate junction (SJ). SJs form regions of close membrane appositions
along the lateral membrane domains, and control paracellular transport. The ZA is an
adhesive belt-like structure around the circumference of the cell, which connects apicolaterally localized actin belts of adjacent cells with each other.
The ZA lies between the apical and basolateral membrane domains and exhibits a
concentration of DE-cadherin (encoded by the shotgun gene), Dα-catenin, βH-spectrin,
Armadillo (a homologue of β-catenin), and phosphotyrosyl proteins. The region just apical of
the ZA, referred to as the subapical complex (SAC), is characterized by an accumulation of
membrane and membrane-associated proteins, such as Crumbs, Discs Lost36, Stardust37,
Bazooka/Par-3 and an atypical protein kinase, aPKC. The cytoplasmic domain of Crumbs
binds to Discs Lost38 and to Stardust37, and it has been suggested that these interactions
initiate the formation of a protein complex, which may contain additional proteins. The
presence of multiple protein-binding domains in Discs Lost and Stardust may provide a
molecular interface that mediates additional protein-protein interactions. The ultimate
function of this apical protein scaffold would be to direct the assembly of DE-cadherin and
other ZA components to the correct position in the membrane.
The correct function and localization of Crumbs is dependent on a number of proteins, for
example Scribble, a cytoplasmic protein that localizes basally of the ZA.
Putative function and interactors of CRB1
Although human CRB1 and Drosophila Crumbs are homologous, their expression patterns
are completely different. Human CRB1 is expressed exclusively in eye and brain, while
Drosophila crumbs is expressed in all epithelia derived from the ectoderm. This totally
different expression pattern indicates that the function of these proteins is not completely
conserved. However, a similarity between epithelia and the human retina is that they are both
highly polarized tissues. In the human retina, the photoreceptors and other cell types are
highly organized, and are in close contact with each other. Drosophila Crumbs is essential for
establishing and maintaining apico-basal polarity in all epithelia derived from the ectoderm.
Perhaps CRB1 has a similar function in the retina, where it establishes and maintains polarity
of the photoreceptor cells. Mutations in crumbs and CRB1 both cause a degeneration of the
tissue where they are expressed, ectodermally-derived epithelia and the retina, respectively.
Crumbs forms an anchor for the formation of a protein scaffold at the SAC, composed of
Discs Lost, Stardust and other, as yet unidentified proteins. The functional conservation of the
cytoplasmic domains of Crumbs and CRB1 suggests that CRB1 may interact with human
126
General discussion
1
2
3
4
5
6
7
8
9
10
11
12
MUPP1
INADL
Figure 4. Expression profile of MUPP1 and INADL in human tissues and RPE cell lines,
determined by a semi-quantitative RT-PCR method2. Lane 1, liver; 2, lung; 3, skeletal muscle; 4,
placenta; 5, heart; 6, brain; 7, spleen; 8, kidney; 9, retina; 10, ARPE-19; 11, D407; 12,
RPE/choroid.
homologues of Disc Lost and Stardust. Discs Lost is a four PDZ-domain protein that interacts
with the cytoplasmic domain of Crumbs36,38. By database searches we identified two putative
homologues of Discs Lost, INADL39 and MUPP140, which are both expressed in the retina as
determined by a semi-quantitative RT-PCR (Figure 4; den Hollander et al., unpublished data).
Stardust is a MAGUK (membrane associated guanylate kinase-) family protein characterized
by three protein-protein interaction motifs, a PDZ-, a SH3- and a guanylate kinase domain,
which also binds to the cytoplasmic domain of Crumbs. Human homologues of Stardust
therefore also represent possible interactors of CRB1. Interestingly, recently a gene encoding
a retina-specific MAGUK protein, MPP4, was cloned that belongs to the same MAGUK
subfamily (p55) as Stardust41. It is therefore tempting to speculate that MPP4 interacts with
the cytoplasmic domain of CRB1. Other possible interactors of CRB1 are human homologues
of other proteins localized at the SAC, for example Bazooka and aPKC, although no
interaction of these proteins has yet been identified with Drosophila Crumbs.
The protein scaffold organized by Crumbs is essential for the correct formation of the ZA.
However, protein scaffolds can also act to maximize the efficiency of signal transduction by
aligning receptors with their signaling cascades. In the fly retina, InaD, a scaffold protein
containing multiple PDZ domains, organizes at least seven proteins of the phototransduction
cascade into a supramolecular signaling complex, or signalplex42,43. This signalplex seems to
promote the termination of the photoresponse and may also facilitate the rapid activation and
amplification of the phototransduction cascade. It has been speculated that CRB1 may play a
role in localizing the proteins of the phototransduction cascade44.
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Chapter 7
7.5 Crb1 is expressed exclusively in the retina and the
brain
By in situ hybridizations in mouse we showed that Crb1 is expressed exclusively in the eye
and the brain45. In the retina, Crb1 is expressed in the outer nuclear layer, representing the
photoreceptors cells, and in some nuclei of the inner nuclear layer, presumably representing
the bipolar cells. Many genes involved in RP and LCA are expressed in the photoreceptors,
the cells that usually undergo primary degeneration.
In the developing nervous system, Crb1 is expressed in the most ventral domains of the neural
tube. Patterning of the ventral neural tube is regulated by secretion of Sonic hedge hog (Shh)
by the notochord and the floor plate. A concentration gradient of Shh controls the generation
of five distinct classes of neurons, each at a different dorsoventral position in the ventral
neural tube46,47. Each distinct class of neurons is generated by a profile of homeodomain
proteins that are either activated or repressed by the Shh gradient48. The most ventral neural
progenitors are the V3 interneurons, situated between the floor plate and the motor neurons.
The identity of V3 interneurons is established by expression of homeodomain transcription
factor Nkx2.2 (ref. 49). Crb1 is expressed in the region between the floor plate and the motor
neurons, coinciding with the expression domain of Nkx2.2. This finding suggests that Crb1 is
expressed by the V3 interneurons during neural tube development.
Although the brain has long been thought to be entirely postmitotic, it is now known that
neural stem cells also exist in the adult nervous system50. In the adult brain, Crb1 is expressed
in the hippocampal dentate gyrus, the subventricular zone, the rostral migratory stream and
the olfactory bulbs. The subventricular zone and the dentate gyrus are areas that contain the
dividing neural stem cells. The progeny migrate rostrally to the olfactory cortex, where they
differentiate into astrocytes, oligodendrocytes, and neurons; this pathway is called the rostral
migratory stream51. The expression of Crb1 in areas where the production or migration of
neurons occurs, suggest that Crb1 has a function in neural stem cell biology. Possibly, Crb1
can be used as a molecular marker for neural stem cells; identifying such markers is important
to devise strategies to obtain a pure population of stem cells, for example for therapeutic
use52.
The significance of Crb1 expression in the brain remains unclear, since RP and LCA patients
with CRB1 mutations do not exhibit obvious neurological symptoms. However, recently brain
alterations were observed in aniridia patients who are heterozygous for PAX6 mutations53.
PAX6 is widely expressed in the central nervous system, and governs proliferation and
128
General discussion
migration of adult neural stem cells. Although patients did not exhibit neurological symptoms,
magnetic resonance imaging (MRI) showed that the anterior commissure was absent or
hypoplastic, the regional brain volumes were smaller, and the corpus callosum was reduced.
Additionally, assessment of the olfactory function revealed mild to moderate hyposmia.
Therefore, it would be interesting to perform MRI imaging in patients with CRB1 mutations
to reveal whether they have brain alterations.
7.6 Future directions
The work described in this thesis provides a basis for future research in several directions.
The retina- and RPE-enriched cDNA libraries represent a unique resource for more potential
candidate genes for retinal dystrophies. Analysis of more cDNA clones from these libraries is
currently being performed. Additionally, cDNA clones from these libraries will be arrayed in
a joint project with Dr. Arthur Bergen in Amsterdam, and will be analyzed for expression
differences in healthy eyes compared to eyes from patients affected by age-related macular
degeneration.
The results described in this thesis indicate that CRB1 is an important cause of autosomal
recessive retinal dystrophies. To substantiate the genotype-phenotype model for CRB1
(Figure 3), additional mutation analysis of a larger group of LCA patients and a group of
‘classic’ RP patients is required. Furthermore, functional assays of individual mutations
should be developed to test our hypothesis.
The identification of the cytoplasmic domain of CRB1 and the overexpression studies
performed in Drosophila, suggest that CRB1 may organize a protein scaffold in the retina.
The interaction of CRB1 with several putative interacting proteins is currently being studied.
Additionally, a yeast two-hybrid screen is performed to isolate CRB1 interactors (Roepman et
al., unpublished). Genes encoding interactors of CRB1 are interesting candidates to screen for
mutations in those patients with RP and PPRPE or Coats-like vasculopathy who do not have
mutations in CRB1. Analogous to our CRB1 findings, such genes are also good candidate
genes for LCA. Human homologues of proteins that influence Crumbs function and
localization, such as Scribble, are also interesting candidate genes for retinal disease.
The work described in this thesis has lead to the establishment of a ‘Crumbs consortium’,
with collaborating partners in Amsterdam (Dr. Jan Wijnholds), Düsseldorf (Prof. Dr.
Elisabeth Knust), Sheffield (Dr. Pen Rashbass), Marseille (Dr. André LeBivic) and Milan (Dr.
Vania Broccoli). The consortium will focus on the elucidation of the function of Crumbs and
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Chapter 7
CRB1, through for example the generation of Crb1 knock-out mice, establishing polarized
cell lines overproducing CRB1, characterization of additional mammalian Crumbs
homologues, isolation of interactors of Crumbs and CRB1, and elucidation of the role of
CRB1 in neural stem cell differentiation and migration. Together, these studies will generate
important novel insights into retinal function and dysfunction and form the basis for future
studies aimed at the development of a therapy for CRB1-associated retinal dystrophies.
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Summary
Samenvatting
Summary / Samenvatting
Summary
Most inherited retinal diseases lead to severe visual impairment early in life. Although more
than 60 genes involved in retinal disease have been identified over the last decade, the genetic
basis of retinal dystrophies is extremely heterogeneous leaving many genes to be identified.
The retina represents the sensory part of the eye dedicated to light perception and signal
transduction. The highly specialized functions of the retina and the retinal pigment epithelium
(RPE) are likely to require a broad spectrum of unique genes. Most genes involved in retinal
disease are expressed exclusively or predominantly in the retina or the RPE. Identification of
novel retina- and RPE-specific genes is crucial to our understanding of the molecular
processes underlying retinal function and pathogenesis.
Through suppression subtractive hybridization we isolated 33 novel genes expressed
preferentially in the retina or the RPE. We characterized the complete gene structure of one of
these genes, CRB1, and showed that it is involved in RP12, a severe form of autosomal
recessive retinitis pigmentosa (RP). RP is a heterogeneous group of progressive retinal
dystrophies that is characterized by night blindness, progressive constriction of the peripheral
visual field, and eventually loss of central vision. RP12 is a specific form of RP characterized
by a preserved para-arteriolar RPE (PPRPE). Mutation analysis in patients revealed both
mutant CRB1 alleles in 17 unrelated cases.
Due to the early onset of symptoms in these patients, we considered CRB1 a good candidate
gene for Leber congenital amaurosis (LCA), which causes blindness from birth. We screened
52 LCA patients and revealed CRB1 mutations in 7 (13%) of these patients, which shows that
CRB1 mutations are an important cause of LCA. To determine the frequency of CRB1
mutations in the Dutch RP population, we screened 97 RP patients for two mutations that
were most frequently encountered in RP12 patients. We revealed mutations in one individual,
who had a Coats-like exudative vasculopathy, a relatively rare complication of RP. We
ascertained 8 additional patients with this complication and detected mutations in 4 of them.
Four patients had developed the complication unilaterally, suggesting that CRB1 mutations
are an important risk factor for the development of the Coats-like reaction and that other
genetic or environmental factors may be involved. An obvious genotype-phenotype
correlation could not be established for mutations found in RP12, RP with Coats-like exudates
and LCA. However, three LCA patients carry null mutations on both CRB1 alleles, which is
consistent with the hypothesis that LCA is the most severe phenotype that can be associated
with mutations in CRB1.
135
Summary / Samenvatting
CRB1 encodes two protein isoforms; both have a signal peptide, 19 EGF-like domains and 3
laminin A G-like domains, and one has a transmembrane region and a 37-aa cytoplasmic
domain. The proteins encoded by CRB1 are homologous to Drosophila Crumbs protein. In
the absence of Crumbs, apico-basal polarity of epithelial cells is lost and the zonula adherens
fails to develop. The cytoplasmic domain of Crumbs organizes an intracellular protein
scaffold that defines the localization of the zonula adherens. The Crumbs mutant phenotype
can be partially rescued by expression of just the cytoplasmic domain, and overexpression of
this domain in a wild-type background results in a multilayered epidermis. Rescuing and
overexpression studies in Drosophila show that the cytoplasmic domains of CRB1 and
Crumbs are functionally related between these distant species.
The expression of Crb1 was studied in mice by in situ hybridizations on adult eyes and on
eyes at several stages of embryonic development. In the developing eye, Crb1 is detected in
the retina primordium (E11.5) and in the proliferative retinoblasts (E12.5, E14.5 and E16.5).
In post-natal and adult eyes Crb1 expression is confined to the outer nuclear layer, the
photoreceptors, some nuclei of the inner nuclear layer and in the iris. In early development of
the central nervous system, Crb1 is expressed in the ventral part of the neural tube in the
domain that corresponds to the V3 interneurons, the expression domain of transcription factor
Nkx2.2. In adult brain Crb1 is expressed in regions that contain neural stem cells.
The results described in this thesis indicate that CRB1 is an important cause of autosomal
recessive retinal dystrophies. The identification of the cytoplasmic domain and the studies in
Drosophila, as well as the mRNA expression studies of Crb1 in mouse form an important
basis to elucidate the function of CRB1 in the future. The retina- and RPE-enriched cDNA
libraries represent an important resource for novel retina- and RPE-specific genes.
136
Summary / Samenvatting
Samenvatting
Erfelijke aandoeningen van het netvlies (de retina) kunnen op jonge leeftijd tot blindheid of
ernstige slechtziendheid leiden. In de afgelopen tien jaar zijn meer dan 60 genen
geïdentificeerd die betrokken zijn bij retina aandoeningen. De genetische basis van deze
aandoeningen is echter zeer heterogeen, waardoor veel genen nog niet ontdekt zijn. De retina
is verantwoordelijk voor lichtperceptie en neurale signaal transductie. Voor de uitoefening
van de gespecialiseerde functies van de retina en het retina pigment epitheel (RPE) is een
grote verzameling genen nodig. De meeste genen die gemuteerd zijn bij patiënten met
erfelijke retina aandoeningen komen alleen tot expressie in de retina of het RPE. Nieuwe
retina- en RPE-specifieke genen zijn daarom kandidaat genen voor retina aandoeningen.
Identificatie van deze genen is ook van belang om onze kennis over de moleculaire processen
in de retina uit te breiden.
Met behulp van een cDNA subtractie methode hebben we 33 nieuwe retina- en RPEspecifieke genen geïsoleerd. Van een van deze genen, CRB1, hebben wij de volledige
genstructuur gekarakteriseerd. We toonden aan dat mutaties in het CRB1 gen RP12
veroorzaken, een ernstige vorm van autosomaal recessieve retinitis pigmentosa (RP). RP is
een verzamelnaam voor een groep aandoeningen gekenmerkt door een progressieve
degeneratie van de retina. De ziekte leidt tot nachtblindheid en perifeer gezichtsveldverlies, en
uiteindelijk kan ook het centrale gezichtsveld verloren gaan. RP12 wordt gekenmerkt door
een behoud van het para-arteriolaire RPE (PPRPE). In 17 niet-verwante patiënten met RP12
vonden wij mutaties op beide allelen van het CRB1 gen.
Door de vroege aanvang van symptomen in RP12 patiënten, beschouwden we CRB1 als een
goed kandidaat gen voor Leber congenitale amaurose (LCA), dat gekenmerkt wordt door
aangeboren blindheid. We vonden CRB1 mutaties in 7 van 52 (13%) LCA patiënten, wat
aangeeft dat CRB1 mutaties een belangrijke oorzaak van LCA zijn. Om de frequentie van
CRB1 mutaties in de Nederlandse RP populatie te bepalen, werden 97 RP patiënten zonder
PPRPE geanalyseerd op de aanwezigheid van twee mutaties die frequent gevonden waren bij
RP12 patiënten. We vonden CRB1 mutaties in één RP patiënt met een Coats-achtige
exudatieve vasculopathie, een relatief zeldzame complicatie van RP. We verzamelden nog 8
RP patiënten met deze complicatie, en vonden CRB1 mutaties in 4 patiënten. Omdat deze
complicatie bij 4 patiënten unilateraal aanwezig is, worden CRB1 mutaties als een belangrijke
risico factor beschouwd, en zijn mogelijk andere genetische of omgevingsfactoren van
invloed op het ontstaan van de complicatie. Hoewel er geen duidelijke genotype-fenotype
137
Summary / Samenvatting
correlatie is tussen CRB1 mutaties gevonden in patiënten met RP12, RP met Coats-achtige
exudaten en LCA, dragen drie LCA patiënten geninactiverende mutaties op beide CRB1
allelen. Dit is in overeenstemming met de hypothese dat LCA het meest ernstige fenotype is
dat veroorzaakt kan worden door CRB1 mutaties.
CRB1 codeert twee eiwit producten; beide hebben een signaal peptide, 19 EGF-achtige
domeinen en 3 laminine A G-achtige domeinen, en één heeft een transmembraan domein en
een cytoplasmatisch domein van 37 aminozuren. De eiwit producten die door CRB1 worden
gecodeerd zijn homoloog aan het Crumbs eiwit van de fruitvlieg (Drosophila). Bij
afwezigheid van het Crumbs eiwit verliezen epitheliale cellen hun apico-basale polariteit, en
ontwikkelen zij geen zonula adherens. Het cytoplasmatisch domein van Crumbs vormt een
anker voor een intracellulair eiwitcomplex dat essentieel is voor het tot stand komen van de
zonula adherens. Het fenotype van de Crumbs mutant kan gedeeltelijk hersteld worden door
alleen het cytoplasmatisch domein tot expressie te brengen. Overexpressie van dit domein in
wild-type Drosophila leidt tot een meerlagige epidermis. Door middel van overexpressie
studies in wild-type Drosophila en in de Crumbs mutant toonden wij aan dat de
cytoplasmatische domeinen van CRB1 en Crumbs functioneel aan elkaar verwant zijn.
Tenslotte hebben we de expressie van Crb1 in volwassen muizen en op verschillende
tijdstippen gedurende de embryonale ontwikkeling bestudeerd met behulp van in situ
hybridisaties. Tijdens de ontwikkeling van het oog werd Crb1 aangetoond in het retina
primordium (E11.5) en in de proliferatieve retinoblasten (E12.5, E14.5 en E16.5). In postnatale en volwassen ogen lokaliseert Crb1 in de buitenste nucleaire laag (ONL), de
fotoreceptoren, sommige kernen van de binnenste nucleaire laag (INL) en de iris. Tijdens de
ontwikkeling van het centrale zenuwstelsel werd Crb1 aangetoond in het ventrale deel van de
neurale buis. Crb1 komt tot expressie in de V3 interneuronen, in het expressiedomein van
transcriptiefactor Nkx2.2. In volwassen hersenen werd Crb1 aangetoond in gebieden die
neurale stamcellen bevatten.
Het onderzoek beschreven in dit proefschrift toont aan dat CRB1 een belangrijke oorzaak is
van autosomaal recessieve retina aandoeningen. De identificatie van het cytoplasmatisch
domein en de studies in Drosophila, alsmede de mRNA expressie studies van Crb1 in muis
vormen een belangrijke basis om de functie van CRB1 in de toekomst op te helderen. De
cDNA subtractie banken vormen een belangrijke bron van nieuwe retina- en RPE-specifieke
genen.
138
Acknowledgements / Dankwoord
Acknowledgements / Dankwoord
Promoveren doe je niet alleen. Dit blijkt alleen al uit het groot aantal co-auteurs die staan
vermeld bij de publicaties opgenomen in dit proefschrift, in totaal 33 mensen van 16
afdelingen en instituten in binnen- en buitenland. Eén voor één zijn deze mensen onmisbaar
geweest voor het tot stand komen van dit boekje, die ik middels dit dankwoord persoonlijk
wil bedanken.
Allereerst Frans, mijn co-promotor. Jou wil ik bedanken voor het vertrouwen dat je in mij
gesteld hebt tijdens mijn promotie, voor jouw enorme inspanning bij het schrijven van alle
publicaties en uiteindelijk dit proefschrift. De snelheid waarmee jij tussentijdse versies hebt
nagekeken is geweldig, als ik ’s avonds een versie inleverde kreeg ik het vaak de volgende
dag alweer nagekeken terug. Ook voor privé-zaken kon ik altijd bij jou terecht, zoals advies
bij het kopen en verkopen van huizen, en ik vind het ook nog steeds geweldig dat je geholpen
hebt bij het leggen van de laminaatvloer in mijn huis aan de Pijkestraat.
Han, tijdens mijn aanstelling ben jij hoogleraar geworden en was het vanzelfsprekend dat je
mijn promotor zou worden. Ik kon altijd bij jou terecht voor een discussie over publicaties,
‘de toekomst’, etc., of voor zomaar een praatje. De snelheid waarmee jij alles hebt nagekeken
is, gezien jouw drukke agenda, echt fantastisch.
Yvette, jouw inbreng in het tot stand komen van dit boekje is enorm geweest. De afgelopen
vier jaar heb jij aan mijn zijde gestaan, en het was dan ook vanzelfsprekend dat jij mijn
paranimf zou worden. Jouw expertise, nauwkeurigheid en geweldige planning zijn voor mij
onmisbaar geweest de afgelopen jaren! Ik heb het altijd erg prettig gevonden om met jou
samen te werken, en ik hoop dat je, ondanks je tweede baan als moeder van Daniël en Elian,
nog lang op ons lab blijft werken! Voor het tot stand komen van hoofdstuk 4 zijn de
geweldige inzet van jou en Saskia onmisbaar geweest. Toen we door kregen dat de concurrent
ons op de hielen zat hebben jullie in een ongelooflijk tempo de SSCP gels gerund en de shifts
gesequenced, zodat het artikel in ± 5 weken tijd de deur uit was. Door alle drukte vergat ik
nog wel eens mijn enthousiasme bij het vinden van ‘weer een mutatie!’ met jullie te delen…
Zonder jullie was dit hoofdstuk er echter nooit gekomen.
Saskia, jouw inzet was overweldigend, jij kwam zelfs als de kinderen naar bed waren terug
om nog meer PCRs in te zetten. Ik hoop dat we nog lang kunnen blijven samenwerken, maar
dan in een iets rustiger tempo.
Marc, heel lang geleden, voordat jij aan je eigen promotie-onderzoek begon, heb je ook nog
een paar maanden aan dit proefschrift gewerkt; jij hebt geholpen bij het tot stand brengen van
hoofdstuk 2. Maar jouw computer expertise was gedurende al die jaren onmisbaar, ik kon
altijd op je rekenen als ik weer eens problemen had met ‘dat ding’, je liet je eigen werk dan
zelfs vallen om hulp te bieden. Ook met privé ‘probleempjes’ kon ik altijd bij jou terecht, en
ik mis onze melige buien nog wel eens sinds jouw vertrek naar het CMBI. Jouw ontwerp van
de kaft van dit proefschrift is echt geweldig geworden! Bedankt!!
Dorien, de basis van dit onderzoek ligt bij jou, namelijk de isolatie van retina en RPE
poly(A)+ RNA. Voor praktische vragen kon ik altijd bij jou terecht, en sinds je vertrek naar
het dierenlab mis ik je wel, ‘lab-mama’!
139
Acknowledgements / Dankwoord
Van de afdeling oogheelkunde wil ik Carel Hoyng, August Deutman en Johan Cruysberg
bedanken. Carel, jouw hulp bij hoofdstuk 4 was onmisbaar. Op de vraag of jij RP patiënten
met Coats kende, trok je het archief open en toverde een paar dossiers tevoorschijn. Hopelijk
wordt het archief bij OHK snel geautomatiseerd zodat het zoeken wat gemakkelijker wordt!
Voorts wil ik Arthur Bergen, Jacoline ten Brink, Simone van Soest, Jan Wijnholds, Ingeborgh
van den Born en Mary van Schooneveld bedanken. Onze samenwerking tijdens mijn promotie
was erg vruchtbaar, en ik hoop dat we deze ook na mijn promotie zullen voortzetten. Annette
Payne, Shomi Bhattacharya, Ulrich Kellner, John Heckenlively, Bernard Jurklies, Anita
Blankenagel, Klaus Rohrschneider, Bernd Wissinger and Eckart Apfelstedt-Sylla; many
thanks for providing your patient samples for chapters 3 and/or 4. John, it was an honor
working with you, I hope we can keep collaborating in the future to identify the ‘second RP12
gene’. Kevin Johnson, Ansgar Klebes and Elisabeth Knust; it’s great when you find a gene
and the people working on the Drosophila homologue are only 121 km away. This was
perfect to set up a collaboration, which has led to Chapter 5 of this thesis, and will certainly
lead to more joint publications. It was great working with you, and our visits to your institute
were very useful. Michela Ghiani, Andrea Ballabio and Vania Broccoli; our visit to your
institute in Milan was the beginning of a fruitful collaboration, which led to Chapter 6 of this
thesis. Vania, many thanks for your tremendous efforts and your great in situ pictures.
En dan natuurlijk de mensen die niet vermeld staan als co-auteur maar wel degelijk een
belangrijke rol hebben gespeeld in het tot stand komen van dit proefschrift. Alle mensen van
het lab: bedankt voor de gezelligheid en de prettige werksfeer. Gerard, wij hebben altijd
contact gehouden, ook toen je op het TIL werkte. Ik ben erg blij dat je weer terug bent, je bent
een echt feestnummer, de ideale paranimf dus! Vincent, jij hebt als stagiaire op mijn project
gewerkt, bedankt voor je inzet!
Martine, Marian, Daphne, Debbie, Thessa, Natasja en Ingrid: bedankt voor de gezellige
kaasfondues en weekendjes weg, ik hoop dat we deze traditie nog lang zullen voortzetten!
Joep, mijn jiu-jitsu maatje, als ik je voor elke rake klap nog een biertje moet geven dan ben ik
nog wel even bezig…
Pap en mam, zonder jullie steun en onvoorwaardelijke liefde was dit boekje er nooit
gekomen. Dit proefschrift heb ik heb daarom aan jullie opgedragen. Kees, Anne-Marie en
Nico, bedankt voor jullie interesse gedurende de afgelopen jaren. Herman en José, hier is het
boekje dan eindelijk! Herman, bedankt voor al je hulp in ons huis, jij was zelfs aan het
klussen terwijl wij aan het werk waren…
Lieve Tim, jouw gedrevenheid heeft me in het laatste jaar van mijn promotie een extra
stimulans gegeven om m’n boekje af te maken. ‘Even tussendoor’ verhuizen naar Lent, zodat
jij je kan richten op jouw promotie. Ik ben ervan overtuigd dat jouw boekje er net zo mooi uit
komt te zien als dat van mij! Love you!
Anneke
Het drukken van dit proefschrift werd mede mogelijk gemaakt door Tramedico BV
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Curriculum vitae
Curriculum vitae
Anneke (Antonia Ingrid) den Hollander werd op 16 oktober 1973 geboren te Sassenheim.
Haar VWO-opleiding volgde zij aan het Eindhovens Protestants Lyceum, waar zij in 1991
haar eindexamen behaalde. Vervolgens studeerde zij Biomedische Wetenschappen aan de
Rijksuniversiteit Leiden. Haar eerste stage liep zij bij de vakgroep Medische Microbiologie,
afdeling Bacteriologie van het Academisch Ziekenhuis Leiden (AZL), waar zij onderzoek
deed naar aminoglycoside-resistentie in Pseudomonas, onder leiding van Dr. Jos van de
Klundert en Drs. Renée van Boxtel. Vervolgens liep zij een klinische stage bij de vakgroep
Nierziekten (AZL) en evalueerde onder leiding van Dr. Peter Chang een aantal kunstnieren
voor hemodialyse en hemodiafiltratie. Haar derde korte stage volgde zij bij de vakgroep
Medische Genetica, afdeling Antropogenetica. Onder leiding van Dr. Dorien Peters en Drs.
Fred Petrij analyseerde zij een gedeelte van het CBP gen, dat betrokken is bij het RubinsteinTaybi syndroom. Voor deze stage ontving zij de derdejaars stageprijs Biomedische
Wetenschappen. Voor haar afstudeerstage keerde zij terug naar de afdeling Bacteriologie,
waar ze het aacC2-operon karakteriseerde in Enterobacteriaceae. Tenslotte volgde ze een
extra stage bij de vakgroep Medische Biochemie, afdeling Moleculaire Carcinogenese waar
zij onder leiding van Dr. Matthieu Noteborn en Drs. Astrid Danen-van Oorschot interacties
van apoptin met cellulaire eiwitten onderzocht. Na haar afstuderen op 22 oktober 1996, begon
zij op 1 november bij de afdeling Antropogenetica van het UMC St Radboud te Nijmegen aan
het in dit proefschrift beschreven onderzoek, onder leiding van Dr. Frans Cremers. In juni
2000 behaalde zij een Individual Research Award van de Foundation Fighting Blindness,
getiteld “Elucidation of the molecular processes underlying a severe form of autosomal
recessive retinitis pigmentosa (RP12)”. Vanaf 1 mei 2002 zal zij werkzaam zijn als
gastonderzoeker bij het Téléthon Institute of Genetics and Medicine (TIGEM) in Napels,
Italië.
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List of publications
List of publications
Danen-van Oorschot AAAM, den Hollander AI, Takayama S, Reed JC, van der Eb AJ and
Noteborn MHM (1997) BAG-1 inhibits p53-induced but not apoptin-induced apoptosis. Apoptosis
2:395-402.
Giles RH, Petrij F, Dauwerse HG, den Hollander AI, Lushnikova T, van Ommen G-JB, Goodman
RH, Deaven LL, Doggett NA, Peters DJM and Breuning MH (1997) Construction of a 1.2-Mb contig
surrounding, and molecular analysis of, the human CREB-binding protein (CBP/CREBBP) gene on
chromosome 16p13.3. Genomics 42:96-114.
Cremers FPM, van de Pol DJR, van Driel M, den Hollander AI, van Haren FJJ, Knoers NVAM,
Tijmes N, Bergen AAB, Rohrschneider K, Blankenagel A, Pinckers AJLG, Deutman AF and Hoyng
CB (1998) Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site
mutations in the Stargardt’s disease gene ABCR. Hum Mol Genet 7:355-362.
den Hollander AI, ten Brink JB, de Kok YJM, van Soest S, van den Born LI, van Driel MA, van de
Pol DJR, Payne AM, Bhattacharya SS, Kellner U, Hoyng CB, Westerveld A, Brunner HG, BleekerWagemakers EM, Deutman AF, Heckenlively JR, Cremers FPM and Bergen AAB (1999) Mutations
in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12). Nature Genet
23:217-221.
den Hollander AI, van der Velde-Visser SD, Pinckers AJLG, Hoyng CB, Brunner HG and Cremers
FPM (1999) Refined mapping of the gene for autosomal dominant retinitis pigmentosa (RP17) on
chromosome 17q22. Hum Genet 104:73-76.
den Hollander AI, van Driel MA, de Kok YJM, van de Pol DJR, Hoyng CB, Brunner HG, Deutman
AF and Cremers FPM (1999) Isolation and mapping of novel candidate genes for retinal disorders
using suppression subtractive hybridization. Genomics 58:240-249.
Müller D, Hoenderop JGJ, Meij IC, van den Heuvel LPJ, Knoers NVAM, den Hollander AI, Eggert
P, García-Nieto V, Claverie-Martín F and Bindels RJM (2000) Molecular cloning, tissue distribution,
and chromosomal mapping of the human epithelial Ca2+ channel (ECAC1). Genomics 67:48-53.
den Hollander AI, Heckenlively JR, van den Born LI, de Kok YJM, van der Velde-Visser SD,
Kellner U, Jurklies B, van Schooneveld M, Blankenagel A, Rohrschneider K, Wissinger B, Cruysberg
JR, Deutman AF, Brunner HG, Apfelstedt-Sylla E, Hoyng CB and Cremers FPM (2001) Leber
congenital amaurosis and retinitis pigmentosa with Coats-like exudative vasculopathy are associated
with mutations in the crumbs homolog 1 (CRB1) gene. Am J Hum Genet 69:198-203.
den Hollander AI, Johnson K, de Kok YJM, Klebes A, Brunner HG, Knust E and Cremers FPM
(2001) CRB1 has a cytoplasmic domain that is functionally conserved between human and
Drosophila. Hum Mol Genet 10:2767-2773.
den Hollander AI, Ghiani M, de Kok YJM, Wijnholds J, Ballabio A, Cremers FPM and Broccoli V
(2002) Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression
pattern in eye and brain. Mech Dev 110:203-207.
Zendman AJW, van Kraats AA, den Hollander AI, Weidle UH, Ruiter DJ and van Muijen GNP
(2002) Characterization of XAGE-1b, a short major transcript of cancer/testis-associated gene XAGE1, induced in melanoma metastasis. Int J Cancer 97:195-204.
den Hollander AI, Knust E and Cremers FPM. Crumbs (CRB) genes. In Encyclopedia of Molecular
Medicine, edited by Creighton, T.E., New York: John Wiley & Sons, Inc. In press.
Cremers FPM, Maugeri A, den Hollander AI and Hoyng CB. The expanding roles of ABCA4 and
CRB1 in inherited blindness. Novartis Found Symp, in press.
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