Transcript Document

The Role of the Clinical Investigator in
Discovering how the Brain Controls
Reproduction?
Evidence from Genetic Approaches in the Human
William F. Crowley, Jr., M.D.
Professor of Medicine, Harvard Medical School
Director, Harvard Reproductive Endocrine Sciences Center
Director of Clinical Research, Mass General Hospital
The People Who Did the Work !
Others
Female Team
Genetics Team
Jan Hall
Jim Gusella
Stephanie Seminara
Corinne Welt
Sue Slaughenhaupt
Nelly Pitteloud
Kathy Martin
Larry Jameson
James Acierno
Judy Adams Cricket & John Seidman
Yousef Bo-Abbas
Yari Jimenez
Eric Lander
Astrid Meysing
David Altshuler
Jenna Sagourny
Pat Sluss
Paradigm Therapeutics
Carl Pallais
Sam Aparicio
Stephen O’Rahilly
William Colledge
Sophia Messager
Male Team
Emmanouella Chatzidaki
Frances Hayes
Nellie Pitteloud
U of Pittsburgh: Tony Plant
Maria Yialamas
U of Washington: Bob Steiner
Andrew Dwyer
Oregon Primate Center: Sergio Ojeda
Today’s Goals
• Use human disease model to address fundamental problem
• Idiopathic hypogonadotropic hypogonadism & Kallmann’s Syndrome
• Genotype/phenotypes of 2 new genes that control GnRH
• We propose to be “gatekeepers” of GnRH secretion, sexual
maturation, & puberty
• Put them in some context
• Biology, Development, Evolution & Opportunities
????
Hypothalamus
GnRH
Pituitary
FSH
LH
+
T/E2 Inhibin B
Leydig cells
T/E2
Testes
+
+
Sperm
Why Study the GnRH Neuronal System?
GnRH is Central, Critical, & Highly Conserved in Reproduction
‘Pilot Light’ of mammalian reproduction
•
•
•
•
•
Initiates all reproductive activity
‘On’ During neonatal and pubertal periods
‘Off’ during the childhood period or anestrus
Initiates puberty/sexual maturation
Rest of system - passively regulated
“The Boss”
Key Evolutionary Role
Links external environment and internal endocrine milieu
Synchronizes nutrition, light/dark cycles, stress, olfaction, & predators &
reproduction
Key to evolutionary success (‘fitness’)
Genetic Control is Highly Species Specific
E.g. Alpha adrenergic modulation of GnRH
Links Between Reproduction & Environment:
Role of Predators, Nutrition & Genes for Selection
Neuroendocrine Activity
(GnRH,LH,FSH,FAS)
Neuroendocrine Activity of the
Reproductive Axis Across Life in Humans
GnRH +mRNA
GnRH +Immunostaining
? Controlling Genes
Neonatal
Years
Childhood
Puberty
Rule #1 - Start with a Patient with a Disease:
= Important Biologic Problem
History (1943)
• good general health
• small genitals; absence of puberty
• absent sense of smell
Exam
• short stature
• arm span > height
• no axillary or pubic hair
• microphallus
• small L testis; no R testis palpable
Rule #2 – Measure Something!
- First Classification of Gonadal Function -
PITUITARY
LH
FSH
LH
FSH LH
FSH
GONADS
Gonadotropins Normal
Gonadal Fxn
Normal
Hyper 
1o Failure
Hypo 
2o Failure
Hypogonadotropic Hypogonadism
? Hypothalamic GnRH Deficiency
An Opportunity for Therapy based on Physiology
Normal
GnRH Deficient
HYPOTHALAMUS
GnRH
Pulsatile
GnRH Rx
PITUITARY
LH
GONADS
FSH
LH
FSH
50
LH (IU/L)
T = 500ng%
Normal Adult Male
40
Pulsatile GnRH Rx:
Re-Constitutes Normal
Normal Adult Male Range
HPG Axis in IHH
(Crowley et al, JCEM, 1980,
30
20
10
Hoffman et, NEJM, 1980)
0
0
240
480
720
960
1200
1440
LH (IU/L)
50
40
IHH Male: Baseline
T = 20ng%
30
Normal Adult Male Range
20
10
0
0
240
480
720
960
1200
1440
(LH (IU/L)
GnRH IV q 2hr
80
T = 500ng%
60
40
+ Generates
Opportunity for
Dose-Response
Curves
(Spratt et al, JCEM)
20
0
0
120
240
360
480
TIME (minutes)
600
720
Infant Male with Terminal Deletion at Xp22.31 
Karyotypic Abnormality
Mother
46XX
shared
terminal
deletion
Xp22.31
• Dysmorphic features
• Cryptorchism & microphallus
• Low LH, FSH, & T levels
• Absent olfactory bulbs/tracks
• Icthyiosis & Calcium Disorder
Son
46XY
Bick et al. Am J Med Gen 1989
Localization of the KAL Gene on Xp 21.3
splice
frameshift
nonsense
missense
deletion
*
1 2 3
Novel
Mutation
4
5
C172R
Anosmin
His-rich
7
6
*
*
*
8
9 10 11
R191X
181
WAP
R457
X
285
FN III
402
FN III
12
13 14
1951delC
540
680
H
FN III
FN III
*MGH
(Georgopoulos 1997)
Migration of GnRH Neurons in Mouse Brain
vno
gt
ob
poa
vomeronasal organ
ganglion terminale
olfactory bulb
preoptic area
Schwanzel-Fukuda & Pfaff, Nature 1989
Olfactory
bulb
Embryology of GnRH
Olfactory
Neuronal Migration
Tract
• Olfactory neurons send
processes  hypothalamus
NORMAL
Cribriform
• GnRH neurons migrate
Plate
along olfactory tract
Olfactory
Placode
• Anosmin in extracellular
matrix
• Kallmann’s Syndrome olfactory bulb & tract
absent in Kallmann’s pts
 anosmia
KALLMANN’S
• Migration defect in KAL-1
deletion/mutation 
GnRH deficiency
Genotype-phenotype:
Males with Confirmed KAL mutations
Inherit TV Crypt Micro LH pulse Other
Cys172Arg
3 Brothers 1-2 mL
-
-
No
Arg 191X
1 sporadic 1-2 mL
1 sporadic 1-2 mL
1
2
+
Tyr328X
X-linked
1-2 mL
-
-
No
11 base del
X-linked
1-2 mL
2
-
No
14 base del
Sporadic
1-2 ml
-
-
No
Arg457X
X-linked
4.5 mL*
1
-
No
Del.
Sporadic
1-2 mL
1
+
No
Synkinesia
No
Hi-Arched palate
color blindness
color blindness
Renal agen.
Oliviera et al: JCEM 2002
The Genetics of GnRH Deficiency:
Role of the Autosome
GnRH
Deficiency
Anosmia
Delayed
Puberty
Fam. History
5
Autosmal Dom.
Autosmal Rec.
X-Linked
IHH/KS alone
32%
47%
21%
+/-anosmia
50%
32%
18%
+/- DP
64%
25%
11%
Waldstreicher et al, JCEM 1996
Mutations in FGFR1 Gene (8p11.2)
cause Kallmann’s Syndrome
• Hardelin et al, Nature Genetics, 2003
•
•
•
•
•
2 Patients with deletions of 8p11.2 & 12 region (GnRH)
1 with hereditary spherocytosis (ANK1) + KS
Multiple congential anomalies (shortened 5th finger, micrognathia)
12% incidence of heterozygous mutations in FGFR1 in 129 KS (91M/28F)
Autosomal Dominant
• Dode et al, Nature Genetics, 2003
• 9% incidence of FGFR1 coding sequence mutations in KS patients (12/129)
• High frequency of cleft lip/palate
• Asymptomatic carriers (female)
• Sato et al, JCEM, 2004
• 2 heterozygous mutations of FGFR1 in KS patients
• 11% incidence
• No Reproductive Phenotypes!!!
FGFR1 Mutations: Genotype-Phenotype Correlations
A.
# 1,3,4,9
Absent Puberty
T = 45 ng/dL
10
5
0
0
Glu274Gly Leu340Ser
4
# 12
B.
Tyr339Cys
2
6
8
10
12 hr
Partial Puberty
E2<20 pg/mL
Gln680X
10
IgI
IgII
IgIII
Arg250Gln
TK
TK
5
0
Arg622X
0
2
4
#10
6
8
10
12 hr
Reversal of KS
T = 368 ng/dL
C.
10
5
0
0
2
4
6
8
10
12 hr
Genotype-phenotype - FGFR1 mutations
(MGH Pitteloud, unpublished)
Sex FH
TV
Crypt. Micro.
LH Ansomia Other
Gly696Ser
M
-
8 mL
-
N
U
+
Tyr337Cys
M
-
3 mL
-
N
U
+
Arg620X
M
+
10mL(S+) -
N
Reversal
+
Ser344Cys M
+
4 mL
-
N
LH3.5/FSH6 +
Arg252Glyn M
-
No puberty -
N
?
+
Gly235Ser
M
+
2 ml*
Bilat
Y
U
+
Glu272Gly
M
+
2 mL
Bilat
Y
U
+
Val271Met
M
+
2 mL
-
Y
U
+
Gly678X
M
+
1 ml
-
N
U
Gly701Ser
M
-
3 mL*
Uni
Y
U+
L>S (exon8) M
-
1mL
Bilat
N
U
F
+
Partial pub. ?
RforQ (ex7)
Y
cleft palate
Normal cleft palate
+
Hypo.
Hirschspr.
cleft palate
cleft palate
FGFR1 Mutation and normosmic IHH
IHH
Anosmia
colorblind
Clues/Lessons re FGFR1 Mutations:
1. AD that Mimics X-linked (? Females)
2. Causes not just KS but IHH s anosmia
3. Confirms cleft palate = part of spectrum
Gln678X
Gln678X
19 yr
28yr
IHH (TV 1mL)
IHH (TV 1mL)
Cleft palate
Missing teeth
Color blindness R/G
Gln678X
19yr
IHH? GHD?
FGFR1 Families
IHH
Pedigree 2
Pedigree 1
12
Delayed Puberty
Anosmia
11
Menarche 15
6
7
10
Clues
re9FGFR1 Mutations:Arg620X
Menarche 16
1. Reproductive
Glu272Glyphenotype quite variable
8
3
2
2. Cause
delayed puberty = first gene
Arg620X
r
3. Females
are attenuated to no phenotype
4
1
5
Glu272Gly
4. ? Why
are males more severely affected
Arg620X
Evidence from the Fgfr1 Knock-out Mouse:
FGFR1 and KAL1 Genes
• both expressed in the
Olfactory Bulb during
development
FGFR1 Knock-out
• Telencephalon does not
develop normally
• Interfers with Olfactory
Bulb development
Herbert Development 2003
FGF, FGFR, and HS are required for receptor dimerization
(Bernfield, 1994)
Anos.
Anos.
FGFR1/HS binding domain
in the Ig II domain
Anos.
Anos.
Pellegrini 2001
Biology of FGFR1
KAL-1 & FGFR1 co-expressed developmentally
Brain & kidney
Conditional K/O of FGFR1 in the telencephalon
No olfactory bulb; no pathway for GnRH neurons
FGFR1-FGF2 interaction with HS
 Role for co-receptor (e.g. TGF beta family)
Hypothesis: Kallmann’s Syndrome Due to
FGFR1 Mutations is a Digenic Disorder?
1.
KAL1/Ansomin binds HSPG “Syndecans”
• Anosmin = ligand or co-receptor for FGFR1 (vs FGF2)
2.
KAL1 = Non-Lyonized X gene
• i.e. Females have 2 copies; Males only 1
3.
? FGFR1 Defects Digenic in nature
• Females have 2 copies; males only 1 ligand
• i.e. males are ‘haploinsufficient’ for anosmin
• Therefore males get the more severe phenotypes
IHH
I:1
II:1
I:2
II:2
II:3
II:4
Anosmia
6
III:1
III:2
III:3
III:4
S/L
S/L
S/L
3
III:5
III:6
III:7
S/L
S/L
S/L
3
IV:9
IV:10 IV:11 IV:12
L/L S/S L/S
IV:1
IV:2
IV:3
L/L
S/S
L/S
IV:4
S/S
IV:13
III:8
IV:14
IV:15
IV:16
L/L L/L
S/S
S/L
IV:5
IV:6
IV:7
S/S
S/S
S/L
IV:8
L/L
IV:17
Chromosome 19
rs7815
rs731804
rs668447
rs10390
REU-1902
rs1006473
rs1006474
rs1006475
rs757331
REU-1903
rs736926
D19S886
REU-1905
Seminara et al,
JCEM, June, 2003
GPR54


Protein
Phosphorylation
Gq/11
GTP
PIP2
DAG
Phospholipase C
IP3
Ca 2+ release
• GPCR
• Rhodopsin-like
• Galanin & SomatoStatin Receptors
(~35-38%)
Cognition
Nociception
Feeding/Nutrition
Reproduction
Genomic Screening of GPR54
Pro
WT
Leu
Arg
Pro
L148S
Ser
Arg
443T>C [L148S]
•Segregates properly
•Absent in control pops.
•Highly conserved
• Nonpolar to polar
Human GPR54
Mouse Gpr54
Rat
Gpr54
SVDRWYVTVFPLRALHRRTPRL
SVDRWYVTVFPLRALHRRTPRL
SVDRWYVTVFPLRALHRRTPRL
Human
Human
Human
Human
Human
Human
Human
Human
SVDRYVAIVHSRRSSSLRVSRN
Galanin Family
SVDRYLAIRYPLHSRELRTPRN
SVDRYLAVRHPLRSRALRTPRN
SVDRYVAVVHPIKAARYRRPTV
SIDRYLAVVHPIKSAKWRRPRT Somatostatin Family
SVDRYLAVVHPTRSARWRTAPV
SVDRYVAVVHPLRAATYRRPSV
SVDRYLAVVHPLSSARWRRPRV
GALR1
GALR2
GALR3
SSTR1
SSTR2
SSTR3
SSTR4
SSTR5
50
LH (IU/L)
T = 500ng%
Normal Adult Male
40
GPR54 Patient:
Baseline Studies
30
Normal Adult Male Range
20
10
0
0
240
480
720
960
1200
1440
LH (IU/L)
50
40
GnRH Deficient Male: Baseline
T = 20ng%
Normal Adult Male Range
30
20
10
0
0
240
480
720
960
1200
1440
10
IHH Patient: +GPR54 Mutations
8
+ Low
Amplitude
LH Pulses
6
4
2
0
0
240
480
720
TIME (minutes)
960
1200
1440
25 ng/kg
75ng/kg
2.5 ng/kg
250ng/kg
LH (IU/L)
60
Dose Response
Studies
40
20
0
0
TIME (hr)
2
6
8
LH Amplitude
60
40
20
0
0
1
2
3
0
1
2
3
LH Amplitude
60
40
20
+ Left Shifted
Dose Response
Curve Compared
To 6 IHH Men
Without GPR54
Mutations
0
LOG (10) GnRH
Seminara SB et al, NEJM, 2003
-/-/-/-
+/+
+/+
B
+/+
+/+
-/-
-/-
GPR54 -/Paradigm Therapetuics
“Harry Potter”
-/-
Genotype
0.4
0.3
0.2
0.1
0
50
20
0
Genotype/Treatment
20
10
0
Genot ype
0
Genotype/cycle stage
f)
8
P <0.01
Genotype/Treatment
Gn -/RH
e)
60
fem -/ale
100
70
P<0.01
+
0.5
100
+ -/PB
S
0.7
120
50
wi
femld-t y
ale pe
200
c)
ma -/le
P<0.01
80
wi
ld
ma -t yp
le e
500
FSH ng/ml
600
P<0.05
w
i
+ ld-t
PB yp
S e
w
+ ildGn t y
RH pe
Genotype
LH ng/ml
0
b)
fe -/m
ale
300
w
pro ildest t yp
r e
w i us
oe ld-t y
str pe
us
w
me ild
toe -t yp
st e
w rus
dio ilde st t yp
rus e
no
n-c -/yc
lin
g
6
w
i
fe ld-t
m yp
ale e
400
Oestradiol pg/m l
P<0.05
m /al
e
0
Gn -/RH
1
+
2
ild
m -t y
a l pe
e
3
+ -/PB
S
4
wi
+ ld-t y
PB p
S e
wi
+ ld-t
Gn yp
RH e
5
Pituitary LH ng/m g tissue
g)
w
0.6
GnRH pg/mg hypothalamus
wi
femld-t y
ale pe
ma
le
P<0.001
fe -/m
ale
w
i
fe ld-t
m yp
ale e
-/-
wi
l
ma d-t y
le pe
Testosterone pg/m l
7
m /al
e
ild
m -t y
a l pe
e
d)
w
LH ng/ml
a)
P<0.01
110
90
40
30
40
20
30
10
10
Genotype
P <0.01
40
7
30
6
5
4
3
2
1
0
Phenotype of GPR54 -/- Mouse
(“Harry Potter”)
• Faithful Recapitulation of Human IHH
• Normal to  GnRH Levels in Hypothalamus
- ? Means GPCR 54 maybe responsible for GnRH
processing
- ? Like prepubertal monkey and rat hypothalamus
• If mutation IHH, Could Antagonists HH
- ? Good candidate for small ligand screening
If GPR54 is the Receptor,
What is the ligand?
Kisspeptin-1
/Metastin
Structure of Metastin
Kisspeptin-1
Signal Peptide
1
19 20
M
G E
Dibasic cleavage sites
67 68
RK
RLSRR GTS
Metastin
1
16
31
46
GTS
121 122
GLRF GKR
GLRF NH2
GTSLSPPPESSGSRQ
QPGLSAPHSRQIPAP
QGAVLVQREKDLPNY
NWNSFGLRF-NH2
15
30
45
54
145
Q
KiSS-1 mRNA
transcripts
in neurons of
arcuate nucleus
in the rodent
(Steiner Lab)
GPR54 & KiSS1
mRNA in overlapping cell
populations
of female monkeys
(Ojeda & Plant)
GPR54
Expression in
Female and
Agonadal Male
Monkeys Across
the Pubertal
Transition
Expression of
GPR54 does not
increase in the
agonadal male
monkey
KISS1
Expression in
Female and
Agonadal Male
Monkeys Across
the Pubertal
Transition
Expression of
KISS1 increases
in both animal
models
Central Administration of Metastin in Juvenile
Orchidectomized Monkeys
Vehicle
30 ug Metastin
100 ug Metastin
Metastin
Plasma LH (ng/ml)
16.0
Acyl+100 ug Metastin
12.0
8.0
4.0
0.0
-30
0
30
60
90
120
Time (min)
150
180
210
240
Metastin
ellicits a brisk
LH response
in juvenile,
agonadal male
monkeys
Physiology of Metastin/Kisspeptin
•
GPR54 is on nearly all GnRH neurons + median eminence
•
Metastin & GPR 54 mRNA  across sexual maturation in monkey,
rat, and mouse
• (Ojeda, Plant, Steiner, Manuel-Tempore)
•
This  appears to be regulated by sex steroids
•
Kisspeptin neurons overlay GPR54 & GnRH neurons
• Steiner; Ojeda
•
Metastin administration induces c-fos in GnRH neurons
•
Metastin administration induces LH pulses
• (rat, mice & monkey)
•
Metastin administration ‘trumphs’ leptin def.
IHH Due to Possible Metastin Mutation?
•
IHH male with homozygous duplication at 5’ end of
metastin gene (Paris [Deroux] + Boston
collaboration [Seminara + Crowley]
•
No mRNA present in peripheral lymphocytes
•
Some normal controls + for heterozygous changes
•
Being evaluated
GPR 54 & Metstatin:
? Site of Action within the Hypothalamus
GnRH
HypophysealPortal
Blood
Supply
(GnRH,LH,FSH,FAS)
Neuroendocrine Activity
Role of GPR54 & Metastin in IHH
IHH (GPR54/Metastain)
Neonatal Childhood
Years
Puberty
Hypothalamic Amenorrhea
(+ stress) (- stress)
(GnRH,LH,FSH,FAS)
Neuroendocrine Activity
? Role of Metastin/GPR54 in
Hypothalamic Amenorrhea?
Neonatal Childhood
Years
Puberty
Adult-Onset IHH
(- stress)
(GnRH,LH,FSH,FAS)
Neuroendocrine Activity
? Role of Metastin/GPR54 in
Adult Onset IHH
Neonatal Childhood
Years
Puberty
(GnRH,LH,FSH,FAS)
Neuroendocrine Activity
? Role of Metastin/GPR54 in
Disorders of Pubertal Timing?
? Precocious Puberty/
Constitutional Delay
Neonatal Childhood
Years
Puberty
Human Disease Model:
Idiopathic Hypogonadotropic Hypogonadism
-Anosmia
+ Anosmia
? Developmental Pathways -? Regulatory GnRH
Kallmann’s Syndrome
KAL1 (X)
FGFR1 (AD)
? FGF 2
? Others
Normosmic IHH
GPR 54
? Kisspeptin
FGFR1
? Others
Karotypic
Abnormality
Contiguous
Gene Syndrome
Linkage
X-p22.1
KAL1
8p11.2
FGFR1
19q13.3
GPR54
LEPR
1p31.2
PIT1
3p11.2
2003 Human Genome Assembly
GnRNR
4q13.2
1
13
2
14
3
15
FGFR1
8p12
? GnRH1
8p 12.2
Common Features
All Monogenic
LEP
4All Prismatic
67q32.1
7
8
10
5
9
PROP1
GPR54
5q35.3
KAL1
None19p13.3
are PolygenicXp22.31
11
19
X
16
17
18
20
21
22
12
DAX1
Xp21.2
Y
Trends in Bedside to Bench
Bedside
1930-60s
1960-00s
2000+
Bench
Universities, AHCs, &
Professional Societies
Thoughtful
Clinicians
Clinical
Investigators
Basic
Investigators
Lessons for the Clinical Investigator
in the Post-Genomic Era
1. Take full advantage of UBOs
• patients or families (espeically
those with contiguous gene
syndromes or chromosomal
translocations) afford Unique
Biologic Opportunities to
locate and identify new genes
Spiral
Staircase
To
Success
Lessons for the Clinical Investigator
in the Post-Genomic Era
1. Take full advantage of UBOs
2. Partnerships with basic
investigators are key for
translational investigators to
establish & are mutually beneficial
• basic research collaborations will be
increasingly bi-directional in the future
• human mutations provide critical
information re: structure-activity
relationships
• accurate, detailed, & quantitative
phenotyping will be key to identifying
new genes & their biology
Spiral
Staircase
To
Success
Lessons for the Clinical Investigator
in the Post-Genomic Era
1. Take full advantage of UBOs
2. Partnerships key & mutually
beneficial
3. Strap on the new tool belt
• Human Genome Project provides
expanding collection of new tools
Spiral
Staircase
To
Success
Lessons for the Clinical Investigator
in the Post-Genomic Era
1. Take full advantage of UBOs
2. Partnerships key & mutually beneficial
3. Strap on the new tool belt
Spiral
4. Access to the DNA is THE rate-limiting Staircase
To
step!
Success
• samples from patients & relatives
are a key resource:
• phenotyping of proband
• complete family histories
• relational databases
GPR 54 & Metstatin:
? Site of Action within the Hypothalamus
?
?
?
?
?
?