Transcript GALT and PAH deletion analysis in galactosaemia and PKU

GALT and PAH deletion analysis in
galactosaemia and PKU patients
Sarah Burton-Jones
Bristol Genetics Laboratory
CMGS Spring Meeting Liverpool 2008
03 April 2008
GALT/PAH Deletions
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Galactosaemia and Phenylketonuria
• ‘Inborn errors of metabolism’
• Autosomal recessive
• Highly variable incidence across Europe
• Majority of cases involve single
nucleotide (point) mutations
• Service at Bristol since 1993 (PKU) and
1996 (galactosaemia)
• Minority of cases involve large deletions
spanning one or multiple exons
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Galactosaemia
Gene / locus
GALT, 9p13 (4kb gDNA, 11 exons)
Enzyme
galactose-1-phosphate uridyl transferase
UK incidence
~1/44000 in UK
(1/500 among Irish travellers)
UK carrier frequency
~1/110
Newborn screen
Scotland and Ireland; not in England, Wales, N.Ireland
Symptoms
Neonate: FTT, vomiting, diarrhoea, hypotonia,
hepatomegaly, bacterial sepsis. Fatal if undiagnosed.
Later: Cataracts, developmental delay, POF in >90%
females
Treatment
Lactose-free diet (developmental delay may persist)
Biochemical test
Measure Gal-1-PUT activity in blood
Molecular testing
(Bristol)
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i) p.Gln188Arg (exon 6 c.563A>G) covers ~75% of UK
GALT mutations (Tyfield,L. et al 1999)
ii) Bi-directional sequencing of GALT
GALT/PAH Deletions
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Phenylketonuria
Gene / locus
PAH, 12q22-24 (90kb gDNA, 13 exons)
Enzyme
phenylalanine hydroxylase
UK incidence
~1/10-12000
UK carrier frequency
~1/50
Newborn screen
Nationwide in UK
Symptoms
Progressive mental retardation, epilepsy, scleroderma.
Maternal PKU
Foetus affected if mother has PKU, regardless of foetal
genotype
Treatment
Restricted phenylalanine diet (minimal quantity is essential)
Biochemical test
Measure free [Phe] in blood
Molecular testing
(Bristol)
Bi-directional sequencing of PAH
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Galactose metabolism
Aldolase
reductase
Glucose
oxidase
Galactonate
Galactose
Galactitol
ATP
Galactokinase
Mg2+
ADP
Galactose
1-phosphate
Gal-1-P uridyl
transferase
UDP-glucose
Glucose
1-phosphate
UDP
galactose 4
epimerase
NAD+
Glucose
6-phosphate
UDPgalactose
Glucose + Pi
Glycolipids,
glycoproteins
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Phenylalanine metabolism
Phenylethylamine
Phenylacetate
L-Phenylalanine
+O2
Phenylacetylglutamine
O-hydroxyphenylacetate
Phenylpyruvate
Phenyllactate
NAD+
DHPR
Tetrahydrobiopterin
(BH4)
NAD + H+
Quininoid
dihydropterin
PAH
4 alphahydroxytetrahydropterin
4 alphacarbinolamine
dehydratase
H2O
L-Tyrosine
Tryptophan
Fumarate
Serotonin
Acetoacetate
TCA cycle
CO2 + H2O
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DOPA
Dopamine
DHPR = Dihydrofolate reductase
PAH = Phenylalanine hydroxylase
GALT/PAH Deletions
Neurotransmitters,
e.g. adrenalin
Melanin
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Context
PAH:
• Bristol lab: A-grade project 2000 investigated PKU patient
alleles with no PAH mutation detected using ‘CMDA’
(PCR/PAGE) and LiCOR QIR dosage analysis. 13/37 showed
deletion of at least one exon
• 10 separate publications 1990-2007 reporting PAH deletions
of one or more exons
GALT:
• Bristol lab: Patient identified 1999 with homozygous
deletion by PCR and Southern blot
• Coffee et al (2006) characterised GALT 5.5kb deletion
• 3 previous reports of 5kb / exons 1-11 deleted, 2001-2006
• Possible ethnic association – Jewish/Hispanic
• Gort et al (2006) described GALT deletion exons 5-10
MLPA kits now available for PAH and GALT (MRC Holland)
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Aims of 2007 GALT/PAH Study
• Evaluate MLPA for use in PAH and GALT
dosage analysis
• Develop and validate PCR methods for
confirmation of large deletions
• Prepare the novel methods for introduction
to laboratory service
• Investigate patients with only one or no
mutations detected by DGGE
• Estimate frequency of gross rearrangements
in PAH and GALT
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Project strategy
1. Identify candidate patients and obtain consent for retesting
Galactosaemia patients
PKU patients
2.
Test for whole gene deletion using
deletion junction fragment PCR
(Coffee et al, 2006)
2.
Test for exon dosage using
MLPA kit P055 (MRC
Holland)
3.
Test all patients using MLPA kit
P156 (MRC Holland)
3.
If single exon deletion
identified, confirm by
long-range PCR
4.
Compare patients with apparent
same deletion by sequencing
across breakpoints
4.
If dosage normal, carry out
bi-directional sequencing
of PAH gene
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Selection of patients
GALT
PAH
(1996 to July 2007)
(1993 to July 2007)
655
949
15
54
Excluded; no consent or no DNA
3
34
DNA available, consent obtained
7
12
(10 alleles)
(13 alleles)
5
8
(7 alleles)
(8 alleles)
12
20
Total individuals tested
Total selected for deletion analysis
‘Control’ deletion carriers
Included in ’07 deletion study
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GALT/PAH Deletions
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GALT junction fragment PCR
• Primer sequences from Coffee et al 2006
• PCR optimised using Megamix double (Microzone Ltd)
• JF-PCR adapted for robotic sequencing set up to
determine breakpoints
F1
F2
R
GALT genomic region (11 exons, 4kb)
• F1/R product (487bp) only if GALT deletion on allele
• F2/R product (629bp) only if GALT exon 11 present
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GALT PCR results
• Patients A, B and C affected with
galactosaemia, no mutations detected prior
Normal
N/Del
Del/Del
Pt A
Pt B
Pt C
blank
Normal allele (629bp)
Deletion allele (487bp)
100bp
ladder
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50bp
ladder
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GALT family study - MLPA
P156 MLPA probe
(GALT)
Normal
control
Grandmother
of X (p)
Father of X
Patient X
Sister of X
Mother of X
Grandmother
of X (m)
Duarte del 4bp
0.92
0.52
0.56
0.00
0.51
0.51
0.48
Exon 1
1.08
0.58
0.55
0.00
0.31
0.59
0.61
Exon 2
0.92
0.52
0.57
0.00
0.21
0.52
0.53
Exon 4
0.98
0.59
0.58
0.00
0.54
0.53
0.59
Exon 5
1.03
0.62
0.59
0.00
0.58
0.60
0.61
Exon 6
0.93
0.58
0.57
0.00
0.55
0.58
0.53
Exon 7
0.89
0.52
0.55
0.00
0.35
0.51
0.52
Exon 8
0.89
0.51
0.56
0.00
0.46
0.49
0.47
Exon 9
1.00
0.59
0.56
0.00
0.42
0.59
0.58
Exon10
0.95
0.54
0.53
0.00
0.60
0.53
0.54
Exon 11
1.07
0.60
0.55
0.00
0.51
0.63
0.61
IL11RA gene
1.14
1.13
1.06
1.16
1.25
1.15
1.08
?
?
Patient X = control homozygote;
deletion identified previously by
Southern blotting
X
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Determining GALT allele structure
• Bi-directional sequencing of deletion allele using ABI3730
• Exported in text format from Mutation Surveyor
• Multiple alignment using ClustalW, visualised using BioEdit
1
2
3
4
5
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GALT allele structure
Normal GALT allele
1
2
3
4
5
6
7
8
9
10
11
(GALT ex on number)
Deletion GALT allele
Deletion 3162bp
Deletion 2294bp
Ins ertion 12bp (GAATAGACCCCA)
Expanded diagram of GALT region structure on deletion allele
 c.1-1040 and 5’ s equenc e
Key
GALT
c oding DNA
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c.754c.820
Unidentified
ins erted s equenc e
c.820+1- Ins
c.820+50 12bp
Non-c oding
DNA
GALT/PAH Deletions
c.1137+793 and 3’ sequence 
Forward
primer
Rev ers e
primer
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PAH MLPA
P055 MLPA
probe (PAH)
Normal control
Control A
(N/Del.ex.6)
Control B
(N/Del.ex.6)
Control C
(N/Del.ex.6)
Test patient
ASCL1 gene
0.96
1.07
1.02
1.07
1.01
Exon 1
0.92
1.20
1.06
1.14
1.03
Exon 2
0.95
1.06
0.98
1.06
1.10
Exon 3
0.99
1.04
1.01
1.02
1.05
Exon 4
1.02
1.03
1.06
1.09
1.03
Exon 5
1.02
1.08
1.02
1.09
1.03
Exon 6
1.00
0.53
0.51
0.60
0.48
Exon 7
0.93
1.07
1.00
1.24
1.08
Exon 8
0.95
1.10
1.03
1.10
1.10
Exon 9
0.92
1.04
1.01
1.08
1.07
Exon10
1.01
1.09
0.99
1.13
1.06
Exon 11
1.06
0.99
1.18
0.93
1.13
Exon12
0.96
1.11
1.04
1.08
1.10
Exon 13
1.03
1.18
1.09
1.12
1.06
IGF1 gene
1.02
1.07
0.97
1.01
1.05
• PAH MLPA less reliable than GALT kit
• Susceptible to DNA quality variation
• Necessary to confirm results by a second method
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PAH exon 6 LR-PCR
• Initial conditions from
Desviat et al 2006
Normal N/Del
N/del N/del
blank
• First primer set 
Normal allele
Exon 6 deletion allele
• Normal allele did not
amplify in two
heterozygous controls
1 kb
ladder
• Intron 5F and intron 6R primers redesigned
• Two long-range Taq polymerases trialled
• Complex touchdown PCR program optimised with
Sigma AccuTaq LA
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PAH exon 6 LR-PCR results
• 3 control heterozygotes, 1 patient with deletion
identified by MLPA
• Sized approximately using GeneTools software
(Syngene)  0.8kb deleted (exon 6 = 197bp)
Normal N/Del
N/del
N/del
Patient blank
Normal allele
(approx. 5.5kb)
Exon 6 deletion
allele (approx. 4.7kb)
1 kb
ladder
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1 kb
ladder
GALT/PAH Deletions
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PAH bi-directional sequencing
• 11 PKU patients in whom one or no mutations
detected by DGGE, CMDA, MLPA*
• PAH exons 1-13 sequenced using ABI3730
• Previously undetected mutation identified in 4
of 11 patients:
Patient
Status
Prior known mutation
Mutation identified
1
Affected
None
Intron 4
c.441+5G>T homozygous
2
Affected
Intron 10
c.1066-3C>T
Exon 13
c.1340C>A (p.Ala447Asp)
3
Affected
Exon 8
c.896T>G (p.Phe299Cys)
*Exon 5
c.500A>T (p.Asn167Ile)
4
Carrier
None
Intron 4
c.441+4A>G heterozygous
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Conclusions
GALT
PAH
• 4 unrelated patients with
identical homozygous
5.5kb deletion
• 5 family members
heterozygous for deletion
allele
• ? Jewish/Hispanic
association upheld
• 13 alleles of 1310 tested
(655 referrals) = ~1%
• MLPA and JF-PCR now
available for detection and
confirmation
• 4 unrelated patients with
heterozygous deletion of
exon 6
• ? (Scottish) founder mutation
• 4 alleles of 1898 tested (949
referrals) = ~0.2%
• Only exon 6 deletion can be
confirmed by LR-PCR as yet
• Mutations identified by
sequencing in 4 patients not
detected by DGGE
• 7 patients with biochemical
diagnosis of PKU only
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Thanks to…
Bristol team
Metabolic clinicians
Mary Gable
Hilary Sawyer
Laura Yarram
Elena Mavraki
Thalia Antoniadi
Maggie Williams
Prof. Benal Buyukgebiz
Dr Paz Briones
Dr Philip Lee
Dr Peter Robinson
Dr Andrew Morris
Dr Ed Blair
Dr Mike Champion
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