03 April 2008 GALT/PAH Deletions 1
GALT and PAH deletion analysis in galactosaemia and PKU patients
Sarah Burton-JonesBristol Genetics Laboratory
CMGS Spring Meeting Liverpool 2008
03 April 2008 GALT/PAH Deletions 2
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)
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
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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 PKUFoetus 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
Galactose 1-phosphate
Glucose 1-phosphate
UDP-glucose
UDP-galactose
Glycolipids, glycoproteins
Galactose GalactitolGalactonate
UDP galactose 4 epimerase
Gal-1-P uridyl transferase
Glucose oxidase
Aldolase reductase
Galactokinase
Glucose 6-phosphate
Glucose + Pi
ATP
ADP
Mg2+
NAD+
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Phenylalanine metabolism
L-Phenylalanine+O2
L-Tyrosine
Fumarate Acetoacetate
Phenylethylamine Phenylacetate Phenylacetylglutamine
Phenylpyruvate
Phenyllactate
O-hydroxyphenylacetate
Tryptophan Serotonin
DopamineDOPANeurotransmitters,
e.g. adrenalin
CO2 + H2O
TCA cycle
Tetrahydrobiopterin (BH4)
4 alpha-hydroxytetrahydropterin
Quininoid dihydropterinPAH
DHPR
4 alpha-carbinolamine dehydratase
H2O
NAD + H+NAD+
MelaninDHPR = Dihydrofolate reductasePAH = Phenylalanine hydroxylase
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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
3. If single exon deletion identified, confirm by long-range PCR
3. Test all patients using MLPA kit P156 (MRC Holland)
4. Compare patients with apparent same deletion by sequencing across breakpoints
4. If dosage normal, carry out bi-directional sequencing of PAH gene
1. Identify candidate patients and obtain consent for retesting
Galactosaemia patients PKU patients
2. Test for exon dosage using MLPA kit P055 (MRC Holland)
2. Test for whole gene deletion using deletion junction fragment PCR (Coffee et al, 2006)
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Selection of patients
GALT(1996 to July 2007)
PAH(1993 to July 2007)
Total individuals tested 655 949
Total selected for deletion analysis 15 54
Excluded; no consent or no DNA 3 34
DNA available, consent obtained 7(10 alleles)
12(13 alleles)
‘Control’ deletion carriers 5(7 alleles)
8(8 alleles)
Included in ’07 deletion study 12 20
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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
GALT genomic region (11 exons, 4kb)
F1 RF2
• 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
Normal allele (629bp)
Deletion allele (487bp)
Normal N/Del Del/Del Pt A Pt B Pt C blank
100bp ladder
50bp ladder
• Patients A, B and C affected with galactosaemia, no mutations detected prior
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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 XGrandmother
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
12345
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GALT allele structure
Deletion 3162bp Deletion 2294bp
c.1-1040 and 5’ sequence c.1137+793 and 3’ sequence c.754-
c.820c.820+1-
c.820+50
Ins
12bp
Expanded diagram of GALT region structure on deletion allele
Deletion GALT allele
Normal GALT allele
Insertion 12bp (GAATAGACCCCA)
1 2 3 4 5 6 7 8 9 10 11 (GALT exon number)
Key GALT
coding DNA
Unidentified
inserted sequenceReverse
primer
Forward
primer
Non-coding
DNA
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PAH MLPA
P055 MLPA probe (PAH)
Normal controlControl 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
• First primer set
• Normal allele did not amplify in two heterozygous controls
Normal N/Del N/del N/del blank
Normal allele
Exon 6 deletion allele
1 kbladder
• 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 allele (approx. 5.5kb)
Exon 6 deletion allele (approx. 4.7kb)
Normal N/Del N/del N/del Patient blank
1 kbladder
1 kbladder
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:
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Patient Status Prior known mutation Mutation identified
1 Affected NoneIntron 4
c.441+5G>T homozygous
2 Affected Intron 10c.1066-3C>T
Exon 13c.1340C>A (p.Ala447Asp)
3 Affected Exon 8 c.896T>G (p.Phe299Cys)
*Exon 5c.500A>T (p.Asn167Ile)
4 Carrier NoneIntron 4
c.441+4A>G heterozygous
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Conclusions
GALT
• 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
PAH
• 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