03 april 2008galt/pah deletions 1 galt and pah deletion analysis in galactosaemia and pku patients...

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


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


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


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


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+


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


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)


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


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)


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


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


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


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


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


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


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


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


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:


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


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


Thanks to…

Bristol team

Mary GableHilary SawyerLaura YarramElena MavrakiThalia AntoniadiMaggie Williams

Metabolic clinicians

Prof. Benal BuyukgebizDr Paz BrionesDr Philip LeeDr Peter RobinsonDr Andrew Morris Dr Ed BlairDr Mike Champion

03 april 2008galt/pah deletions 1 galt and pah deletion analysis in galactosaemia and pku patients...

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