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GALT and PAH deletion analysis in galactosaemia and PKU patients

GALT and PAH deletion analysis in galactosaemia and PKU patients. Sarah Burton-Jones Bristol Genetics Laboratory CMGS Spring Meeting Liverpool 2008. Galactosaemia and Phenylketonuria. ‘Inborn errors of metabolism’ Autosomal recessive Highly variable incidence across Europe

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GALT and PAH deletion analysis in galactosaemia and PKU patients

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  1. GALT and PAH deletion analysis in galactosaemia and PKU patients Sarah Burton-Jones Bristol Genetics Laboratory CMGS Spring Meeting Liverpool 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 GALT/PAH Deletions

  3. Galactosaemia GALT/PAH Deletions

  4. Phenylketonuria 03 April 2008 GALT/PAH Deletions 4

  5. Aldolase reductase Glucose oxidase Galactonate Galactose Galactitol ATP Galactokinase Mg2+ ADP Galactose 1-phosphate Gal-1-P uridyl transferase UDP-glucose Glucose 1-phosphate Glucose 6-phosphate UDP galactose 4 epimerase UDP- galactose NAD+ Glucose + Pi Glycolipids, glycoproteins Galactose metabolism GALT/PAH Deletions

  6. Phenylethylamine Phenylacetate Phenylacetylglutamine L-Phenylalanine +O2 O-hydroxyphenylacetate Phenylpyruvate Phenyllactate DHPR NAD+ Tetrahydrobiopterin (BH4) NAD + H+ Quininoid dihydropterin PAH 4 alpha- hydroxytetrahydropterin 4 alpha- carbinolamine dehydratase H2O L-Tyrosine Tryptophan Serotonin Fumarate Acetoacetate Neurotransmitters, e.g. adrenalin DOPA Dopamine TCA cycle Melanin CO2 + H2O DHPR = Dihydrofolate reductase PAH = Phenylalanine hydroxylase Phenylalanine metabolism GALT/PAH Deletions

  7. Context PAH: • Bristol lab: A-grade project 2000 investigated PKU patient alleles with no PAHmutation 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) GALT/PAH Deletions

  8. 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 GALT/PAH Deletions

  9. Project strategy • Identify candidate patients and obtain consent for retesting • Galactosaemia patients PKU patients • Test for exon dosage using MLPA kit P055 (MRC Holland) • Test for whole gene deletion using deletion junction fragment PCR (Coffee et al, 2006) • If single exon deletion identified, confirm by long-range PCR Test all patients using MLPA kit P156 (MRC Holland) • If dosage normal, carry out bi-directional sequencing of PAH gene Compare patients with apparent same deletion by sequencing across breakpoints GALT/PAH Deletions

  10. Selection of patients GALT/PAH Deletions

  11. 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 GALT/PAH Deletions

  12. 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 50bp ladder GALT/PAH Deletions

  13. ? ? X GALT family study - MLPA Patient X = control homozygote; deletion identified previously by Southern blotting GALT/PAH Deletions

  14. 1 2 3 4 5 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 GALT/PAH Deletions

  15. GALT allele structure GALT/PAH Deletions

  16. PAH MLPA • PAH MLPA less reliable than GALT kit • Susceptible to DNA quality variation • Necessary to confirm results by a second method GALT/PAH Deletions

  17. Normal N/Del N/del N/del blank Normal allele Exon 6 deletion allele 1 kb ladder PAH exon 6 LR-PCR • Initial conditions from Desviat et al 2006 • First primer set  • Normal allele did not amplify in two heterozygous controls • Intron 5F and intron 6R primers redesigned • Two long-range Taq polymerases trialled • Complex touchdown PCR program optimised with Sigma AccuTaq LA GALT/PAH Deletions

  18. Normal N/Del N/del N/del Patient blank Normal allele (approx. 5.5kb) Exon 6 deletion allele (approx. 4.7kb) 1 kb ladder 1 kb ladder 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) GALT/PAH Deletions

  19. 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: GALT/PAH Deletions

  20. 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 Conclusions GALT/PAH Deletions

  21. Thanks to… Bristol team Mary Gable Hilary Sawyer Laura Yarram Elena Mavraki Thalia Antoniadi Maggie Williams Metabolic clinicians Prof. Benal Buyukgebiz Dr Paz Briones Dr Philip Lee Dr Peter Robinson Dr Andrew Morris Dr Ed Blair Dr Mike Champion GALT/PAH Deletions

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