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Diagnostic Testing for Familial Pancreatitis

Diagnostic Testing for Familial Pancreatitis. Emma McCarthy Clinical Scientist Merseyside & Cheshire Regional Molecular Genetics Laboratory. Aims. Overview of pancreatitis both hereditary and idiopathic Genes involved & diagnostic service PRSS1 dosage analysis. Pancreatitis.

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Diagnostic Testing for Familial Pancreatitis

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  1. Diagnostic Testing for Familial Pancreatitis Emma McCarthy Clinical Scientist Merseyside & Cheshire Regional Molecular Genetics Laboratory

  2. Aims • Overview of pancreatitis both hereditary and idiopathic • Genes involved & diagnostic service • PRSS1 dosage analysis

  3. Pancreatitis • Inflammatory disease • Severe abdominal pain • Nausea • Vomiting fever • Acute or chronic • Premature activation digestive enzymes • Common triggers • Excessive alcohol consumption • Smoking • Gallstones

  4. activation TRYPSINOGEN TRYPSIN AUTODIGESTION CHRONIC PANCREATITIS PERMANENT LOSS OF FUNCTION DIABETES / PANCREATIC CANCER premature activation enzymes multiple attacks irreversible scarring advanced stages

  5. Reproduced from: Rosendahl et al.Orphanet Journal of Rare Diseases 2007 2:1

  6. Diagnostic service • PRSS1 • Fluorescent sequencing exons 2 + 3 • SPINK1 • p.N34S mutation (pyrosequencing) • CFTR • 28 mutations (Elucigene CFHT) • EUROPAC:www.liv.ac.uk/surgery/europac.html

  7. Hereditary Pancreatitis (HP) • Autosomal dominant • Reduced penetrance • Childhood: • recurrent episodes acute pancreatitis • Adulthood: • chronic pancreatitis • Mutations of cationic trypsinogen gene (PRSS1)

  8. Cationic Trypsinogen • PRSS1 gene (protease serine 1) • Trypsin • Activates digestive enzymes • Self regulating • Autocatalytic activation • Feedback inhibition - cleaves at Arg122 • R122H mutation - “super trypsin”

  9. TCR PRSS1 TRY5 TRY6 TRY7 PRSS2 TCR 4kb initial PCR product

  10. N29I L41L C48C p.N29I c.86A>T p.L41L c.121C>T p.C48C c.144T>C p.N54S c.161A>G p.V59V c.177A>G p.G62G c.186C>T

  11. Laboratory Data • Prior 2007: • PCR / restriction digest - p.A16V, p.N29I, p.R122H • Feb 2007: • fluorescent sequencing exons 2 & 3 • Pathogenic mutations detected - 7/77 (9.1%) • 2/77 unclassified variants • Total sequence changes identified - 9/77 (11.7%)

  12. Idiopathic Chronic Pancreatitis (ICP) • 30% pancreatitis cases - unknown cause • Susceptibility genes: • SPINK1 - Serine Protease Inhibitor Kazal type 1 • CFTR – mild mutations; ICP specific mutations? • ?polygenic model

  13. SPINK1 • Inhibits 20% pancreatic trypsin • p.N34S mutation • 22% ICP patients • 2.5% general population • ?disease modifier

  14. Pyrosequencing assay c.101A>G; p.N34S 9/60 mutation carriers 0/8 CFTR mutations no CF testing on 1 positive case SPINK1 Analysis Normal 2 copies N34S 1 copy N34S

  15. PRSS1 Dosage Analysis • Triplication of trypsinogen locus reported • Le Maréchal et al., Nature Genetics 2006; 38:1372-4 • Fluorescent dosage analysis PRSS1 exon 1 • 40 families (EUROPAC) • Duplication in mother & daughter

  16. Normal Sample Proband PRSS2 exon 4 Normal Sample Proband PRSS1

  17. Pedigree 70 84 78 Awaiting sample DUP 72 nmd nmd 44 51 DUP DUP 21 19 22 nmd DUP DUP

  18. Roger Mountford Victoria Blake DNA Laboratory Staff Chris Grocock Bill Greenhalf John P. Neoptolemos Acknowledgements

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