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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 • Inflammatory disease – – – – Severe abdominal pain Nausea Vomiting fever Acute or chronic • Premature activation digestive enzymes • Common triggers – Excessive alcohol consumption – Smoking – Gallstones TRYPSINOGEN activation TRYPSIN premature activation enzymes AUTODIGESTION multiple attacks CHRONIC PANCREATITIS irreversible scarring PERMANENT LOSS OF FUNCTION advanced stages DIABETES / PANCREATIC CANCER normal pancreas inherited pancreatitis Trypsinogen Trypsinogen Mesotrypsin Trypsin SPINK1 AP Trypsin ? Mesotrypsin Trypsin SPINK1 Trypsin AP ? CFTR enzyme cascade autodigestion CFTR enzyme cascade autodigestion pancreatitis Reproduced from: Rosendahl et al. Orphanet Journal of Rare Diseases 2007 2:1 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 Hereditary Pancreatitis (HP) • Autosomal dominant • Reduced penetrance • Childhood: – recurrent episodes acute pancreatitis • Adulthood: – chronic pancreatitis • Mutations of cationic trypsinogen gene (PRSS1) Cationic Trypsinogen • PRSS1 gene (protease serine 1) • Trypsin – Activates digestive enzymes – Self regulating • Autocatalytic activation • Feedback inhibition - cleaves at Arg122 • R122H mutation - “super trypsin” TCR PRSS1 TRY5 TRY6 TRY7 4kb initial PCR product PRSS2 TCR N29I L41L p.N29I c.86A>T p.C48C c.144T>C C48C p.L41L c.121C>T p.N54S c.161A>G p.V59V c.177A>G p.G62G c.186C>T 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%) 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 SPINK1 • Inhibits 20% pancreatic trypsin • p.N34S mutation – 22% ICP patients – 2.5% general population – ?disease modifier SPINK1 Analysis • Pyrosequencing assay – c.101A>G; p.N34S Normal • 9/60 mutation carriers – 0/8 CFTR mutations • no CF testing on 1 positive case 2 copies N34S 1 copy N34S 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 Normal Sample Proband PRSS2 exon 4 Normal Sample Proband PRSS1 Pedigree 70 Awaiting sample 21 DUP 84 DUP 72 nmd 44 51 DUP DUP 19 nmd 22 DUP 78 nmd Acknowledgements • Roger Mountford • Victoria Blake • DNA Laboratory Staff • Chris Grocock • Bill Greenhalf • John P. Neoptolemos
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