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

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

Diagnostic service PRSS1 –Fluorescent sequencing exons SPINK1 –p.N34S mutation (pyrosequencing) CFTR –28 mutations (Elucigene CFHT) EUROPAC:

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”

PRSS1PRSS2TRY5TRY7TRY6TCR 4kb initial PCR product

N29IL41LC48C p.N29I c.86A>T p.C48C c.144T>C 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 9/60 mutation carriers –0/8 CFTR mutations no CF testing on 1 positive case Normal 2 copies N34S 1 copy N34S

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

Normal Sample Proband PRSS2 exon 4 Proband Normal Sample PRSS1

Pedigree DUP Awaiting sample nmd

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