Diagnostic Testing for Familial Pancreatitis

Emma McCarthyClinical 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 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”

PRSS1 PRSS2TRY5 TRY7TRY6 TCRTCR

4kb initial PCR product

N29I L41L C48C

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; 38:1372-4

• 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

DUP

DUP

DUPDUP

Awaiting sample

nmdnmd

nmd

44

72

51

21 19

70

22

7884

Acknowledgements

• Roger Mountford• Victoria Blake• DNA Laboratory Staff

• Chris Grocock• Bill Greenhalf• John P. Neoptolemos