Transcript 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
• 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