Detection of a large genomic deletion in the pancreatic secretory trypsin inhibitor (SPINK1) gene

Abstract

Mutations and polymorphisms in the SPINK1 gene, which encodes trypsin's physiological inhibitor, pancreatic secretory trypsin inhibitor, have been found to be associated with chronic pancreatitis. However, to date, all currently reported SPINK1 variants are either single-nucleotide substitutions or microinsertions/deletions. It is possible that large genomic rearrangements at this locus may underlie certain cases of chronic pancreatitis. However, such events, if indeed they exist, may have been overlooked by conventional PCR-based techniques. Here we attempted to screen all four exons as well as the promoter region of the SPINK1 gene for large genomic deletions by means of quantitative high-performance liquid chromatography analysis. Of the 47 pancreatitis families (not carrying any known PRSS1, SPINK1 and CFTR variants/mutations after screening the coding regions by our previously established denaturing highperformance liquid chromatography methods), one family was suggested to carry a large genomic deletion in the SPINK1 gene. The aberrant chromosomal junction was encapsulated by long-range PCR and the breakpoints were determined by direct sequencing of the rearranged fragment. A 2-bp short direct repeat was present at the deletion breakpoints; this simple deletion (c.1 -320_c.55 þ 961del1336 bp) can thus in principle be explained by replication slippage. Identification of this lesion has not only expanded the SPINK1 mutational spectrum but also served to identify a novel mutational mechanism causing chronic pancreatitis.

Signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor (SPINK1) cause autosomal dominant hereditary pancreatitis

“…This is the first clear genetic-biochemical demonstration that severe loss-of-function SPINK1 alterations are associated with a high risk of CP; therefore, these alterations should be regarded as disease-causing rather than diseasemodifiers. Potential examples of other severe SPINK1 alterations in association with hereditary pancreatitis have been described previously [Witt et al, 2000;Le Maréchal et al, 2004;Masson et al, 2006]. The results presented here support the notion that this group of SPINK1 alterations should be classified as ''severe'' and should be differentiated from ''mild'' alterations, particularly from the relatively frequent, archetypal N34S variant [Witt et al, 2001a;Le Maréchal et al, 2004;Masson et al, 2006].…”

“…Potential examples of other severe SPINK1 alterations in association with hereditary pancreatitis have been described previously [Witt et al, 2000;Le Maréchal et al, 2004;Masson et al, 2006]. The results presented here support the notion that this group of SPINK1 alterations should be classified as ''severe'' and should be differentiated from ''mild'' alterations, particularly from the relatively frequent, archetypal N34S variant [Witt et al, 2001a;Le Maréchal et al, 2004;Masson et al, 2006]. Mild alterations confer a smaller risk of pancreatitis, are typically found in association with idiopathic pancreatitis, and additional genetic and environmental risk factors are believed to influence their expression to a significant degree.…”

Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene

“…12 Last, a diverse range of variations including apparent 'loss of function' mutations (ie c.2T4C (p.M1? ), 13 c.98_99insA, 14 c.27delC, 15 c.87 þ 1G4A, 15 c.194 þ 2T4C, 13 and a large genomic deletion 16 ) in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor (PSTI); MIM no. 167 790) have also been identified in subjects with chronic pancreatitis (for an up to date list of variations, see http://www.unileipzig.de/pancreasmutation/db.html).…”

CFTR Gene Mutation in Patients with Apparently Idiopathic Pancreatitis: Lack of Phenotype-Genotype Correlation

“…Screening of the coding region and flanking intronic sequences of PRSS1 and SPINK1 genes was performed in the C Ferec’s unit by DGGE and by quantitative high-performance liquid chromatography analysis for point mutations and for large genomic deletions, respectively, as previously described [39,40]. The complete method is described elsewhere [41].…”