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Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.

ABSTRACT: Two common haplotypes of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been shown to increase the risk for chronic pancreatitis. A haplotype comprising the c.101A>G (p.N34S) missense variant and four intronic alterations has been found worldwide, whereas a second haplotype consisting of the c.-215G>A promoter variant and the c.194+2T>C intronic alteration has been observed frequently in Japan.In the present study, the functional significance of the intronic variants in the pathogenic SPINK1 haplotypes was examined by utilising minigenes, which harbour individual introns placed in the appropriate context of the full-length SPINK1 cDNA. Cells transfected with the SPINK1 minigenes secrete active trypsin inhibitor, thereby allowing evaluation of mutational effects simultaneously on transcription, splicing, translation and secretion.It was found that the c.194+2T>C intronic alteration abolished SPINK1 expression at the mRNA level, with consequent loss of inhibitor secretion, whereas the p.N34S-associated intronic variants had no detectable functional effect.Taken together with previous studies, the results indicate that all known variants within the p.N34S-associated haplotype are functionally innocuous, suggesting that an as yet unidentified variant within this haplotype is responsible for the pathogenic effect. The marked negative impact of the c.194+2T>C variant on SPINK1 expression supports the notion that SPINK1 variants increase the risk of chronic pancreatitis by diminishing protective trypsin inhibitor levels.

SUBMITTER: Kereszturi E

PROVIDER: S-EPMC2677899 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.

Kereszturi E E Király O O Sahin-Tóth M M

Gut 20081031 4

<h4>Background and aims</h4>Two common haplotypes of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been shown to increase the risk for chronic pancreatitis. A haplotype comprising the c.101A>G (p.N34S) missense variant and four intronic alterations has been found worldwide, whereas a second haplotype consisting of the c.-215G>A promoter variant and the c.194+2T>C intronic alteration has been observed frequently in Japan.<h4>Methods</h4>In the present study, the functional signifi ...[more]

PMID: 18978175

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Project description:BackgroundThe clinical significance of SPINK1 intronic variants in chronic pancreatitis has been previously assessed by various approaches including a cell culture-based full-length gene assay. A close correlation between the results of this assay and in silico splicing prediction was apparent. However, until now, a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking. Herein, we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants.ResultsWe confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants. We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants. The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese chronic pancreatitis patients by sequencing the entire intronic sequence of the SPINK1 gene. The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients. Employing a minor allele frequency of > 5% as a population frequency filter, 6 of the 16 deep intronic variants were immediately classified as benign. In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant, c.194 + 5G > A, was likely to be of functional significance. Employing the cell culture-based full-length gene assay, we functionally analyzed c.194 + 5G > A, together with seven predicted non-functional variants, thereby validating their predicted effects on splicing in all cases.ConclusionsWe demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants. Based upon these findings, we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.

Dataset Information

Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.

Publications

Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.

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