N34S mutation in the SPINK1 gene is not associated with alternative splicing

Atsushi Masamune, Kiyoshi Kume, Yasuhiko Takagi, Kazuhiro Kikuta, Kennichi Satoh, Akihiko Satoh, Tooru Shimosegawa

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

OBJECTIVES: Previous studies have shown an association between chronic pancreatitis (CP) and mutations, especially the N34S mutation, in the serine protease inhibitor Kazal type 1 (SPINK1) gene. But the underlying molecular mechanisms are unknown. The aberrant splicing caused by the cosegregating intronic mutations might play a role, but this hypothesis has not been tested. We here examined the messenger RNA sequences of the SPINK1 gene in patients carrying the mutations. METHODS: RNA was isolated from the surgically resected pancreas of 2 CP patients carrying the homozygous N34S mutation and from the gastric biopsy specimen of a CP patient carrying the heterozygous [-215G>A; IVS3+2T>C] mutation. The entire coding region of the SPINK1 gene was amplified by reverse transcription-polymerase chain reaction, subcloned, and sequenced. The level of the wild-type SPINK1 transcript was assessed by real-time polymerase chain reaction. RESULTS: Alternative splicing was not associated with the N34S mutation. On the other hand, the [-215G>A; IVS3+2T>C] mutation caused skipping of whole exon 3, where the trypsin binding site is located. This mutated protein was predicted to consist of 63 amino acids: deletion of amino acid sequence from residues 30 to 64 and shifting of reading frame at amino acid 65 with a novel stop codon. The expression of the wild-type SPINK1 transcript was decreased to 62% of the healthy control in the CP patient carrying the heterozygous [-215G>A; IVS3+2T>C] mutation. CONCLUSIONS: Splicing mutation might represent a mechanism for SPINK1-associated CP, but the N34S mutation is not associated with alternative splicing.

Original languageEnglish
Pages (from-to)423-428
Number of pages6
JournalPancreas
Volume34
Issue number4
DOIs
Publication statusPublished - 2007 May

Keywords

  • Alternative splicing
  • Exon skipping
  • Pancreatic secretory trypsin inhibitor
  • Pancreatitis
  • Serine protease inhibitor Kazal type 1
  • Trypsin

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Hepatology
  • Endocrinology

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