Masamune Atsushi, Kume Kiyoshi, Takagi Yasuhiko, Kikuta Kazuhiro, Satoh Kennichi, Satoh Akihiko, Shimosegawa Tooru
Division of Gastroenterology, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
Pancreas. 2007 May;34(4):423-8. doi: 10.1097/mpa.0b013e3180335fd0.
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.
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.
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.
Splicing mutation might represent a mechanism for SPINK1-associated CP, but the N34S mutation is not associated with alternative splicing.
既往研究表明，慢性胰腺炎（CP）与丝氨酸蛋白酶抑制剂Kazal 1型（SPINK1）基因的突变，尤其是N34S突变之间存在关联。但其潜在分子机制尚不清楚。共分离的内含子突变引起的异常剪接可能起作用，但这一假设尚未得到验证。我们在此检测了携带这些突变的患者中SPINK1基因的信使核糖核酸序列。
从2例携带纯合N34S突变的CP患者手术切除的胰腺以及1例携带杂合[-215G>A; IVS3+2T>C]突变的CP患者的胃活检标本中分离RNA。通过逆转录-聚合酶链反应扩增SPINK1基因的整个编码区，亚克隆并测序。通过实时聚合酶链反应评估野生型SPINK1转录本的水平。
可变剪接与N34S突变无关。另一方面，[-215G>A; IVS3+2T>C]突变导致整个外显子3缺失，胰蛋白酶结合位点位于该外显子。预测这种突变蛋白由63个氨基酸组成：从第30至64位氨基酸序列缺失，阅读框在第65位氨基酸处移位并带有一个新的终止密码子。在携带杂合[-215G>A; IVS3+2T>C]突变的CP患者中，野生型SPINK1转录本的表达降至健康对照的62%。
剪接突变可能是SPINK1相关CP的一种机制，但N34S突变与可变剪接无关。
