N34S mutation in the SPINK1 gene is not associated with alternative splicing
N34S mutation in the SPINK1 gene is not associated with alternative splicing
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DOI：
10.1097/mpa.0b013e3180335fd0
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发表时间：
2007-05-01
期刊：
影响因子：
2.9
通讯作者：
Shimosegawa, Tooru
中科院分区：
文献类型：
--
作者：
Masamune, Atsushi;Kume, Kiyoshi;Shimosegawa, Tooru
目的：先前的研究表明慢性胰腺炎（CP）与丝氨酸蛋白酶抑制剂Kazal 1型（SPINK 1）基因突变，特别是N34 S突变之间存在关联。但其潜在的分子机制尚不清楚。由共分离基因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。我们在这里研究了携带突变的患者的SPINK 1基因的信使RNA序列。方法：从2例携带纯合N34 S突变的CP患者的手术切除的胰腺和1例携带杂合[-215G > A; IVS 3 + 2 T> 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SPINK 1基因的整个编码区，亚克隆并测序。野生型SPINK 1转录水平通过实时聚合酶链反应进行评估。结果：选择性剪接与N34 S突变无关。另一方面，[-215G > A; IVS 3 + 2 T> C]突变导致胰蛋白酶结合位点所在的整个外显子3的跳跃。预测该突变蛋白由63个氨基酸组成：从残基30至64的氨基酸序列的缺失和在氨基酸65处的阅读框用新的终止密码子移位。在携带杂合子[-215G > A; IVS 3 + 2 T> C]突变的CP患者中，野生型SPINK 1转录本的表达降低至健康对照的62%。结论剪接突变可能代表SPINK 1相关CP的机制，但N34 S突变与选择性剪接无关。
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 intromic 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.