Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.
Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis.
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DOI：
10.1136/gut.2008.164947
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发表时间：
2009-04
期刊：
影响因子：
24.5
通讯作者：
Sahin-Tóth M
中科院分区：
文献类型：
--
作者：
Kereszturi E;Király O;Sahin-Tóth M
丝氨酸蛋白酶抑制剂Kazal 1型（SPINK1）基因的两种常见单倍型已被证明会增加慢性胰腺炎的风险。一个单倍型包括c.101A>G（p.N34S）错义变异和四个内含子改变已在世界范围内发现，而第二个单倍型包括c.−215G>A启动子变异和c.194+2T>C内含子变异在日本经常被观察到。在本研究中，我们研究的功能意义的内含子变异的致病性SPINK1单倍型，通过利用minigene，其中含有单独的内含子放置在适当的情况下的全长SPINK1 cDNA。用SPINK1小基因转染的细胞分泌活性胰蛋白酶抑制剂，从而允许同时评估突变对转录、剪接、翻译和分泌的影响。我们发现，c.194+2T>C内含子的改变在mRNA水平上消除了SPINK 1的表达，随之而来的是抑制剂分泌的损失，而p.N34S相关的内含子变体没有可检测到的功能效应。与以前的研究一起，结果表明，p.N34S相关单倍型内的所有已知变体在功能上是无害的，这表明该单倍型内尚未鉴定的变体是致病作用的原因。c.194+2T>C变异体对SPINK1表达的显著负面影响支持了SPINK1变异体通过降低保护性胰蛋白酶抑制剂水平而增加慢性胰腺炎风险的观点。
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 we examined the functional significance of the intronic variants in the pathogenic SPINK1 haplotypes by utilizing minigenes, which harbor 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. We 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 a 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.
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影响因子：
3.5
作者：
Kaneko, K;Nagasaki, Y;Horii, A
通讯作者：
Horii, A
影响因子：
2.9
作者：
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通讯作者：
Kammesheidt, Anja
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3.8
作者：
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Ferec, Claude
DOI：
10.1111/j.1440-1746.2007.05291.x
发表时间：
2008-03-01
影响因子：
4.1
作者：
Shimosegawa, Tooru;Kume, Kiyoshi;Masamune, Atsushi
通讯作者：
Masamune, Atsushi
影响因子：
6.3
作者：
Masamune, Atsushi;Kume, Kiyoshi;Shimosegawa, Tooru
通讯作者：
Shimosegawa, Tooru