在mRNA水平验证SGLT2基因剪切突变的新方法  

A Convenient New Method For Verifying Splice Mutations in SGLT2 mRNA

作　　者：于磊 刘国平 

机构地区：[1]内蒙古自治区人民医院肾内科,内蒙古呼和浩特010017

出　　处：《内蒙古医学杂志》2011年第3期257-260,F0003,共5页Inner Mongolia Medical Journal

基　　金：内蒙古自然科学博士基金(2010BS1102)

摘　　要：目的:由于实验技术的限制,在以往SGLT2基因突变筛查过程中未能对剪切突变在mRNA水平进行验证,因此本研究拟建立一种方便的验证SGLT2基因剪切突变的方法。方法:收集家族性肾性糖尿患者临床及家系资料,55名健康献血员作为对照。聚合酶链反应(PCR)扩增SGLT2基因14个外显子及其周围内含子区和启动子区,PCR产物直接测序法筛选突变。发现可能的剪切突变后,本研究用EB病毒转染B淋巴细胞的方法建立肾性糖尿患者的永生细胞系,提取永生细胞总RNA,应用逆转录-聚合酶链反应(RT-PCR)扩增SGLT2编码的mRNA,经PCR扩增后直接测序的方法分析和验证剪切突变,从而确证剪切突变对mRNA的影响。结果:在家族性肾性糖尿患者中,发现了2个剪切突变位点(IVS 1-16 C>A,IVS 11+1 G>C),通过本研究建立的验证SGLT2基因剪切突变的方法,在SGLT2基因mRNA水平验证了剪切突变对mRNA的影响(IVS 1-16 C>A:外显子3缺失,11+1 G>C:外显子11缺失)。结论:本研究解决了SGLT2基因mRNA在外周血有核细胞中表达量少造成mRNA获取困难的问题,建立了一种方便的验证SGLT2基因剪切突变的方法,为进一步探讨FRG的分子致病机制奠定了基础。Objective：Due to technical constraints,SGLT2 splice mutation has not been verified in mRNA.The present study aimed to establish a convenient procedure to verify splice mutation in FRG patients.Method：Patients were recruited in our renal division because of persistent glucosuria,and the clinical data of patients and their family members were recored.Fifty-five healthy Chinese were served as controls.The entire coding region and adjacent intronic segments of SGLT2 were sequenced in patients and their family members.cDNA was reverse transcribed from total RNA extracted from epstein-barr virus transformational permanent lymphoblastoid cell lines.Splice mutations were detected by cDNA-sequencing.Resuts：Through the method which were established in our study,two splice mutations were successfully verified in SGLT2 mRNA level（IVS 1-16 CA： Del exon3,IVS 11＋1 GC： Del exon11）.Conclusion：In this study,a convenient methods wildly used for verifying splice mutation in SGLT2 mRNA was established.It provides a valuable clue for further investigations of SGLT2 molecular mechanism.

关 键 词：家族性肾性糖尿 SGLT2基因 剪切突变 

分 类 号：R587.1[医药卫生—内分泌] R691[医药卫生—内科学]