摘要
目的通过对候选基因SH3GL1的核酸序列进行比对分析,确定其是否为青少年特发性脊柱侧凸(AIS)的致病基因。方法采取以家系为基础的"病例同胞对"研究方案,提取基因组DNA,根据候选基因SH3GL1核酸序列,以10个外显子为重点设计引物对,进行PCR扩增、克隆和测序,用GeneTool软件对外显子测序结果进行序列比对分析,判断是否存在碱基变异,并进行蛋白质结构预测分析。结果候选基因SH3GL1中的10个外显子成功地在AIS"同胞对"基因组DNA中得到扩增和克隆,且序列测定均取得阳性结果。通过比对分析,在SH3GL1的10个外显子中共发现12处碱基变异,分别位于第2(3处)、4、5(4处)、6、8和10(2处,非编码区)六个外显子上。其中先证者mRNA第515位碱基如果为T,则形成终止密码子,导致蛋白阅读框发生改变,蛋白质序列预测分析显示编码截短蛋白,从而影响蛋白质的一级结构。结论SH3GL1是AIS的致病基因之一。
Objective To determine whether the candidate gene SH3GL1 is the pathogenic gene of adolescent idiopathic scoliosis （AIS） by the comparative study on sequence alignment of its exons. Methods The affected sib-pair （ASP） method based on family constellation was adopted. A sib-pair was chosen, blood samples were drawn, and genome DNA was extracted. Primer pairs were designed with 10 exons as emphasis according to nueleotide sequence of SH3GL1, and then PCR amplification, cloning, and sequencing were performed. The exons sequencing results were comparatively analyzed between the sib pairs, and between the sib pair and NCBI with Gene- Tool software to find base variation, and the prediction analysis of protein structure was performed. Results All the 10 exons of candidate gene SH3GL1 were successfully amplified and cloned, and positive results were obtained in sequencing. Sequence comparative analysis showed there were 12 base mutations in 10 exons which located in the 2nd （3 mutations）, 4th, 5th （4 mutations）, 6th, 8th, and 10th （2 mutations, noneoding region） exon. If the 515th base of mRNA in proband mutated to T, the stop codon （TAG） would be formed and result in alternation of open reading frame, and the prediction of the sequence of proteinum showed brachytmema proteinum which could cause changes of primary structure. Conclusion SH3GL1 is one of the pathogenic genes to AIS.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第7期848-850,共3页
Medical Journal of Chinese People's Liberation Army
