实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.

Application of real-time quantitative PCR in testing repeat expansion in dystrophia myotonica 1

Objective To identify the pathogenic mutation for a Chinese family with dystrophy myotonica ( DM) and explore the potency of real-time quantitative PCR in detecting the trinucleotide repeat expansions causing DM1. Methods Genomic DNA was extracted from peripheral blood samples of family members including 3 patients. Routine PCR were used to amplify the CTG repeats in the DMPK gene and the CCTG repeats in the CNBP gene, the expansion of which can cause DM1 and DM2 respectively. Real-time quantitative PCR were used to determine the relative copy number of these two repeat regions. Furthermore, microsatellite markers from DM1 and DM2 loci were analyzed to verify the diagnosis. Results when subjected to agarose gel elec-trophoresis, the routine PCR product of the DM1 CTG repeats showed that all three patients have a 250 bp band and a smear over 2 kb, while the normal family members had only the 250 bp band. However, with the PCR products of the DM2 CCTG repeat, all family members, including three patients, showed just a band of about 250 bp. The quantitative PCR analysis for the CTG repeat showed that all three patients had a relative copy number (RCN) of about 0.5, but all normal family members have a RCN of about 1. For the CCTG repeat, the RCN is about 1 for both the patients and the normal members. Allele sharing was detected for all markers spanning the DM1 locus, but not for most markers from the DM2 locus, suggesting that the DM1 be the disease locus in this family. Conclusion The pathogenic mutation in the DM family is the CTG repeat expansions in DM1 locus. Real-time quantitative PCR can help to establish the DM1 diagnosis by detecting the copy number losing, which is caused by the amplification failure of large CTG repeat allele.