荧光PCR法在非综合征型遗传性耳聋基因诊断中的应用研究

Application of fluorescence PCR technique in gene diagnosis of non-syndromic hearing loss

目的分析GJB2、SLC26A4和mt DNA12SrRNA基因热点突变在非综合征型遗传性耳聋人群中的突变谱和突变频率。方法采用荧光PCR法,针对本院收集的126例非综合征型耳聋患者进行中国人群常见的3个耳聋致病基因GJB2、SLC26A4和mt DNA 12SrRNA的10个热点突变的筛查,分析总结突变数据。阳性结果进一步采用直接测序法进行验证。结果应用荧光PCR技术在126例非综合征型耳聋(non syndromic hearing loss,NSHL)患者中检测出携带基因突变的患者31例,阳性率为24.6%(31/126),其中GJB2双等位基因突变、SLC26A4双等位基因突变和12SrRNA的均质突变分别占该人群分子病因的6.35%(8/126)、2.33%(3/126)和3.17%(4/126)。此外,IVS7-2A>G单等位基因突变的检出率高达11.11%(14/126)。GJB2 c.235del C和SLC26A4IVS7-2A>G是本研究中最为常见的热点突变。进一步采用直接测序法验证阳性位点,其结果与荧光PCR法一致。结论 GJB2双等位基因突变是本研究人群最为常见的分子致病因素,其次为SLC26A4双等位基因突变和12SrRNA的均质突变。GJB2 c.235del C和SLC26A4 IVS7-2A>G是本研究中最为常见的热点突变。

Objective To analyze the mutation frequency and spectrum of GJB2, SLC26A4 and mtDNA12SrRNA gene hotspot mutations in non-syndromic hearing loss （NSHL） population. Methods Mutation screening was performed on 126 cases with NSHL collected in our hospital by fluorescence PCR technique. Ten common mutations of three deafnessrelated genes GJB2, mtDNA12srRNA and SLC26A4 were included. Then the mutation data were analyzed and summarized, and the positive results were further validated by Sanger sequencing. Results Allelic variants were observed in 31 of the 126 patients with a positive rate of 24.6% （31/126）. The incidences of biallelic mutations in GJB2, SLC26A4 and homogeneous mutations in 12SrRNA were 6.35% （8/126）, 2.33% （3/126） and 3.17% （4/126） respectively. In addition, the prevalence of IVS7 - 2A 〉 G single allele mutation was 11.11% （ 14/126 ）. GJB2 c. 235delC and SLC26A4 IVS7 -2A 〉 G were the most common hotspot mutations in our study. The positive results further validated by Sanger sequencing showed consistent with those by fluorescent PCR method. Conclusion In the NSHL population studied, GJB2 biallelic mutations are the most common molecular risk factors followed by SLC26A4 biallelic mutations and 12SrRNA homogeneous mutations. GJB2 c. 235delC and SLC26A4 IVS7 -2A 〉 G are the most common hotspot mutations,