线粒体DNA1555位点和GJB2基因及SLC26A4基因的诊断方法及临床应用

目的建立常见耳聋基因如线粒体DNA（mtDNA）1555位点、GJB2基因、SLC26A4（Pendren’s syndrome gene，PDS gene）基因突变的临床检测方法。方法来自门诊的散发耳聋患者367例，有母系家族遗传史耳聋患者60例（27个家系），来自聋哑学校的先天性聋患者20例，来自门诊经高分辩CT证实双侧前庭水管扩大患者3例，无感音神经性聋病史的对照个体50例。应用线粒体基因A1555G突变检测试剂盒检测线粒体基因1555位点的突变情况；针对20例语前聋患者进行GJB2全序列分析；针对3例大前庭水管综合征的患者，应用变性高效液相色谱技术进行SLC26A4基因的全部外显子筛查，出现异常波形之外显子行序列分析。结果在26个家系的59例患者和5例散发患者中发现mtDNA A1555G突变；20例先天性聋中发现2例GJB2 235delC纯合突变，酶切加测序发现1例235delC＋299-300delAT复合突变，均为先天性聋的肯定原因，另外2例具有109G-A杂合突变；3例大前庭水管综合征患者的变性高效液相色谱技术筛查均发现包含第7、8外显子的扩增子具有异常波形，测序证实1例为杂合的SLC26A4 G316X突变；另2例为919-2 A-G纯合突变。结论耳聋基因诊断具有显著的临床意义，可操作性强，在不远的将来耳聋的基因诊断可能会正式列为耳科临床检测项目。

Diagnostic methods and clinic application for mtDNA A1555G and GJB2 and SLC26A4 genes in deaf patients

OBJECTIVE: To establish the method of clinic genetic testing for common deaf genes such as mtDNA nt1555, GJB2 gene and SLC26A4 (Pendren's syndrome gene, PDS) gene. METHODS: Three hundred and sixty seven sporadic patients with hearing loss from out-patient department of General Hospital of Chinese People's Liberation Army, 60 patients with history of maternal inherited hearing loss from 27 family, 20 congenital deaf patients from special educational school for deaf and dumb, 3 deaf patients with enlarged vestibular aqueduct (EVA) confirmed by CT scan, 50 control individuals with normal bone conductive hearing were analyzed. The genetic testing kit for mtDNA A1555G mutation was used to detect mtDNA A1555G mutation. The whole gene sequencing were accomplished in 20 congenital deaf patients. In 3 patients with EVA, fragments covering all exons of PDS gene were analyzed by denatured high productive liquid chromatogram and special exons were sequenced when DHPLC showed abnormal wave patterns of amplicons covering these exons. RESULTS: Fifty nine patients from 26 family and 5 sporadic patients were found to carry mtDNA A1555G mutation. Among 20 congenital deaf patients, 2 cases were found to have homozygous GJB2 235 del C mutation, 1 case had compound 235del C and 299-300 del AT mutation. Other 2 cases carried heterozygous 109 A-G mutation. Among 3 patients with EVA, 1 case was found to have heterozygous PDS G316X mutation and other 2 cases had homozygous 919-2 A-G mutation. CONCLUSIONS Genetic testing for deafness is feasible procedure with remarkable clinic significance.