实时荧光定量聚合酶链反应诊断面肩肱型肌营养不良

Genetic diagnosis of facioscapulohumeral muscular dystrophy by real-time fluorescent quantitative polymerase chain reaction

目的建立面肩肱型肌营养不良（FSHD）的实时荧光定量PCR（FQ—PCR）检测方法。方法常规酚-氯仿法抽提基因组DNA，通过EcoRI酶切及琼脂糖凝胶电泳，回收38kb以下DNA作为模板，根据4号染色体上D424序列设计特异的引物和探针，对115例研究对象进行FQ—PCR检测，根据荧光曲线与阳性对照的比较判断结果。结果16例已知EcoRI片段大小的FSHD患者FQ—PCR检测结果为13例阳性，78名健康人检测结果除3例阳性外其余均为阴性，16例经临床诊断的新FSHD患者和5名高危者中分别有15例和3例检测结果为阳性。统计学分析FQ—PCR方法与传统印迹杂交方法（K=0．765，P=0．002）及临床诊断（k=0．844，P=0．000）之间的一致性，结果有统计学意义。结论我们创建了以FQ—PCR技术对FSHD进行基因诊断的新方法。该方法能克服印迹杂交方法费时费力、有放射性污染的缺点，且能较好解决由于4q-10q易位及p13E-11探针结合部位缺失造成传统杂交基因诊断方法欠准确的问题，具有较好的临床应用价值。

Objective To develop a convenient, rapid and specific method using real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） for detection of facioscapulohumeral muscular dystrophy（FSHD） . Methods Genomic DNA was extracted and digested by restricted endonuclease EcoR I, followed by agarose electrophoresis. The DNA （ 〈 38 kb） was retrieved from agarose electrophoretic gels. The primers and probe were designed in D4Z4 gene in chromosome 4. One hundred and fifteen subjects were examined by FQ-PCR using the retrieved DNA （ 〈 38 kb） as a template and the result was analyzed by fluorescent curve comparing with positive control. Results The results by FQ-PCR showed that 13 cases were positive in 16 FSHD cases whose EeoR I fragment sizes were known, 75 cases were negative in 78 cases of normal controls, 15 cases were positive in 16 FSHD cases diagnosed clinically whose EcoR I fragment sizes were unknown, and 3 cases were positive in 5 cases of relatives of FSHD patients. Consistency was checked using Kappa index between the 2 gene diagnostic tests for FSHD （ FQ-PCR test and the traditional Southern blotting test）, and between the 2 diagnostic criterions （gene diagnosis by FQ-PCR and clinical diagnosis）. The results were statistically significant （ K = 0. 765, P = 0. 002 ; K = 0. 844, P = 0. 000）. Condusions A new genetic diagnostic method of FSHD by FQ-PCR was developed, which was more simplified and reliable compared to the time-consuming, radioactive Southern blotting. It could also detect the D4Z4 arrays in cases having deletion of pl3E-11 as well as the interchromosomal exchange between 4q35 and 10q26. The new method of FQ-PCR for FSHD may be extended to utilize clinically in future.