Genetic diagnosis of facioscapulohumeral muscular dystrophy by real-time fluorescent quantitative polymerase chain reaction
10.3760/cma.j.issn.1006-7876.2009.08.013
- VernacularTitle:实时荧光定量聚合酶链反应诊断面肩肱型肌营养不良
- Author:
Quanxi SU
;
Wanyi LI
;
Cheng ZHANG
;
Fu XIONG
;
Benchang SHEN
;
Mingfan HONG
;
Xilin LU
- Publication Type:Journal Article
- Keywords:
Muscular dystrophy;
facioscapulohumeral;
Polymerase chain reaction;
Translocation;
genetic
- From:
Chinese Journal of Neurology
2009;42(8):555-558
- CountryChina
- Language:Chinese
-
Abstract:
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 D4ZA 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 EcoR 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 (κ = 0. 765, P = 0. 002 ; κ = 0. 844, P = 0. 000). Conclusions 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 p13E-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.
