应用双酶切/Southern杂交方法诊断面肩肱型肌营养不良

Diagnosis of facioscapulohumeral muscular dystrophy using double enzyme digestion associated Southern blotting method

目的 探讨面肩肱型肌营养不良 (FSHD)的基因诊断方法。方法 抽取我院 1 997～2 0 0 0年收治的 37例FSHD患者的静脉血 ,常规抽提gDNA ,以EcoRI/HindⅢ、EcoRI/BlnⅠ分别酶切gDNA ,0 6 %琼脂糖凝胶电泳分离 ,p1 3E 1 1探针Southern杂交 ,应用ImageMasterTotalLabv1 1 1分析软件判断杂交片段大小。结果 正常对照只检测到 1个 4q35来源的、大于 33kb的EcoRI +HindⅢ /p1 3E 1 1片段 ,而所有FSHD患者中有 33例检测到 2个 4q35来源的EcoRI +HindⅢ /p1 3E 1 1片段 ,其中包含 1个小于 33kb的小片段 ;3例患者中可检测到 2个小于 33kb的片段 ,但其中 1个来自 1 0q2 6 ;另1例散发性患者中检测到 3个 4q35 型片段 ,且其中 2个小于 33kb。在 1名无症状 9岁男孩中检测到1个与其患病父亲一致的小片段 ,提示为症状前患者。结论 双酶切 /Southern杂交的方法可用于中国人面肩肱型肌营养不良患者的基因诊断及症状前诊断。本文结果首次提示我国FSHD患者中也存在 4q 1

Objective To develop an operational gene diagnosis method for Chinese Facioscapulohumeral muscular dystrophy (FSHD) patients Methods Genomic DNAwas double digested with restriction enzymes EcoRⅠ/HindⅢ and EcoRI/BlnI,respectively The digested fragments were separated on a 0 6% agarose gel After transferred to a Nytran SuperCharge Membrane, the fragmented DNAs were hybridized with the probe p13E 11 The hybridizing fragments were analyzed by the software ImageMaster Total Lab v1 11 and the size of each band was then given Results Only a 4q35 EcoRI+HindIII/P13E 11 fragment larger than 33 kb was detected in each of the controls Two fragments were detected in each of the 33 FSHD patients, one of which was smaller than 33 kb Although there was also presence of two small alleles in the 3 other FSHD cases, either of them turned out to be 10q26 derived owing to its BlnI sensitivity Interestingly, we found a sporadic patient who carried three 4q35 type fragments and, strikingly, two of them were smaller than 33 kb In the analysis of FSHD family members, a 9 year old boy with no clinical signs was found to share the small fragment with his affected father, indicating that he may be a pre symptomatic patient Conclusion The double digestion associated Southern blotting method we developed can be applied to both the diagnosis of FSHD patients and the prediction of pre symptomatic patients Furthermore, by the gene detection using this method, we first got the evidence of translocation between 4q and 10q in Chinese FSHD patients, which may be helpful to the elucidation of the pathogenesis of FSHD