上海、江苏、浙江血友病甲基因突变的检测

Molecular characterization of genetic defects in hemophilia in Shanghai

目的：对血友病甲进行基因诊断。方法：采用多聚酶链反应（PCR），变性梯度凝胶电泳（DGGE）和DNA测序等方法对上海地区血友病甲基因突变进行检测。50例无内含子22倒位的血友病甲患者（其中重型24例，中型9例，轻型17例，无亲缘关系患者45例）先用PCR扩增基因组DNA，扩增范围包括所有外显子［编码B区的外显子14（氨基酸783～1598）部分除外，但包含凝血酶切割位点AA740和1689］及其侧翼内含子序列，然后对扩增片段进行DGGE分析，发现异常条带则进行DNA测序。结果：11例无亲缘关系患者中检出突变11种，其中无义突变5种，均为重型；错义突变5种，均为轻、中型；小缺失1例。其中，466Lys（AAG）－Thr（ACG），719Tyr（TAC）－Stop（TAG）及312Ile（ATC）－xxC为新发现的突变。结论：除内含子22倒位外，绝大部分血友病甲基因突变为单碱基置换导致的点突变。有亲缘关系的患者都有相同的基因突变。基因突变与临床表现基本相符。

Objective:To detect gene mutations in hemophilia A in Shanghai. Methods:PCR,denatur ing gradient gel electrophoresis (DGGE) atd DNA sequencing were used. Fifty Chinese cases of hemophilia A without intron 22 inversion, including 24 severe, 9 moderate and 17 mild cases, were screened. Genomic DNA was amplified using GC-clamped primers covering all the exons and flanking in tron regions,excluding the middle portion of extron 14 encoding the B domain,but including the thrombin cleavage sites at AA740 and 1689. The amplified GC-clamped PCR fragments were then electrophoresed on DGGE. The abnormal bands were sequenced. Results: Eleven different mutations were identified, in cluding 5 nonsense mutations, 5 missense mutations and one small deletion. Among them, 3 mutations,466Lys (AAG )-Thr (ACG ), 719Tyr (TAC )-Stop (TAG ) and 312Ile (ATC )-xxC have not been reported be fore. Conclusion: Apart from intron 22 inversion,most gene mutations in hemophilia A were point mu tations resulted from single base substitution. Generally the genetic defects correspond to the c1inical man ifestations.