应用脉冲场电泳技术研究中国人4q35与10q26亚端粒区EcoRⅠ片段的结构多态性

Study on EcoRⅠ fragment polymorphism of the subtelomeric domains within 4q35 and 10q26 with pulsed field gel electrophoresis in the Chinese population

目的研究中国人4q35与10q26同源性EcoR片段的结构多态性,探讨该区域的可塑性、易位和体细胞嵌合现象及其与D4Z4重复单位缺失的关系。方法研究对象包括110名无血缘关系的健康成年人。低熔点胶包埋法抽提基因组DNA。同一样品分别进行EcoR酶切和EcoR/Bln双酶切,脉冲场电泳分离,p13E-11探针Southern杂交,曲线拟合法计算分析EcoR片段的长度,SPSS11.0统计软件分析数据。结果77.3%(85/110)的个体为标准构型,4q35EcoR片段的长度均值为(87.9±3.3)kb,中位数为78.5kb;10q26同源性片段的长度均值为(90.1±4.1)kb,中位数为73.0kb;经t检验,两者差异无显著性(P>0.05)。19.1%(21/110)的个体检测到易位构型,其中4q→10q易位和10q→4q易位分别为9.1%(10/110)和10.0%(11/110),经卡方检验,两种易位形式的频率基本相等(χ2=0.053,P>0.05)。3.6%(4/110)的个体检测到体细胞嵌合片段。此外,14.5%(16/110)的个体检测到小于35kb的10q26EcoR短片段。结论正常中国人4q35和10q26EcoR片段具有多态性和动态性变化特征,两者具有高度同源性。4q35-10q26易位是导致4q35区不稳定和D4Z4缺失的重要因素,体细胞嵌合现象的出现提示4q与10q区D4Z4的互换重组可能发生于有丝分裂过程中。

Objective To elucidate the structural polymorphism of EcoRⅠ fragment within chromosomes 4q35 and 10q26 in the Chinese population and investigate the relationship of plasticity, translocation and somatic mosaicism in these domains with deletion of D4Z4 repeated units. Methods One hundred and ten unrelated healthy individuals from a random Chinese population were investigated. The genomic DNA was extracted from peripheral blood lymphocytes according to the specific procedure designed to minimize DNA shearing, then digested with EcoRⅠ or double digested with EcoRⅠ and BlnⅠ. The cleaved DNA was separated by pulsed field gel electrophoresis (PFGE) and Southern blotted with the probe p13E 11. The sizes of EcoRⅠ fragments were calculated by 'curve fitting' according to the MidRange PFG marker and the alleles were assigned to their respective chromosomes based on their BlnⅠ sensitivity. Data were analyzed using a commercially available statistical package (Version 11.0 SPSS). Results Seventy seven point three per cent (85/110) of the unrelated healthy individuals displayed a standard configuration distribution. The mean and median of 4q35 repeat arrays are (87.9±3.3) kb and 78.5 kb respectively, whereas the mean and median of 10q26 homologous arrays are (90.1±4.1) kb and 73.0 kb. Repeat size distributions between both of them were of no significance according to the t test ( P > 0 05). 19.1% (21/110) of the individuals displayed a translocation repeat array configuration on chromosomes 4 and 10. No significant difference was detected between 4q→10q translocation and 10q→4q translocation according to Chi square test (χ 2=0.053, P >0.05). Somatic mosaicism was observed in 3 6% (4/110) of the subjects and less than 35 kb 10 type array was found in 14.5% (16/110) of the individuals. Conclusion The structural polymorphism and dynamic behaviors of EcoRⅠ fragments within 4q35 and 10q26 were demonstrated in this study using PFGE. The occurrence of frequent translocations and somatic mosaicism between 4q35 and 10q26 subtelomeric domains in the Chinese population further confirmed that mitotic interchromosomal gene conversion or translocation might be a major mechanism relating to the deletion of D4Z4 units.