使用经固定细胞的DNA检测Prader-Willi综合征

doi:10.3969/j.issn.1673-8640.2017.011.015

摘要/Abstract

摘要：

目的探讨以外周血染色体检查或荧光原位杂交（FISH）检查剩余的固定细胞作为DNA来源,采用甲基化特异性（MS）聚合酶链反应（PCR）与甲基化特异性多重连接探针扩增（MS-MLPA）方法进行Prader-Willi综合征（PWS）分子检测的可行性,为采用少量全血液样本同时开展染色体、FISH和分子检测奠定基础。方法将13例受试样本分为正常对照组（3例）、确诊组（2例）和疑似组（8例）3个组。将所有的冻存固定细胞进行离心,弃固定液,采用基因组DNA提取试剂盒进行DNA提取,13例PWS样本同时采用MS-PCR与MS-MLPA进行检测。结果 13例DNA样本的A₂₆₀/A₂₈₀比值平均为1.94±0.1、浓度平均为（64.0±10） ng/ μL、片段大小均在10 kb以上;MS-PCR检测发现正常对照组父源与母源条带均存在,确诊组仅存在母源条带、父源条带缺失,疑似组父源与母源条带均存在;MS-MLPA结果表明除确诊组2例提示为父源缺失型PWS外,其余2个组结果都提示为正常。结论来源于固定细胞的基因组DNA在纯度、浓度及完整性方面均可满足MS-PCR以及MS-MLPA检测的实验要求,可用于PWS临床分子检测及其相关实验。

关键词: Prader-Willi综合征, DNA提取, 甲基化特异性聚合酶链反应, 甲基化特异性多重连接探针扩增, 固定细胞

Abstract:

Objective To investigate the feasibility of molecular determination for Prader-Willi syndrome（PWS） through methylation-specific（MS）-polymerase chain reaction（PCR） and MS-multiplex ligation-dependent probe amplification（MLPA）,with remaining fixed cells from peripheral blood chromosome examination or fluorescence in situ hybridization（FISH） being treated as the source of DNA,so as to lay a foundation for carrying out chromosomal,FISH and molecular determination simultaneously with a little amount of whole blood sample. Methods A total of 13 samples were collected and classified into normal control group（3 cases）,confirmed group（2 cases） and suspected group（8 cases）. All frozen fixed cells were centrifuged,the stationary liquid was discarded,DNA extraction was conducted with genome DNA extraction kit,and MS-PCR combined with MS-MLPA was performed to determine PWS in the 13 samples. Results The average A₂₆₀/A₂₈₀ ratio of the 13 extracted DNA samples was 1.94±0.1,the average concentration was （64.0±10） ng/μL,and all the fragment sizes were >10 kb. The results of MS-PCR indicated that both paternal and maternal bands existed in normal control group and suspected group,while only maternal band existed in confirmed group. The results of MS-MLPA demonstrated that the results were normal except for 2 cases in confirmed group,which showed paternal deletion type PWS. Conclusions Genome DNA from fixed cells satisfies the experimental requirements of MS-PCR and MS-MLPA in the aspects of purity,concentration and integrity,and it could be used for clinical molecular determination for PWS as well as relevant experiments.

Key words: Prader-Willi syndrome, DNA extraction, Methylation-specific-polymerase chain reaction, Methylation-specific-multiplex ligation-dependent probe amplification, Fixed cell

中图分类号:

R446.1

杜成坎, 孙恒娟, 兰小平, 杨永臣, 孙嘉鹏, 许无恨, 张泓. 使用经固定细胞的DNA检测Prader-Willi综合征[J]. 检验医学, 2017, 32(11): 1008-1012.

DU Chengkan, SUN Hengjuan, LAN Xiaoping, YANG Yongchen, SUN Jiapeng, XU Wuhen, ZHANG Hong. Fixed cell DNA for determining Prader-Willi syndrome[J]. Laboratory Medicine, 2017, 32(11): 1008-1012.

图/表 4

图1 使用固定细胞所提取的基因组DNA凝胶电泳检测其完整性电泳结果注：M为Marker;1为使用正常血液样本提取的基因组DNA;2~14为使用固定细胞所提取的基因组DNA

图2 MS-PCR电泳结果注：M为Marker;1为正常血液样本对照;2为PWS患者血液样本对照;3~5为正常对照组;6~7为确诊组;8~15为疑似组

图3 MS-MLPA分析结果注：(a)、(b)为正常对照分析结果;(c)、(d)为确诊组分析结果;疑似组MS-MLPA分析结果同(a)、(b),PWS患者分析结果同(c)、(d)

表1 MS-MLPA检查结果

病例	甲基化检测结果	拷贝数检测结果
PWS	存在甲基化	缺失
AS	不存在甲基化	缺失
母源单亲二倍体型PWS	存在甲基化	未缺失
父源单亲二倍体型AS	不存在甲基化	未缺失
正常	存在甲基化	未缺失

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