国际生殖健康/计划生育杂志

国际生殖健康/计划生育 ›› 2017, Vol. 36 ›› Issue (4): 276-279.

• 论著 • 上一篇    下一篇

X-连锁慢性肉芽肿病的植入前遗传学诊断方法学研究

吴海涛，沈晓婷，刘瑜亮，钟依平，王静，曾艳红，丁晨晖，周灿权   

  1. 510080  广州，中山大学附属第一医院生殖医学中心（吴海涛，沈晓婷，刘瑜亮，钟依平，王静，曾艳红，丁晨晖，周灿权）；广东省江门市中心医院生殖医学中心（吴海涛）
  • 收稿日期:2017-05-22 修回日期:2017-06-24 出版日期:2017-07-15 发布日期:2017-07-15
  • 通讯作者: 周灿权，E-mail：zhoucanquan@hotmail.com E-mail:zhoucanquan@hotmail.com
  • 基金资助:
    国家自然科学基金（81370765），广州市科技计划项目（201300000097），广东省科技计划项目（2013B021800271）

Methodology Study on Preimplantation Genetic Diagnosis of X-linked Chronic Granulomatous Disease

 WU Hai-tao，SHEN Xiao-ting，LIU Yu-liang，ZHONG Yi-ping，WANG Jing，ZENG Yan-hong，DING Chen-hui，ZHOU Can-quan   

  1. Reproductive Medicine Center，The First Affiliated Hospital，Sun Yat-sen University，Guangzhou 510080，China（WU Hai-tao，SHEN Xiao-ting，LIU Yu-liang，ZHONG Yi-ping，WANG Jing，ZENG Yan-hong，DING Chen-hui，ZHOU Can-quan）；Reproductive Medicine Center，Jiangmen Central Hospital，Jiangmen 529000，Guangdong Province，China（WU Hai-tao）
  • Received:2017-05-22 Revised:2017-06-24 Published:2017-07-15 Online:2017-07-15
  • Contact: ZHOU Can-quan，E-mail：zhoucanquan@hotmail.com E-mail:zhoucanquan@hotmail.com

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摘要： 目的：采用多重置换扩增（MDA）建立一种可靠、准确的植入前遗传学诊断（PGD）方法，可应用于X-连锁慢性肉芽肿病的PGD。方法：采用16个位于CYBB基因两侧的短串联重复序列（STR）位点对X-连锁慢性肉芽肿病家系进行单体型分析，采用MDA对单细胞进行全基因组扩增，对其产物采用在家系分析中具有多态性的STR位点及特异性扩增CYBB基因进行分析，同时采用位点Amel进行性别诊断。结果：家系分析结果显示共有7个STR位点具有多态性。共进行了10个单淋巴细胞及10个单卵裂球的MDA，全部扩增成功。对致病基因CYBB外显子8的聚合酶链反应（PCR）扩增效率为100%，其中1个单淋巴细胞发生致病位点的等位基因脱扣（ADO），10个单卵裂球诊断结果均为215 bp，未检测出异常的204 bp条带，ADO率为10%（1/10）；对于7个有多态性的STR位点和位点Amel，PCR扩增效率为96.9%（155/160），ADO率为11.3%（13/115）。结论：采用MDA结合致病基因特异性扩增及单体型分析在单细胞水平对X-连锁慢性肉芽肿病进行检测，两者相结合可避免污染、ADO等导致的误诊，可提高PGD的诊断效率，为进一步的临床应用奠定了基础。

关键词: X-连锁慢性肉芽肿病, 系谱, 植入前诊断, 多重置换扩增, 单体型分析

Abstract: Objective：To develop a reliable and exact protocol for the multiple displacement amplification (MDA)-based preimplantation genetic diagnosis (PGD) of X-linked chronic granulomatous disease (X-CGD). Methods：The haplotype analysis of X-CGD was performed for 16 short tandem repeats (STR) loci on both sides of the CYBB gene. Whole-genome amplification of single cell was carried out by MDA. The STR loci with polymorphism as demonstrated by the linkage analysis and the specific amplification of CYBB gene were analyzed. The locus Amel was used for sex determination. Results：Seven STR loci were found to be polymorphic in the linkage analysis. MDA was performed in 10 single lymphocytes and 10 single blastomeres. The efficiencies of PCR amplification of the pathogenic CYBB exon 8 was 100%. ADO of the mutant allele of CYBB was detected in one of the lymphocytes, the ADO rate 10% (1/10). For those single blastomeres, the 215 bp band corresponding to the normal allele was detected in all MDA products, while the abnormal 204 bp band was not detected. For the 7 polymorphic STR loci and AMELs, the PCR amplification efficiency and the ADO rate were 96.9% (155/160) and 11.3% (13/115), respectively. Conclusions：In this study, MDA combined with pathogenic gene specific amplification and haplotype analysis was performed to detect chronic granulomatous disease in a single cell level. The combination of the two techniques can improve the efficiency of PGD for CGD by reducing the misdiagnosis caused by contamination and ADO.

Key words: X-linked chronic granulomatous disease, Pedigree, Preimplantation diagnosis, Multiple displacement amplification, Haplotype analysishaplotype analysis