摘要
目的建立可有效监测污染和等位基因脱扣(allele dropout,ADO)的可靠的葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症单细胞基因诊断体系。方法获取正常G6PD基因型个体及G6PD基因突变杂合子的单个淋巴细胞,在全基因组扩增(whole genome amplification,WGA)的基础上,应用多重SNaPshot技术检测25个已报道的中国人群G6PD基因突变位点,同时联合针对Xq28上6个短串联重复序列(short tandem repeat,STR)位点的单倍型分析技术辅助分析结果。结果共检测60个正常和96个杂合子单淋巴细胞,WGA扩增效率为94.23%,成功扩增的WGA产物在后续基因检测的总扩增效率和总ADO率分别为89.37%和13.74%;8个基因位点的扩增效率和ADO率均有统计学差异(P总<0.05);发生G6PD基因ADO的15例G6PD杂合子单淋巴细胞中,均未同时出现6个STR位点全部扩增失败或ADO现象;所有样本的STR位点检测结果除目的片段的信号峰外,均未出现其他峰信号。结论联合SNaPshot和单倍体分型技术所建立的单细胞G6PD基因诊断体系,在G6PD基因分型的同时,还可提示是否存在污染或ADO,最大程度保证诊断结果的可靠性,减少误诊率,可望取代传统的性别选择,实现真正意义上的G6PD缺乏症植入前遗传学诊断。
Objective： To establish a reliable genetic diagnosis system for glucose-6-phosphate dehydrogenase （G6PD）deficiency based on a single cell, which can monitor the contamination and allele dropout （ADO） effectively. Methods： A single lymphocyte was acquired from the individuals with normal G6PD genotype and the heterozygote of G6PD gene mutation. After the whole genome amplification（WGA）,multiplex SNaPshot assay was applied to detect 25 mutation sites in G6PD gene, which have been reported in Chinese population. The haplotyping of 6 short tandem repeat（STR）loci located on Xq28 was performed to assist the interpretation of results simultaneously. Results： Sixty lymphocytes from normal people and 96 lymphocytes from heterozygote were detected. The amplification efficiency of WGA was 94.23%. Total amplification efficiency and total ADO rate of subsequent gene detections by those WGA products amplified successfully were 89.37% and 13.74% respectively. Both amplification efficiency and rate of ADO showed statistically different among 8 gene loci （Ptotal〈0.05）. Fifteen cases of single lymphocyte from G6PD heterozygo none of them showed completely amplification failure or ADO of a Furthermore, no extra signal peak except target fragment was observed samples. tes had ADO of G6PD gene,but he 6 STR loci simultaneously. the STR loci detection results of Conclusions： The established G6PD genetic diagnosis system in a single cell by SNaPshot combined with and haplotyping technique can gonatype G6PD and indicate whether there exists contamination and ADO effectively. This system guarantees a maximum reliability of the diagnosis results and reduces the rate of misdiagnosis. It may be expected to replace the traditional gender selection, and achieve the preimplantation genetic diagnosis of G6PD deficiency with real meaning.
出处
《生殖医学杂志》
CAS
2015年第11期928-937,共10页
Journal of Reproductive Medicine
基金
深圳市科技计划项目(201202200)
深圳市基础研究项目(JCYJ20140415114532535)
