摘要
【目的】采用多重置换扩增建立一种可靠、准确的植入前遗传学诊断方法,应用于X-连锁严重联合免疫缺陷病(X-SCID)的植入前遗传学诊断(PGD)。【方法】选择5个位于致病基因IL-2受体γ链(IL2RG)基因两侧的短串联重复序列(STR)位点对X-SCID家系进行单体型分析,采用多重置换扩增(MDA)对单细胞进行全基因组扩增,对扩增产物采用在家系分析中具有多态性的STR位点进行单体型分析,结合位点Amel进行性别诊断,STR位点均采用单重荧光PCR,同时对IL2RG基因exon5进行测序分析。【结果】对家系分析中,共有3个STR位点具有多态性。对10个单淋巴细胞及10个单卵裂球进行了预实验:MDA扩增成功率为100%,对致病基因IL2RG exon5的行基因测序的检测效率为100%;对于3个有多态性的STR位点和AMEL,PCR扩增效率为96.3%(77/80),等位基因脱扣率(ADO)为11.5%(7/61)。对该家系进行了一个周期的PGD,对7个胚胎进行了诊断,其中2个为正常胚胎,移植后获得双胎妊娠,早产活产一个健康男婴及一个健康女婴。【结论】采用多重置换扩增结合致病基因特异性扩增测序检测及单体型分析在单细胞水平对X-连锁严重联合免疫缺陷病进行检测,两者相结合可避免污染、等位基因脱扣等导致的误诊,提高了PGD诊断效率。
【Objective】To establish a reliable and accurate preimplantation genetic diagnosis(PGD)method using multiple displacement amplification(MDA), which can be applied to the diagnosis of X-linked severe combined immunodeficiency disease(XSCID).【Methods】Haplotype analysis for the X-SCID family was performedusing five short tandem repeats(STR) markers flanking the both sides of the interleukin-2(IL-2) receptor gamma chain(IL2 RG) gene. MDA technique was used for single-cell whole genomic amplification. The products were used as template in polymerase chain reaction(PCR) of informative STR markers found by linkage analysis for haplotype analysis as well as sequencing of the IL2 RG gene exon 5.The amelogenin(AMEL) locus was used to do sex diagnosis.【Results】Linked analysis revealed 3 STR markers were informative. The method was evaluated with 10 single lymphocytes and10 single blastomeres. MDA was successful in all single cell. The detection efficiency of gene sequencing of pathogenic IL2 RG exon5 was 100%. The PCR efficiency of 3 STR informative markers and AMEL was 96.3%(77/80)and the average allele drop-out(ADO) rate was 11.5%(7/61). A cycle of PGD was performed on the family, and seven embryos were diagnosed, two of which were normal embryos. Twin pregnancy occurred after transplantation which were given a healthy baby boy and a healthy baby girl.【Conclusion】In this study, multiple displacement amplification combined with specific amplification/sequencing of pathogenic gene and haplotype analysis in the single cell level of X-linked severe combined immunodeficiency disease were performed. The protocol can avoid misdiagnosis caused by contamination and ADO, and improve the diagnostic efficiency of PGD.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2017年第6期955-960,共6页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学博士启动基金(S2013040014613)
广州市科技计划项目(201300000097)
关键词
X-连锁严重联合免疫缺陷病
植入前遗传学诊断
多重置换扩增
短串联重复序列
单体型分析
X-linked severe combined immunodeficiency
preimplantation genetic diagnosis(PGD)
multiple displacement amplification(MDA)
short tandem repeat(STR)
haplotype analysis
