SNP单体型分析在单基因病植入前遗传学检测中的应用

Application of SNP haplotype analysis in preimplantation genetic testing of monogenetic diseases

目的探讨基于二代测序的植入前单体型分析在单基因病植入前遗传学检测中的应用概况。方法对发育至第5天或者第6天的囊胚行活检,对活检细胞进行全基因组扩增,将致病基因突变作为目标区域,在该基因突变上、下游选择数十至上百个单核苷酸多态性位点作为连锁遗传标记,通过二代测序技术所获胚胎行植入前单体型分析和染色体非整倍体筛查,胚胎结果经一代测序和妊娠中期羊水穿刺结果验证。结果22例患者一共获卵421枚,成熟卵子355枚,受精305枚,卵裂301枚,共活检囊胚143枚,经植入前单体型分析和植入前遗传学筛查,40枚胚胎为可移植胚胎,移植周期数为17例,临床妊娠12例,活产9例婴儿(其中1例患者分娩2名婴儿,为单卵双胎),随访均健康,4例患者继续妊娠中,妊娠率为70.6%。所有单体型分析结果经过Sanger测序验证,均一致。结论基于二代测序的植入前单体型分析方法是切实可行的,通过此技术的应用可以帮助具有高遗传风险的单基因病家庭获得健康子代,最终达到阻断遗传病发生的目的。

Objective To explore the application of preimplantation genetic haplotyping based on next generation sequencing in process of preimplantation genetic diagnosis of monogenetic diseases.Methods The whole genome of the biopsied trophoblast cells from blastocysts was amplified by multiple displacement amplification method. The pathogenic gene mutation was selected as the target region, and some SNPs were selected as the genetic markers in the upstream and downstream of the target mutation. Preimplantation genetic haplotyping and aneuploidy screening were carried out for embryos by next generation sequencing. The results were confirmed by Sanger sequencing.Results A total of 421 oocytes were obtained from 22 patients, 355 oocytes matured, 305 oocytes fertilized, 301 oocytes cleaved and 143 blastocysts were biopsied. After haplotype analysis, 40 embryos were transplantable, there were 17 transplantation cycles, which lead to 12 clinical pregnancies and 9 live births(1 patient gave birth to 2 infants, which were monozygotic twins). All the infants were followed up in good health. The pregnancy rate was 70.6%. All haplotype analysis results were confirmed by Sanger sequencing and the results were consistent.Conclusion The preimplantation genetic haplotyping method based on the next generation sequencing is feasible, which can help families with high genetic risk to obtain healthy offspring, so as to achieve the goal of eugenics.