单精子测序技术在一个脊髓性肌肉萎缩症家系胚胎植入前遗传学检测中的应用

Application of single sperm sequencing for the preimplantation genetic testing of a Chinese family affected with Spinalmuscularatrophy

目的探讨单精子测序技术在脊髓性肌肉萎缩症(SMA)家系胚胎植入前单基因遗传病检测(PGT-M)中的应用价值。方法选取2020年6月于江西省妇幼保健院就诊的一个生育过2例SMA患儿(均已夭折)的家系作为研究对象,利用机械制动法分离11份单精子样本并进行全基因组扩增,采用荧光定量PCR与Sanger测序法对扩增产物进行SMN1基因变异检测。选取女方、女方父母和男方的基因组DNA,以及1份携带和2份未携带SMN1基因变异的单精子扩增产物,在变异位点上下游2 Mb区域内选取100个单核苷酸多态性位点,设计引物进行靶向捕获高通量测序,通过连锁分析确定男女双方与SMN1基因致病变异连锁的染色体单体型。经卵胞浆内单精子显微注射技术受精获取囊胚,活检滋养外胚层细胞,经全基因组扩增后行高通量测序检测胚胎的单体型,判断胚胎的致病性。对野生型胚胎进行染色体非整倍体检测,选择SMN1基因型为正常的整倍体胚胎进行移植。于孕18周行羊水穿刺产前诊断,确认胎儿的基因型。新生儿出生后进行跟踪随访。结果基因检测发现夫妇双方均携带SMN1基因第7、8外显子缺失杂合变异,其中女方携带的变异遗传自其父亲,男方为新发变异。利用单精子测序技术成功构建出男方单体型。PGT检测发现5枚胚胎携带SMN1基因杂合变异,4枚为野生型,其中3枚为整倍体。移植1枚野生型整倍体胚胎,妊娠中期羊水检测证实胎儿未携带SMN1基因第7、8外显子缺失变异。新生儿足月出生,随访9个月未见异常。结论应用单精子测序技术为1对夫妻双方均携带SMN1基因第7、8外显子杂合缺失变异且男方为新发变异的夫妇构建出男方致病变异的连锁单体型,并为该夫妇提供PGT检测,成功避免了SMA患儿的出生。

ObjectiveeTo assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease(PGT-M).Methods A Chinese couple with two children whom had died of Spinal muscular atrophy(SMA)and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject.Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification.Real-time PCR and Sanger sequencing were used to detect the SMNl variants in the single sperm samples.Genomic DNA of the wife,her parents and the husband,as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis.Targeted capture and high-throughput sequencing were carried out to test 1o0 single nucleotide polymorphisms distributed within 2 Mb up-and downstream the variant site.The haplotypes linked with the SMNl variants were determined by linkage analysis.Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection.Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status.Chromosomal aneuploidy of wild-type embryos was excluded.An euploid embryo of high quality was transferred.Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus.Results Genetic testing showed that the couple both had deletion of exons 7~8 of the SMNl gene.The wife has inherited the deletion from her father,while the husband was de novo.The haplotypes of the husband were successfully constructed by single sperm sequencing.Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant,4 embryos were of the wild type,among which 3 were euploid.Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion.Conclusion By single sperm sequencing and PGT-M,the birth of further affected child has been successfullyavoided.