单精子测序结合PCR-反向点杂交技术在1例东南亚缺失型α地中海贫血患者胚胎植入前遗传学检测中的应用

Application of single sperm sequencing combined with PCR-reverse dot blot in preimplantation genetic testing of Southeast Asian deletion typeαthalassemia

目的探讨单精子测序结合PCR-反向点杂交(PCR-reverse dot blot,PCR-RDB)技术在东南亚缺失型α地中海贫血胚胎植入前遗传学检测中的应用价值。方法选取2020年4月24日就诊于广州医科大学附属第三医院妇产科的一对东南亚缺失型α地中海贫血携带者夫妇,针对男方无法提供家系成员样本的情况,本案例采用上游法结合显微操作移液管挑选其5份单精子样本并进行全基因组扩增,通过PCR-RDB技术确定男方单精子样本的地中海贫血基因分型,在--^(SEA)区域的上下游1 Mb范围内选择单核苷酸多态性位点(single nucleotide polymorphism,SNP)作为遗传标记,进行二代测序分析构建双亲单体型。对6份囊胚活检样本进行全基因组扩增后行二代测序,通过单体型分析胚胎是否携带致病变异,并选取非患病囊胚进行移植。孕20周抽羊水进行产前诊断,验证与单基因病胚胎植入前遗传学检测(preimplantation genetic testing for monogenetic disease,PGT-M)结果是否一致。结果女方的致病变异来源于其母亲,男方5份单精子样本的地中海贫血基因分型结果显示有4份为野生型。单精子测序筛选出男方有效位点10个,女方家系连锁分析得到女方有效位点6个,PGT-M结果显示4枚囊胚为αα/--^(SEA),2枚为--^(SEA)/--^(SEA),产前诊断结果显示胎儿基因型为αα/--^(SEA),与PGT-M结果一致,并于孕40周分娩一健康女婴。结论对于家系不全的男性东南亚缺失型α地中海贫血携带者,可采用单精子测序结合PCR-RDB技术筛选SNP位点,通过连锁分析行PGT-M。

Objective To investigate the value of single sperm sequencing combined with PCR-reverse dot blot(PCR-RDB)technology in preimplantation genetic testing(PGT)of Southeast Asian deletion type α thalassemia.Methods A couple of Southeast Asian deletion type α thalassemia carrier was selected in this case,who asked for assisted reproduction in the Department of Obstetrics and Gynecology of the Third Affiliated Hospital of Guangzhou Medical University on April 24,2020.The male carrier was subjected to single sperm isolation by the swim-up method combined with micropipette due to his incomplete pedigree.Five single sperm samples were isolated and their whole-genome were amplified.The genotype of thalassemia of single sperm samples were determined by PCR-RDB.SNPs were selected as genetic markers in the range of 1 Mb upstream and downstream of --^(SEA) region to construct chromosomal haplotype.Next,we performed whole-genome amplification on six blastocyst biopsy samples,and then next-generation sequencing was carried out to detect the carrier status of the embryos.Finally,the non-pathogenic blastocysts were selected for transplantation,and the preimplantation genetic testing for monogenic(PGT-M)results were confirmed by the prenatal genetic diagnosis during pregnancy.Results The pathogenic variant in the female was inherited from her mother,and four of the five single sperm samples were wild-type.Ten male informative SNP markers were identified by single sperm sequencing,and six female informative SNP markers were identified by linkage analysis of female family.PGT-M results indicated that four blastocysts were αα/--^(SEA) and two were --^(SEA)/--^(SEA).The result of prenatal diagnosis revealed that the fetus was a carrier of --^(SEA),which was consistent with PGT-M result,and a healthy girl was delivered at 40 weeks of gestation.Conclusion For male carriers of Southeast Asian deletion typeαthalassemia with incomplete pedigree,single sperm sequencing combined with PCR-RDB technology can be used to select SNP sites,and PGT-M can be performed by linkage analysis.