新发突变家系植入前遗传学检测的单倍型构建方法探索  

Exploration of haplotype construction method for preimplantation genetic testing in family with de novo mutation

作　　者：徐惠玲 吴正中[1] 付志红[1] 张华坤[1] 范晶 李雪梅[1] XU Hui-ling;WU Zheng-zhong;FU Zhi-hong;ZHANG Hua-kun;FAN Jing;LI Xue-mei(Department of Reproductive Medicine,Shenzhen Maternity&Child Health Care Hospital Affiliated to Southern Medical University,Shenzhen 518048)

机构地区：[1]南方医科大学附属深圳市妇幼保健院,深圳518048

出　　处：《生殖医学杂志》2022年第12期1699-1705,共7页Journal of Reproductive Medicine

基　　金：深圳市医学重点学科(2020-2024年)(SZXK031);深圳市科创委重点项目(2020231557);深圳市科创委基础研究(面上)项目(JCYJ20210324142811030)。

摘　　要：目的在单基因病胚胎植入前遗传学检测(PGT-M)中采用2种单倍型构建策略,判断3例新发突变家系中胚胎是否携带致病变异。方法收集3例新发突变家系,其中2例为男方新发突变,1例为女方新发突变。经过促排卵、体外受精及胚胎培养后,对所获得的囊胚进行滋养层细胞活检,活检细胞利用多次退火环状循环扩增技术(MALBAC)进行全基因组扩增(WGA),分别利用单精子测序技术及胚胎互推策略进行家系的单倍型构建,判断胚胎是否携带致病变异,最后选择健康的胚胎进行移植。妊娠中期进行胎儿羊膜腔穿刺产前诊断验证PGT-M结果准确性。结果3个家系的单倍型构建成功,移植后均获得了妊娠且得到了产前诊断。其中1例已分娩未携带亲本致病变异新生儿,2例仍在妊娠中。结论对于无法采用常规的单倍型分析方法的家系而言,利用单精子测序及胚胎互推策略可以使新发突变的夫妻从源头上阻止患儿的妊娠。Objective:To determine embryos’genotypes of three families with de novo mutations by using two haplotyping construction methods in preimplantation genetic testing for monogenic(PGT-M).Methods:Three families with de novo mutation were included,2 cases of mutation in male and 1 case in female.After controlled ovarian hyperstimulation and in vitro fertilization&embryo culture,the trophoblast cells of blastocysts were biopsied and amplified by whole genome amplification(WGA)with multiple annealing and looping based amplification cycles(MALBAC).The haplotype of the families was constructed by using single sperm sequencing technology and embryo mutual inference strategy respectively to judge whether the embryo carried pathogenic variation,and finally healthy embryos were selected for transplantation.Amniocentesis was conducted to verify the results of PGT-M in the second trimester of pregnancy.Results:Three families’haplotypes were successfully constructed,and pregnancy and prenatal diagnosis were obtained after transplantation.Among them,one successfully delivered healthy baby without parental pathogenic variation,and the remaining two were still pregnant.Conclusions:For families unable to use conventional haplotype analysis methods,the use of single sperm sequencing and embryo mutual inference strategy can prevent the birth of children with pathogenic variation from the source of newly mutated couples.

关 键 词：新发突变 单倍型分析 胚胎植入前遗传学检测 单基因疾病 

分 类 号：R715.5[医药卫生—妇产科学] R394.3[医药卫生—临床医学]