基于二代测序技术的常染色体隐性遗传性多囊肾病家系胚胎植入前遗传学分析

Preimplantation genetic testing of infantile polycystic kidney disease by next generation sequencing

目的探讨二代测序(NGS)技术在常染色体隐性遗传性多囊肾病(ARPKD)家系胚胎植入前遗传学检测(PGT)中的应用价值。方法选取1个ARPKD家系,采用Sanger测序调查家系成员多囊肾/多囊肝病变1(PKHD1)基因突变情况。以PKHD1基因编码区为目标区域,在基因上下游2 M区域内选择120个高密度紧密连锁的单核苷酸多态性(SNP)位点作为遗传连锁标记,采用多重聚合酶链反应(PCR)和NGS选择有效SNP位点构建家系成员的SNP单倍型,确定夫妇双方携带基因突变的风险染色体。对活检获得的滋养层细胞进行全基因组扩增,采用NGS对胚胎的PKHD1基因突变位点进行直接测序,构建胚胎SNP单倍型进行连锁分析。采用Sanger测序验证胚胎PKHD1基因突变位点NGS结果。对正常和携带杂合突变的胚胎进行低深度的染色体非整倍性筛查。结果家系成员中,父亲携带PKHD1基因c.5935G>A,为杂合子;母亲携带PKHD1基因c.10058T>G,为杂合子;先证者携带PKHD1基因c.5935G>A和c.10058T>G双重杂合突变。用于活检的5个胚胎中有2个未检测到突变,有3个携带杂合突变。低深度的染色体非整倍性筛查显示5个胚胎中3个为整倍体,2个为非整倍体。选择未检测到突变且发育良好的整倍体胚胎植入母体子宫后,足月分娩一健康婴儿。结论应用NGS对ARPKD家系进行PGT,可阻断此单基因病在该家系中的再发风险,同时还可避免选择非整倍体胚胎而导致的流产问题。

Objective To investigate the application value of next generation sequencing technology in preimplantation genetic testing(PGT)of autosomal recessive polycystic kidney disease(ARPKD)family.Methods A case of ARPKD family was selected,and the mutation of polycystic kidney and hepatic disease 1(PKHD1)gene in family members was investigated by Sanger sequencing.The coding region of PKHD1 gene was selected as target region,120 high-density closely linked single nucleotide polymorphisms(SNP)were selected as the genetic linkage markers in the upstream and downstream 2 M regions of the gene.The SNP haplotypes of family members were constructed by selecting effective SNP sites by multiplex polymerase chain reaction(PCR)and next generation sequencing,and the risk chromosomes of gene mutation were determined.After the whole genome amplification of the trophoblast cells obtained by blastocyst biopsy,the mutation sites of embryonic PKHD1 gene were sequenced directly,and the embryonic SNP haplotypes were constructed for linkage analysis by next generation sequencing.Sanger sequencing was used to verify the next generation sequencing results of embryonic PKHD1 gene mutation sites.Low depth chromosome aneuploidy screening was carried out both in the normal and the heterozygous embryos.Results The father of the family members carried PKHD1 gene c.5935G>A,which was heterozygous.The mother carries PKHD1 gene c.10058T>G,which was heterozygous.The proband carried double heterozygous mutations of PKHD1 gene c.5935G>A and c.10058T>G.Among the 5 embryos used for biopsy,2 cases were undetected mutations,and 3 cases carried heterozygous mutations.Low depth chromosome aneuploidy screening showed that the 3 of 5 embryos were euploidy,and 2 embryos were aneuploidy.A healthy baby was delivered at full term after a well-developed euploid embryo with no mutation,which was implanted into the mother's uterus.Conclusions The application of next generation sequencing in PGT of ARPKD can block the risk of recurrence of this single gene disease in the family,and avoid the abortion caused by the selection of aneuploid embryos.