Application research of PGT in blocking the inheritance of novel mutations in the PKHD1 gene in autoso-mal recessive polycystic kidney disease pedigrees / 实用医学杂志

ABSTRACT

Objective To investigate the application value of single nucleotide polymorphism(SNP)linkage analysis based on next-generation sequencing(NGS)technology in preimplantation genetic testing(PGT)of families with autosomal recessive polycystic kidney disease(ARPKD).Methods A family with ARPKD was selected,where the female member had a pregnancy ultrasound revealing polycystic kidney in the fetus.Genetic testing showed compound heterozygous mutations of the polycystic kidney/polycystic liver disease 1 gene(PKHD1),c.10444C>T(paternal)and c.4303del(maternal),with the c.4303del mutation being reported for the first time.Targeting the coding region of the PKHD1 gene,335 high-density tightly linked SNP sites were selected in the upstream and downstream 2M regions using multiplex polymerase chain reaction(PCR)and NGS.The couple′s SNP risk haplotypes carrying gene mutations were constructed.After in vitro fertilization,blastocyst culture was performed.Trophoblastic cells obtained from the biopsy were subjected to whole-genome amplification,and NGS was used for linkage analysis and low-depth chromosomal aneuploidy screening of the embryos.Sanger sequencing was used to verify the results of embryo linkage analysis.Results Among the 6 biopsied embryos,4 were mutation-free and euploid,1 exhibited heterozygous for the mutation and mosaic while another unstable sequencing data,making it impossible to judge.One of the mutation-free and developmentally healthy euploid embryos was implanted into the maternal uterus,resulting in the full-term delivery of a healthy baby.Conclusion Application of NGS-based SNP linkage analysis in PGT can effectively blocking the vertical transmission of ARPKD within families,while avoiding abortion issues caused by aneuploid embryos.This study is also the first PGT report target-ing the PKHD1 gene c.4303del mutation.