Abstract:

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.

Key words: Polycystic kidney and hepatic disease 1 gene, Autosomal recessive polycystic kidney disease, Next generation sequencing, Preimplantation genetic testing