The Study On Preimplantation Genetic Diagnosis And Birth Defect Prevention For Polycystic Kidney Disease

Objective: Polycystic kidney disease(PKD)is a common hereditary kidney disease,which can be divided into autosomal dominant polycystic kidney disease(ADPKD)and autosomal recessive polycystic kidney disease(ARPKD).ADPKD mostly occurs in the young and middle-aged period,manifested as bilateral renal fluid cyst formation.ARPKD mostly occurs in the neonatal and infant period,manifested as renal and hepatic portal venous system hypoplasia.At present,there is no effective therapy for PKD.In order to prevent birth detects,it is necessary to conduct genetic counseling and prenatal diagnosis for high-risk population in time.Preimplantation genetic diagnosis(PGD)is a kind of prenatal diagnosis method based on assisted reproductive technology(ART),which uses molecular biology technology to carry out embryo biopsy before implantation and selects normal embryos for transplantation.The objective of this study is to explore the application of next generation sequencing(NGS)based on preimplantation genetic haplotyping(PGH)in PKD for preimplantation genetic diagnosis and birth defect prevention.Material and Methods: Nine pedigrees of PKD were enrolled in this study.Next generation sequencing and Sanger sequencing were used to analyze the mutations of PKD1 gene and PKHD1 gene.The whole genome of biopsied trophectoderm cells was amplified by multiple displacement amplification(MDA)method,and dozens to hundreds of single nucleotide polymorphisms(SNPs)in the upstream and downstream of the pathogenic gene were selected as genetic markers.Preimplantation genetic diagnosis was completed by NGS-PGH method and the results were verified by Sanger sequencing.Results: A total of 136 oocytes were obtained from 6 pedigrees.Among them,109 were mature(maturation rate 80.1%),84 were successfully fertilized(fertilization rate77.1%),82 were at cleavage stage(cleavage rate 97.6%),50 were biopsied(biopsy rate61.0%),8 were normal(normal embryo accounted for 16.0%).The results of biopsy were consistent with Sanger sequencing results.The incidence of allele drop out(ADO)ranged from 0% to 62.5%,with an average of 5.8%.72.9% of the SNPs had no ADO occurence,22.5% had only one drop-out,3.4% had two drop-out,and only 1.2% had more than two drop-out.Five patients have been transferred,and all of them have successfully obtained clinical pregnancy.Among them two patients have delivered healthy babies.In terms of mutation identification,a total of 10 pathogenic mutations were detected,including missense mutation,nonsense mutation,frameshift mutation,splicing mutation and large deletion mutation.Among them,6 novel mutations were identified,including c.9447C>A,c.10051-2A>C,c.6915+1G>C,c.6902＿6903del in PKD1 gene and c.8467G>T,c.8705＿8707del in PKHD1 gene.Conclusions:In this study,6 novel mutations were identified for the first time,which expands the mutation spectrum of PKD1 genes and PKHD1 genes.Preimplantation genetic diagnosis technology platform for ADPKD and ARPKD was established.5patients got successful pregnancies,and 2 of them have delivered healthy infants through NGS-PGH method,which provides effective measures for preventing the occurrence of PKD birth defects.