Identification Of Novel PKD1 Mutations And Preimplantation Genetic Diagnosis In Two Chinese Families With Autosomal Dominant Polycystic Kidney Disease

Objective: Autosomal dominant polycystic kidney disease?ADPKD?is a common inherited kidney disease with an estimated incidence of 1 in 1000.It is a late-onset systemic disorder characterized by the development and progressive enlargement of cysts in the kidney,eventually leading to renal failure.Due to high incidence,poor prognosis and absence of effective therapy,it is of important theorical and practical significance to clarify the molecular mechanism on ADPKD and perform presymptomatic diagnosis and genetic counseling for the high-risk individuals.ADPKD is a heterogeneous monogenic disorder resulting from PKD1?85%?and PKD2?15%?mutations.However,the presence of a large number of exons,no mutation hot spots,complex genomic replicated region and high GC content make the mutation analysis difficult and challenging.ADPKD shows delayed dominance with high penetrance and the children of affected parents have a 50% chance of developing ADPKD.Therefore,preimplantation genetic diagnosis?PGD?is the most effective way to block the transmission of genetic disorders from the parents to their offspring,which will benefit early prevention and intervention of the disease.A single cell is often used for the genetic analysis in PGD and the small amount of genetic material in a single embryonic cell hinders the successful PGD.Whole genome amplification?WGA?such as multiple displacement amplification?MDA?and multiple annealing and looping-based amplification cycles?MALBAC?can amplify the genomic sequences with the minimum amplification bias to provide enough DNA templates for the subsequent genetic analysis.Unfortunately,any WGA method exhibits false-positive and false-negative errors due to the cumulative effects of allele dropout or preferential amplification,which will have an impact on the precision of PGD.Linkage analysis using short tandem repeats or single nucleotide polymorphisms?SNPs?has been usually applied to eliminate these errors.Recently,next generation sequencing?NGS?has shown considerable potential in the gene testing for inherited diseases and PGD because it can simultaneously perform the aneuploidy screening in the whole genome and detect the single nucleotide variations.In the present study,mutation analysis was performed in two Chinese families with a definite diagnosis of ADPKD and two pathogenic mutations were identified in the relevant families,respectively.Meanwhile,the preimplantation genetic diagnosis platform for ADPKD was established successfully.These findings will not only enrich the existing ADPKD mutation database,but also match the needs of preimplantation genetic diagnosis for ADPKD,which will provide new clues for future precision medicine and have important theoretical and clinical significance.Methods: Two pedigrees that included 14 individuals with ADPKD and 19 normal individuals were recruited from Dalian Municipal Women and Children's Medical Center,with written informed consent obtained from all subjects prior to their enrollment in this study.Genomic DNA of two probands was extracted from peripheral blood routinely and sheared into 200-250 bp fragments with a Covaris LE220 ultrasonicator,following purification using AMPure Beads.The purified DNA fragments were further processed via ending repair,A-tailing and linker ligation to generate complete DNA libraries.Samples were hybridized on a custom capture array at 47? for 64-72 h in order to capture the targeted sequences.Sequencing was carried out on Illumina HiSeq2500 Analyzer for 90 cycles in both directions and base calling was performed using Illumina Pipeline software to generate primary data.Raw reads were scored in order to remove the sequences with poor quality or linker contamination.The remaining sequences termed as clean reads were then aligned with human reference genome in order to identify the pathogenic mutations.The candidate variants were validated by Sanger sequencing and co-segregation analysis after polymerase chain reaction?PCR?.Bioinformatic analysis were performed to predict the function of mutations.Before PGD,the strategy for single cell WGA was optimized by evaluating the uniformity and allele dropout rate between MDA and MALBAC.NGS with different sequencing depth was done in deserted embryos in order to assess the best condition of preimplantation genetic screening for aneuploidy.The couple who wanted assisted reproductive treatment underwent a routine controlled ovarian hyperstimulation and testicular sperm extraction,respectively.Fertilization was done by intracytoplasmic sperm injection.Embryos were cultured and developed to the blastocyst stage in vitro,following trophectoderm biopsy on day 5 or 6 after fertilization.The whole genome amplification of each embryo biopsy sample was performed using MALBAC method.Six SNPs within 3Mb upstream or downstream from the targeted mutation were selected for linkage analysis.The mutant site and the six SNPs were amplified by regular PCR using MALBAC WGA products as templates,and the PCR products were then mixed with the MALBAC WGA products,following NGS to measure copy number variations?CNVs?and SNPs in the same run.Sanger sequencing was applied to detect the mutant site.Disease-associated haplotype was constructed by SNP linkage analysis.Results: 1.A novel c.6491C>A mutation in exon 15 of PKD1 gene was detected in pedigree 1 and a known c.12608₁2635del mutation in exon 46 of PKD1 gene was detected in pedigree 2 by the targeted NGS.2.There were a C to A transversion at c.6491 site in the proband of pedigree 1 and 28 bp deletion?5'-GGCTGGGGACAAGGTGTGAGCCTGAGCC-3'?at c.12608_c.12635 site in the proband of pedigree 2.These two heterozygotic mutations were validated by Sanger sequencing,which were consistent with the NGS results.3.The heterozygotic c.6491C>A mutation was detected in all the nine affected members but was absent in eleven asymptomatic members in pedigree 1.Similarly,the heterozygotic c.12608_c.12635 del mutation was detected in all the five affected members but was absent in eight asymptomatic members in pedigree 2.These two mutations displayed their co-segregation with ADPKD in the relevant families.4.Bioinformatic analysis showed that protein sequences corresponding to c.6491 and c.12608₁2635 site exhibited high evolutionary conservation among different species such as human,rhesus and mouse.c.6491C>A mutation was predicted to form a premature truncated protein with 2140 amino acid shorter than the wild PKD1 protein,so that the novel c.6491C>A mutation in PKD1 gene was a nonsense mutation and named as p.Ser2164 Ter mutation.c.12608₁2635del mutation was predicted to change the open reading frame from the mutant site and form a prolonged abnormal protein with 44 amino acid longer than the wild PKD1 protein,so that the known c.12608₁2635del mutation in PKD1 gene was a frameshift mutation and named as p.Arg4203Profs*146 mutation.5.Genome coverage of MALBAC?90%?was higher than that of MDA?70%?,but the allele dropout rate of MALBAC?13%?was lower than that of MDA?40%?,suggesting that MALBAC was better than MDA in the whole genome amplification of a single cell.6.Out of 22 embryos which were identified as aneuploidy by routine NGS?average 1.0M PE reads?,9 embryos were identified as normal diploidy by deep NGS?average 3.5M PE reads?.Random sampling based on different sequencing data?including 0.3M,0.6M,1.5M and 3.5M PE reads?indicated that the false-positive rate of CNV detection was decreased significantly with the increase of sequencing data,especially for the chromosomal abnormalities from 1Mb to 10 Mb in size.The above results suggested that the increase of sequencing depth could impove the precision of aneuploidy screening in PGD.7.Genetic verification before PGD on genomic DNA from the key members?II1,II2,III1,III2?in pedigree 1 revealed that the proband?III2?carried the heterozygotic c.6491C>A?p.Ser2164Ter?mutation inherited from his affected mother?II2?while his father?II1?and his wife?III1?did not carry the targeted c.6491C>A?p.Ser2164Ter?mutation.8.After controlled ovarian hyperstimulation,eleven matured oocytes at metaphase-II stage were collected and fertilized by intracytoplasmic sperm injection.Five embryos?named Embryo 1,Embryo 2,Embryo 3,Embryo 4 and Embryo 5,respectively?developed to the blastocyst stage.Trophectoderm biopsy and whole genome amplification were applied for PGD successfully.9.Targeted mutation detection by Sanger sequencing in five embryos revealed that two embryos including Embryo 2 and Embryo 5 were carriers of the targeted heterozygotic c.6491C>A?p.Ser2164Ter?mutation and unsuitable for embryo transfer.SNP linkage analysis in five embryos identified that two embryos?Embryo 2 and Embryo 5?inherited the paternal disease-associated haplotype CCGAAC and were not suitable for embryo transfer.CNV detection in five embryos revealed that two embryos were aneuploidy and unsuitable for embryo transfer,including Embryo 1?46,XN/47,XN,+9?and Embryo 3 [(46,XN/46,XN,del?6??p22.1?].Embryo 4 was diploid and free of the targeted pathogenic c.6491C>A?p.Ser2164Ter?mutation,which was suitable for embryo transfer and selected to be frozen for future use.Conclusions: 1.A novel nonsense mutation c.6491C>A?p.Ser2164Ter?and a known frameshift mutation c.12608₁2635del?p.Arg4203Profs*146?in PKD1 gene are the pathogenic mutations in the relevant ADPKD pedigrees.2.Targeted next generation sequencing is a rapid and effective method in the gene testing for ADPKD.3.SNP haplotype analysis combined with next generation sequencing and Sanger sequencing is a new preimplantation genetic diagnosis strategy for ADPKD.4.MALBAC is suitable for the whole genome amplification of a single cell in preimplantation genetic diagnosis.5.The increase of sequencing depth can improve the precision of aneuploidy screening in preimplantation genetic diagnosis.