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A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.

ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) study and monogenic diagnosis study for ADPKD patients bearing de novo mutations. The innovation of our strategy is to use the gamete (polar body or single sperm) as proband for single-nucleotide polymorphism (SNP) linkage analysis to detect an embryo's carrier status. Nine ADPKD couples with either de novo mutation or without a positive family history were recruited and a total of 34 embryos from 13 PGT-M cycles were examined. Within these nine couples, two successfully delivered healthy babies had their genetic status confirmed by amniocentesis. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients who lack essential family members for linkage analysis.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8490604 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.

Wang Yuqian Y Zhai Fan F Guan Shuo S Yan Zhiqiang Z Zhu Xiaohui X Kuo Ying Y Wang Nan N Zhi Xu X Lian Ying Y Huang Jin J Jia Jialin J Liu Ping P Li Rong R Qiao Jie J Yan Liying L

Journal of assisted reproduction and genetics 20210503 9

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation gene ...[more]

PMID: 33939064

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Project description:BackgroundDespite the gradual application of third-generation long read sequencing (LRS) or reference embryo establishment to preimplantation genetic testing for structural rearrangement (PGT-SR) without familial involvement, there are still limitations to their extensive clinical application yet. This study developed a novel virtual NGS-based long read method (ViLR) and preliminarily evaluated its clinical feasibility of breakpoint characterization and direct SNP haplotyping for de novo chromosomal structural rearrangements (CSR).MethodsA total of 10 families with de novo CSR risk were enrolled in this study for ViLR analysis. In contrast to LRS, ViLR is a virtual long read solution that used the same barcoded labeling and assembly of different long gDNAs differently barcoded. Notably, ViLR could generate an average fragment length of over 30 Kb, with an N50 block size of up to 16 Mb in a single assay, allowing to achieve accurate breakpoint mapping and direct carrier's haplotyping. An approximately 2 Mbp region flanking upstream and downstream of each breakpoint was selected for informative SNP collection. Embryo haplotype determination was based on the established carriers' haplotypes after whole genome amplification and sequencing. To confirm PGT-SR results, we performed prenatal genetic diagnosis.ResultsThis study achieved an average mapping rate of 99.5%, > 90% coverage depth (> 10X), an average number of effective barcode (> 5 kb length) counts of 11,000,000 and an average fragment length of 40 kb, which generated sufficient informative SNPs for breakpoint characterization and haplotype phasing. ViLR analysis of 10 de novo PGT-SR carriers precisely identified breakpoints and haplotypes. Seven families obtained 18 euploid embryos, in which 10 were euploid/normal embryos, 7 were euploid/balanced carrier embryos, and the remaining one unknown was due to homologous recombination of the breakpoint region. Prenatal genetic diagnosis was performed for four women, and the outcomes coincided with the results from embryo PGT-SR. At the time of writing this paper, four healthy babies had been delivered uneventfully.ConclusionHere, we demonstrated the clinical potential of ViLR as a novel solution for breakpoint identification and direct SNP haplotyping in de novo PGT-SR families without proband involvement.Clinical trial numberNot applicable.

Project description:BACKGROUND:We have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported. METHODS:Full-length PKD1 cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family. RESULTS:Five PKD1 mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly de novo. CONCLUSION:The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of de novo PKD1 mutations indicates that this gene is prone to mutations.

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A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.

Publications

A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.

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