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. 2023 Apr;40(4):783-792.
doi: 10.1007/s10815-023-02722-1. Epub 2023 Feb 11.

Blocker displacement amplification-based genetic diagnosis for autosomal dominant polycystic kidney disease and the clinical outcomes of preimplantation genetic testing

Tingting Lin  1   2   3 , Junfeng Luo  4 , Haibing Yu  1   2 , Bohao Dong  4 , Qi Zhang  1   2   3 , Wei Zhang  5 , Ke Chen  1   2 , Yezhou Xiang  1   2 , Dongyun Liu  6   7 , Guoning Huang  8   9   10
Affiliations

Blocker displacement amplification-based genetic diagnosis for autosomal dominant polycystic kidney disease and the clinical outcomes of preimplantation genetic testing

Tingting Lin et al. J Assist Reprod Genet. 2023 Apr.

Abstract

Objective: Given that the molecular diagnosis of autosomal dominant polycystic kidney disease (ADPKD) is complicated, we aim to apply blocker displacement amplification (BDA) on the mutational screening of PKD1 and PKD2.

Methods: A total of 35 unrelated families with ADPKD were recruited from the Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University (Chongqing, China), from October 2018 to October 2021. Long-range PCR followed by next-generation sequencing were applied for resequencing of PKD1 and PKD2, and the putatively disease-causative variants were verified with BDA. The effects of ADPKD on male and female infertility and the factors influencing the clinical outcomes of preimplantation genetic testing (PGT) for ADPKD were investigated.

Results: A total of 26 PKD1 variants and 5 PKD2 variants were identified, of which 13 were newly discovered. The BDA system worked effectively for eliminating the interference of pseudogenes in genetic testing of PKD1 (1-33 exons) with different concentrations of genome DNA. The females with ADPKD have no specific infertility factors, while 68.2% of the affected men were with abnormal sperm concentration and/or motility with an indefinite genotype-phenotype relationship. As for PGT, the fertilization rate of couples with the male partner having ADPKD was relatively lower compared to those with the female partner being affected. The ADPKD patients receiving PGT usually achieved high rates of live births.

Conclusion: These findings expanded the variant spectrum of PKD genes and emphasized the application prospect of blocker displacement amplification on PKD1-related genetic diagnosis.

Keywords: ADPKD; Blocker displacement amplification; PKD1; PKD2; Preimplantation genetic testing.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Mutation analysis of PKD1 and PKD2. (a) The diagrammatic images of PKD1 and PKD2, and the location of primers for long-range PCR. (b) The result of agarose gel electrophoresis of LR-PCR products. (c) Primer and blocker sequences designed for BDA to detect the sequence of exon 15 of PKD1 gene. The location of reverse primer is highlighted in yellow. The PKD1 gene bears a cytosine nucleotide, and the six pseudogenes bear a thymine nucleotide. The 4 A sequence at the 3′ end of the blocker serves as a termination sequence to prevent the blocker from being elongated by the Taq polymerase during PCR. (d) The ΔRn of BDA-based PCR for different doses of genomic DNA. (e) The results of Sanger sequence for BDA-PCR products. The red nucleotide bases and arrowheads showed the variants of PKD1 gene in ADPKD patients, while the blue nucleotide bases and arrowheads showed the differential bases between PKD1 gene and the pseudogenes
Fig. 2
Fig. 2
Flowchart showing the preimplantation genetic testing cycles for patients with ADPKD. PGT: preimplantation genetic testing; ADPKD: autosomal dominant polycystic kidney disease; ICSI: intracytoplasmic sperm injection; OR: oocyte retrieval; FET: frozen–warmed embryo transfer
See this image and copyright information in PMC

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