New perspectives in genetically unresolved ADPKD by long-read genome sequencing: a pilot study

Autosomal dominant polycystic kidney disease (ADPKD) is primarily caused by mutations in the PKD1 and PKD2 genes. However, 7–10% of atypical cases remain genetically unresolved (GUR), likely due to variants in less-characterized genes or in non-coding regions not detectable by conventional short-read sequencing methods. This study explores the utility of long-read whole genome sequencing (WGS) using Oxford Nanopore Technologies (ONT) to identify cryptic genetic variants in three ADPKD patients negative for possible causative mutations. Whole Genome Sequencing (WGS) was performed with 15X average coverage on the ONT PromethION P2 Solo platform, generating approximately 4-6 million reads per sample with a mean N50 of 20 kb approximately for each sample. A targeted analysis focused on 23 candidate genes implicated in kidney diseases or nephropathy diseases (ALG5, ALG8, ALG9, BICC1, DNAJB11, DZIP1L, GANAB, HNF1B, IFT140, JAG1, LOC129992813, LRP5, MIR1225, MIR6511B1, NOTCH2, PKD1, PKD1-AS1, PKD2, PKHD1, PRKCSH, SEC63, TSC2, UMOD), selected through the MalaCards database. Variant calling included both single nucleotide and structural variants, with prioritization based on predicted pathogenicity and low population frequency. Bioinformatic analysis identified high-impact variants, intronic variants within the first 150 nucleotides of exon boundaries, and structural variants across 23 candidate genes. Some variants have been previously associated with other kidney diseases but have not yet been linked to ADPKD. We evaluated with AlphaMissense predictor tool the missense mutations in exonic variants, but no potentially pathogenic missense variants were detected. Genome-wide analysis identified an average of 4.6 million single nucleotide polymorphisms (SNPs) per sample, with an average transition/transversion (Ts/Tv) ratio of 1.77. Although slightly below the expected range for high-quality WGS (2.0–2.1). Also, the comparison of variants detected by Illumina and ONT, after harmonizing Illumina coordinates from GRCh37 to GRCh38, revealed a remarkable level of genotypes concordance with 98% on both platforms, providing key validation for the reliability of ONT long-read sequencing. Across the three subjects, 109, 104, and 100 shared variants passed quality filtering (PASS variants). Nanopore long-read WGS revealed novel candidate variants in ADPKD cases unresolved by standard genetic testing. This technology enables comprehensive detection of structural and non-coding variants, providing new opportunities to advance the genetic understanding of ADPKD. Functional validation and broader population studies are necessary to clarify their pathogenic significance.