Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder and is due to disease-causing variants in PKD1 or PKD2. Strong genotype-phenotype correlation exists although diagnostic sequencing is not part of routine clinical practice. This is because PKD1 bears 97.7% sequence similarity with six pseudogenes, requiring laborious and error-prone long-range PCR and Sanger sequencing to overcome. We hypothesised that whole-genome sequencing (WGS) would be able to overcome the problem of this sequence homology, because of 150 bp, paired-end reads and avoidance of capture bias that arises from targeted sequencing. We prospectively recruited a cohort of 28 unique pedigrees with ADPKD phenotype. Standard DNA extraction, library preparation and WGS were performed using Illumina HiSeq X and variants were classified following standard guidelines. Molecular diagnosis was made in 24 patients (86%), with 100% variant confirmation by current gold standard of long-range PCR and Sanger sequencing. We demonstrated unique alignment of sequencing reads over the pseudogene-homologous region. In addition to identifying function-affecting single-nucleotide variants and indels, we identified single- and multi-exon deletions affecting PKD1 and PKD2, which would have been challenging to identify using exome sequencing. We report the first use of WGS to diagnose ADPKD. This method overcomes pseudogene homology, provides uniform coverage, detects all variant types in a single test and is less labour-intensive than current techniques. This technique is translatable to a diagnostic setting, allows clinicians to make better-informed management decisions and has implications for other disease groups that are challenged by regions of confounding sequence homology.

Figure 1

Variant pathogenicity classification algorithm. Algorithm used to classify pathogenicity of every variant identified. Modified from guidelines issued by American College of Medical Genetics and Genomics.

Figure 2

Coverage and Mapping Quality (MQ) for PKD1, PKD2 and pseudogenes. (a) Coverage across PKD1 and PKD2. The percentage of bases covered with at least 15 unique reads for each exon for PKD1 and PKD2 is shown ±95% confidence interval. The bars are coloured proportionally to the percentage covered. The average GC content of each exon is also shown (red line) on the same scale. (b) MQ for PKD1, PKD2 and pseudogenes. MQ is a measure of the likelihood that a particular read is aligned to the correct segment of the genome. For BWA MEM, the sequence aligner that we used, the maximum possible MQ is 60. The average mapping quality ±95% confidence interval, from n=6 patients are shown (patient ids: 506, 547, 609, 610, 625, 626).

Figure 3

Types of variants detected in PKD1 and PKD2 via whole-genome sequencing. The pathogenicity rating is based on current ACMG guidelines, described in more detail in Figure 1. VOUS, variant of uncertain significance.

Figure 4

Single-exon PKD2 deletion detected with whole-genome sequencing. A 5461 bp base deletion, which overlaps exon 3 of PKD2. Three lines of evidence support the heterozygous deletion: the reduction in read depth to ~50% of the surrounding regions; the presence of three spanning read pairs where each read aligned to either side of the deletion; and the presence of four split reads, where the start and end of the sequencing reads are on either side of the breakpoint.