Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing

Abstract

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE).

Methods: To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants.

Results: Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene.

Conclusions: These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.

Trial registration: ClinicalTrials.gov NCT00792155.

Graphical abstract

Figure 1.

WGS shows a deep and consistent coverage of PKD1 and PKD2 exons. The horizontal axis represents genomic coordinates. The vertical axis indicates median WGS read coverage across 90 cysts for the PKD1 (top panel) and PKD2 (bottom panel) regions. Exonic regions are colored in purple whereas intronic regions are in green. Segmental duplicated regions of PKD1 (exons 1–33) and previously known low-complexity genomic regions are marked. WGS reached consistent coverage of all exons, including segmentally duplicated regions of PKD1.

Figure 2.

Somatic alterations of PKD1 and PKD2 were detected in 90 cysts by WGS. The somatic short mutations were distributed throughout the entire length of the PKD genes, without a hotspot region (top panel). Taking somatic structural alterations into account, 33% were deletion mutations, 20% were insertions/duplications, 29% were substitutions (SNVs), and the remaining 18% were LOH—either copy number neutral (15%) or hemizygous deletions (3%) (bottom panel).

Figure 3.

Large-scale somatic structural alterations disrupt PKD1 and PKD2 in 90 cysts. Top panel: genome-wide patterns of large-scale somatic alterations in 90 cysts. The horizontal axis represents the genome. The vertical axis indicates the frequency of somatic copy number neutral LOH events (green) or deletions (blue), and chromosomal amplifications (red). Recurrently affected somatic regions where PKD1 and PKD2 genes reside are indicated. Bottom panel: shift in VAFs of germline pathogenic PKD1 and PKD2 variants in somatic LOH regions of cysts. In 17 cysts (purple) with somatic LOH events at PKD1 and PKD2 loci, the VAFs of the germline pathogenic variants shifted away from heterozygosity observed in matched PBLs (red). In all except one cyst, the LOH events led to increased VAFs of the pathogenic allele, suggesting biallelic inactivation of PKD1 or PKD2.

Figure 4.

Large chromosome fragment rearrangement involved in LOH. The size and location of the chromosomal fragments involved in the rearrangement are marked on the chromosomes encompassing PKD1 and PKD2. The black lines indicate the positions where the chromosome rearrangement occurred. The CFSs are highlighted with red lines. The original schematics for chromosomes 4 and 16 were obtained and modified from https://ghr.nlm.nih.gov/chromosome/4#idiogram_chromosome_4 and https://ghr.nlm.nih.gov/chromosome/4#idiogram_chromosome_16.