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We read with interest the paper by Urribarri et al 1 which describes that metalloprotease hyperactivity plays an important role in cyst expansion and that metalloprotease inhibition reduces cyst proliferation. As such, these results help to identify potential drug targets.2 We hypothesise that the expansion and maintenance of the cyst is preceded by mutational events that trigger cytogenesis, and we used genetic analysis to provide additional insight into this process. The majority of polycystic diseases are autosomal dominant disorders where every patient cell possesses one germ line mutation (first hit).3 As somatic second-hit mutations play an important role in liver and renal cyst formation,4–6 it was hypothesised that patients with polycystic disease have a DNA repair defect and accumulate somatic mutations.7 This was supported by a comparative genomic hybridisation study where renal cysts harboured multiple chromosomal aberrations, similar to cancers.8 ,9 In addition, patients without a germ line mutation can still develop sporadic cysts, which begs the question if and how somatic mutations contribute here.10 Therefore, we used a genome-wide approach to determine the type and extent of somatic mutations in solitary cysts and polycystic livers.
We collected cyst epithelium of 23 cysts (22 liver cysts, 1 kidney cyst) from 7 patients with different underlying diseases (table 1). All patients were initially screened for germ line mutations in PRKCSH, SEC63 and PKD2. Cyst epithelial cells were collected from fresh tissue samples using EDTA and a CK19 staining was used to analyse the purity of each sample. We assessed copy-number variations and loss of heterozygosity (LOH) regions through genome-wide high resolution cytogenetic array analysis (CytoScan HD, Affymetrix) and searched for intragenic mutations by sequencing.6
In the patients with autosomal dominant polycystic liver disease and autosomal dominant polycystic kidney disease (Patient#1, #2 and #3), we found that 13/18 cysts had acquired a somatic second-hit mutation (table 1). Using genome-wide data we could map the regions affected by LOH and determine the overall genome stability in the cysts (see online supplementary figure S1). LOH was caused by either terminal copy-number neutral (CNN) LOH (5–17 Mb) or interstitial deletions (16–45 Mb), depending on the patient. In Patient#2 we did not detect a germ line mutation in PRKCSH, SEC63 or PKD2, but in 2/6 liver cysts we detected telomeric CNN LOH of respectively 5 Mb and 17 Mb on chromosome 16p suggesting that the affected gene may be located here (see online supplementary figure S1C). This region includes PKD1, the main gene for autosomal dominant polycystic kidney disease, however, sequencing of PKD1 did not reveal any germ line mutation in genomic DNA of this patient. In Patient#3 we identified a novel PKD2 c.1536_1538delTGT germ line mutation. In addition to the liver cysts, we also analysed one kidney cyst of this patient and found loss of function of PKD2 through telomeric CNN LOH (104 Mb).
As it is still unknown what drives cyst formation in sporadic liver cysts we also included 5 sporadic cysts in our analysis (table 1). Single (sporadic) hepatic cysts are found in 10% of the normal population with a clear age dependency. In one cyst we identified a complex biallelic deletion on chromosome 4q resulting in a homozygous loss of a 2.6 Mb region containing PKD2 (see online supplementary figure S1E). We did not detect any genomic aberrations in other sporadic cysts.
In conclusion, loss of both wild type alleles seems sufficient to drive cyst formation, and consistent with the benign growth of the cysts we detected absence of genomic instability or additional large genomic aberrations. Therefore, the genetic changes that we see in these cysts fit with a somatic single-step model of cystogenesis. Finally, we found that a homozygous deletion of the PKD2 genomic region in a solitary cyst provides the long-awaited support for the hypothesis that somatic loss of a polycystic disease gene contributes to the formation of sporadic cysts.
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Contributors MJJ: Study concept and design, acquisition of data, analysis and interpretation of data, statistical analysis and drafting and revising of the manuscript. JS: Acquisition of data, analysis and interpretation of data, technical support and revising the draft paper. CB: Acquisition of data, technical support and revising the draft paper. RP: Acquisition of data, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, technical and material support. JPHD: Study concept and design, critical revision of the manuscript for important intellectual content, obtained funding and supervised the study.
Funding Funding for this study was provided by the Institute for Genetic and Metabolic Diseases (Radboudumc).
Competing interests CB is an employee of Bioscientia/Sonic Healthcare.
Patient consent Obtained.
Ethics approval Commissie Mensgebonden Onderzoek (CMO) regio Arnhem-Nijmegen.
Provenance and peer review Not commissioned; internally peer reviewed.
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