Peer-review started: December 13, 2017
First decision: December 27, 2017
Revised: December 31, 2017
Accepted: February 4, 2018
Article in press: February 4, 2018
Published online: March 6, 2018
Processing time: 82 Days and 12.9 Hours
The primary cilia and polycystins plays an important role in the regulating the severity of autosomal dominant polycystic kidney disease (ADPKD). While the loss of cilia or polycystins alone results in the development and progression of renal cysts, renal cilia involution reduces the progression of cyst growth induced by the inactivation of polycystins. The epithelial cells of renal cysts usually show various structural deformities of the primary cilium involve an absence, shortening or elongation. These structural changes can be caused by genetic defects of ciliary proteins. Mutation profile of ciliary genes in human ADPKD tissues is unknown. Revealing a genetic basis for ciliogenesis defects may identify causative factors of disease progression and the potential molecular targets for the development of new therapies of ADPKD.
Genetic defects of various ciliary genes whose inactivation leads to the development and progression of ADPKD have been identified in mouse models. However, recent studies have confirmed that the animal models of ADPKD incompletely mimic the human disease. Therefore, it is important to detect genetic abnormalities that can affect ciliogenesis directly in ADPKD human tissues.
The main objectives of this study is to identify the genetic defects of ciliary genes causing the loss of primary cilium in ADPKD human tissues. The results of our study are important indicators for directing further analysis.
In our study, we analyzed 191 structural and functional ciliary genes using next-generation sequencing analysis. The tissue samples used in this study were obtained from 7 patients with ADPKD who underwent nephrectomy. Each sample contained polycystic kidney tissue and matched normal kidney tissue. All analyzed samples were formalin-fixed and paraffin-embedded. The germline origin of the identified variants was excluded by analysis of DNA extracted from peripheral blood.
We identified unique of mutation profile in each of analyzed ADPKD samples, which was characterized by the presence of pathogenic variants in 5 to 15 ciliary genes. The most frequently identified defects were found in genes encoding centrosomal proteins and kinesin family member 19, which are important for ciliogenesis. In addition, pathogenic variants in the PCM1 and KIF19 genes were found in all ADPKD samples.
Our study had found that the human ADPKD tissues are characterized by the presence of several genetically altered ciliary proteins that plays an important role in ciliogenesis. The structural and functional disturbance of the primary cilium can be induced by mutations of these proteins. An interesting finding of our study was that the mutations in genes encoding the proteins of intraflagellar transport (IFT) were rarely mutated. These genes are considered candidate genes related to ADPKD in mouse models. Centrosomal proteins and kinesin family member 19 are the most commonly mutated ciliary proteins in renal epithelial cells derived from human ADPKD cysts. Consistent with finding of recent studies, we can confirm that animal models not completely mimic the human disease. Mouse models induce polycystic kidney disease by genetic inactivation of one ciliary gene especially genes encoding IFT proteins. However, the genes encoding the proteins of IFT are rarely mutated in the human renal cystic cells. This study offered new insight into comprehensive mutation profile of ciliary genes in human ADPKD tissues. The results of our study suggested that the loss of the primary cilia in the human ADPKD tissues may be predominantly caused by defects of centrosomal proteins and kinesin family member 19. The defects of centrosomal proteins may be also the cause of chromosome imbalances, which are often present in human ADPKD tissues. The genetic defects of the KIF19 gene may be cause of the primary cilium elongation, which is a characteristic feature of the end-stage renal disease. This is the first study used of archived formalin-fixed and paraffin embedded tissues (FFPE) of ADPKD in order to determine mutation profile of ciliary genes by next-generation sequencing methods. An interesting finding was the presence of only one somatic pathogenic variant in each individual ciliary gene. To determine whether they are “hotspot” mutations representing secondary somatic events in the development of the disease will require further analysis. Somatic mutations in genes encoding centrosomal proteins and KIF19 were present in all analyzed ADPKD samples. These mutations were present exclusively in polycystic kidney tissues and did not occur in matched control so we can assume their effect on cystogenesis. If our hypotheses will be confirmed by further studies, the identified ciliary proteins may represent potential molecular targets for the development of new treatments.
Mutation profile of ciliary genes can be analyzed directly from archived FFPE tissues of ADPKD by NGS. The first step, it is necessary to verify the results on a larger number of samples and matched tissues controls. Further research should be focused on the functional analysis of identified genetic variants. Consequently, it will be necessary to determine whether the inactivation of these genes will lead to a change in the structure of renal cilia and affect the development or progression of ADPKD.