Analysis of PKD1 gene variations at RNA level in polycystic kidney disease

Polycystic kidney disease (PKD) is the most common hereditary kidney disease. It is a genetic disorder characterized by the progressive growth of kidney cysts, leading to kidney failure and hypertension, occurring in 1 in 500 to 1,000 births. PKD can manifest from newborns to the elderly, resulting in numerous cysts in the kidneys. These cysts progressively grow, eventually leading to the kidney being entirely composed of cysts and losing its function. Approximately 85% of PKD cases are associated with the PKD1 gene (Polycystin1). Assessing mutations in the PKD1 gene presents certain methodological challenges. The region between exons 1 and 32 of the gene is located within a segmental duplication region that has repeated six times at a distance of 13-16 Mb from the main gene. The Long- Range PCR (LR-PCR) method, which covers a wide area specific to PKD1, is used to avoid false positives in the analysis of this region. In our study, we aimed to evaluate the detectability of PKD1 mutations by sequencing cDNA obtained from RNA to overcome the challenges posed by segmental duplications in the analysis of the PKD1 gene at the DNA level. For this purpose, among 24 cases referred to the Genetic Diseases Assessment Center at Trakya University Medical Faculty with a preliminary diagnosis of PKD, 9 cases with pathogenic/likely pathogenic variants in the PKD1 gene were identified, and variations determined at the DNA level were examined at the RNA level in six of these cases. Additionally, the gene expression levels of RNA encoded by the PKD1 gene were investigated in six cases and ten controls using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). In our study, variations previously identified at the DNA level were confirmed at the RNA level in five cases. Among the six variations identified in the included cases, three (50%) were novel variations not present in the literature and databases at the time of the study. The novel NM_001009944(PKD1).2853+2_2853+10del splice region variation observed in our case series led to the recognition of a new splice site by losing the canonical splice region in the PKD1 gene. In the analyses of gene expression levels of the PKD1 gene in peripheral blood, although there was a general tendency for lower expression in cases, no statistically significant expression difference was found between cases and controls. In our study, low-level Sanger sequencing analysis of RNA revealed that the PKD1 regions within segmental duplication areas partially contained gene sequences. According to our Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction results, no significant expression difference was observed between cases and controls. While the limited number of cases and controls included in our study presents a limitation, our findings highlight the importance of functional analyses with RNA in determining the effects of new splice region variations on gene expression levels. However, the low level of PKD1 gene expression in blood and the presence of peaks related to segmental duplications indicate that RNA-based analysis isolated from peripheral blood in cases of polycystic kidney disease cannot independently serve as an alternative to diagnostic long-range DNA sequencing approaches.