SO094HUMAN URINE-DERIVED RENAL EPITHELIAL CELLS PROVIDE INSIGHTS INTO THE PATHOGENICITY OF PKHD1 VARIANTS

Abstract

Abstract Background and Aims A 26 year old woman was referred for the investigation of polycystic kidney disease. There was no family history of renal disease and no obvious extra-renal manifestations. A GEMINI ciliopathy gene panel was performed which identified two heterozygous sequence changes (segregating from each parent) in PKHD1: a missense variant c.7964A>C, p.(His2655Pro), listed as a pathogenic variant in association with ARPKD on the Human Gene Mutation Database and a synonymous variant c.6900C>T, p.(Asn2300Asn). The pathogenicity of the synonymous PKHD1 variant is not clear. It is reported as a variant of unknown significance on ClinVar and has a low population frequency in the gnomAD cohort. The altered nucleotide is weakly conserved, but the same heterozygous change has been previously reported in an individual prenatally presenting with multicystic kidneys together with a missense variant on the other allele. We aim to confirm the pathogenicity of this variant in our patient, analysing its effects on the splicing of PKHD1 mRNA. Method PKHD1 is expressed at low levels in the leukocytes, therefore it can be difficult to analyse the splicing of PKHD1, using routine methods that involve RNA extraction from the blood. Urine-derived renal epithelial cells were isolated and cultured from two wild type individuals and from the patient and total RNA was extracted from these cells. RT-PCR and RT-qPCR were carried out to analyse the effects of the synonymous variant on the splicing of the PKHD1 gene in renal epithelial cells. Results RT-PCR revealed that PKHD1 is alternatively spliced both in the controls and in the patient and Sanger sequencing following T-cloning of PCR products revealed that both controls and the patient express an in-frame transcript and a shorter transcript with a 47 nucleotide loss in the exon 43 of PKHD1, that leads to a frame-shift and to the formation of a premature stop codon. We hypothesized that, although both mRNA isoforms are expressed in controls as well as in the patient URECs, the variant p.(Asn2300Asn) may shift the expression ratio between the two transcript isoforms in favour of the shorter, out-of-frame transcript. We designed and tested two sets of transcript-specific primers and performed a SYBR-green based RT-qPCR on controls and patient URECs cDNA. RT-qPCR, revealed that PKHD1 is expressed at lower levels in patient URECs, compared to controls. Specifically, the in-frame PKHD1 isoform is expressed at lower levels in patient URECs, compared to controls, whereas the levels of the shorter transcript leading to a frame-shift are higher in patient cells. Conclusion Using urine-derived renal epithelial cells as a source of kidney-specific RNA, we confirmed the pathogenicity of the PKHD1 synonymous variant p.(Asn2300Asn), which leads to an increased expression of an out-of-frame PKHD1 transcript, predicted to result in a truncated protein and expressed at lower levels also in control cells. The significance of this isoform in control renal epithelial cells is unclear.