Dr Laura Wedd1, Yvonne Hort1, Dr Chirag Patel3, Prof John Sayer4,9, Prof Andrew Mallett5,7,8, Dr Denny Cottle6, Prof Ian Smyth6, Dr Timothy Furlong1, Prof John Shine1, Dr Amali Mallawaarachchi1,2
1Garvan Institute Of Medical Research, Sydney, Australia, 2Clinical Genetics Service, Royal Prince Alfred Hospital, Sydney, Australia, 3Genetic Health Queensland, Royal Brisbane and Women’s Hospital, Brisbane, Australia, 4Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom, 5Department of Renal Medicine, Townsville University Hospital, Townsville, Australia, 6Development and Stem Cells Program, Monash Biomedicine Discovery Institute & Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia, 7College of Medicine and Dentistry, James Cook University, Townsville, Australia, 8Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia, 9Renal services, The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom
Biography:
Dr Mallawaarachchi is a Clinical Geneticist and Nephrologist, and Staff Specialist at Royal Prince Alfred Hospital, Sydney and Head of the Molecular Genetics of Inherited Kidney Disorders Laboratory at the Garvan Institute of Medical Research. Her current research focuses on understanding the molecular basis of polycystic kidney disease and in genetic diagnostics in inherited kidney disease. She is also passionate about education and is the co-lead of Kidney School and Chair of the ANZSN Education and Training Committee.
Background:
ADPKD, caused by pathogenic variants in PKD1 and PKD2, is the most common monogenic cause of kidney failure. Approximately 10% of ADPKD patients remain undiagnosed after coding-region focused genomic testing. Non-coding variants in regulatory regions are not an established cause of disease in ADPKD.
Methods:
We performed regulatory region analysis on short-read whole genome sequencing data, in a cohort of undiagnosed ADPKD patients (n=20). We extended this analysis to patients with undiagnosed cystic kidney disease within the Australian KidGen cohort (n=42) and the Genomics England 100,000 Genomes rare disease cohort (n=1320). We then developed a PKD1 5’UTR-luciferase translation assay to functionally characterise candidate variants identified through these genomic studies. Constructs were designed to model candidate variants.
Results:
We identified two rare, potentially disease-causing variants in the 5’ UTR of PKD1. Our PKD1 5’UTR-luciferase translation assay showed that one of these variants (PKD1 c.-69dupG), reduced the translation efficiency of the main PKD1 open reading frame by 87% compared to wildtype (p<0.0001), establishing this novel variant as disease-causing. We were then able to rescue the effect of this pathogenic variant by introducing novel downstream sequence alterations. Finally, using our model, we found that knocking-out the two upstream open reading frames (uORFs) of the wildtype PKD1 5’UTR sequence, resulted in a 2.3x increase in wildtype polycystin-1 expression (p<0.0001).
Conclusions:
We show that PKD1 5’-UTR variants are an overlooked rare cause of ADPKD and that analysis of this region should be included in genomic-analysis pathways. We also show that manipulation of the wildtype 5’UTR sequence can increase polycystin-1 expression, providing insights into regulation of PKD1 and suggests new approaches for therapeutic intervention in this haplo-insufficient disease.
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