Dr VICKI ATHANASOPOULOS1, Ms JEAN CAPPELLO1, Dr TOM LEA-HENRY1, Ms BRIDIE MOY1, Dr SIMON H JIANG1,2
1John Curtin School Medical Research, Australian National University, Acton, Australia, 2The Canberra Hospital, Garran, Australia
Aim: To develop a high-throughput platform to map the functional consequence and pathogenicity of genetic variants that contribute to CKD in Indigenous populations.
Background: Indigenous Australian populations have the highest recorded rates of CKD worldwide with genetics identified as a major contributor. We have established a national collaboration to sequence and define the genetic contribution to the high rates of CKD in Indigenous communities.
Methods: We designed a high-throughput screening platform utilising cell lines, each capable of assaying four biochemically relevant immunological pathways simultaneously (8 total), utilising distinct luciferase gene emission spectra. The lines were generated by stably transfecting human embryonic kidney (HEK293) cells with a Multiplex Luciferase Reporter (MLR) plasmid. Luciferase activity correlates with gene expression driven by promoters responsive to major disease-relevant pathways (eg. NFkB, TGFβ, IFNβ, NFAT; or IFNα, STAT1-GAS, STAT3-site, AP1). Reporter lines expressing wild-type or variant genes are treated with a range of stimuli and biochemical pathway signalling differences determined from luciferase readouts.
Results: We identified rare, potentially deleterious variants in CKD-relevant genes at high frequencies in patients from the Tiwi Islands including A20/TNFAIP3, AIRE, BLK, IRF5 as well variants in IRAK3, LYN, TRAF5 and RELB which are unique to the Tiwi. Variants in A20, LYN and AIRE were validated for altered function. Whilst the LYN variant had no impact, one variant in AIRE and two in A20 impaired IFNβ and NFkB expression respectively, highlighting the feasibility of multiplexing to rapidly screen for deleterious variants in inflammatory genes within the Indigenous population.
Conclusions: This multiplex system allows mass-testing of genetic variants in our Indigenous cohorts and identifies unique pathogenic variants in these communities.
Biography:
Dr Vicki Athanasopoulos’ (PhD.) research focuses on discovering genetic causes of autoimmunity, and kidney disease, and in understanding the molecular and pathogenic mechanisms of genetic variants so as to refine diagnosis and aide development of effective and targeted therapies for patients. She has worked on several autoimmune models, including the san roque mouse, and currently codirects research on the molecular underpinnings of autoimmune and kidney disease in the laboratory of Dr Simon Jiang (ANU).