Co-Inheritance of Functional Podocin Variants with Heterozygous Collagen IV Mutations Predisposes to Renal Failure

<b><i>Background/Aims:</i></b> A subset of patients who present with proteinuria and are diagnosed with focal segmental glomerulosclerosis (FSGS) have inherited heterozygous <i>COL4A3/A4</i> mutations and are also diagnosed with thin basement membrane nephropathy (TBMN-OMIM: 141200). Two studies showed that co-inheritance of <i>NPHS2</i>-p.Arg229Gln, a podocin variant, may increase the risk for proteinuria and renal function decline. <b><i>Methods:</i></b> We hypothesized that additional podocin variants may exert a similar effect. We studied genetically a well-characterized Cypriot TBMN patient cohort by re-sequencing the <i>NPHS2</i> coding region. We also performed functional studies in cell culture experiments, investigating the interaction of podocin variants with itself and with nephrin. <b><i>Results:</i></b> Potentially disease-modifying podocin variants were searched for by analyzing <i>NPHS2</i> in 35 ‘severe' TBMN patients. One non-synonymous variant, p.Glu237Gln, was detected. Both variants, p.Arg229Gln and p.Glu237Gln, were tested in a larger cohort of 122 TBMN patients, who were categorized as ‘mild' or ‘severe' based on the presence of microscopic hematuria alone or combined with chronic renal failure and/or proteinuria. Seven ‘severe' patients carried either of the 2 variants; none was present in the ‘mild' patients (p = 0.05, Pearson &#x03C7;²). The 7 carriers belong in 2 families segregating mutation <i>COL4A3</i>-p.Gly1334Glu. Inheritance of the wild-type (WT) and mutant alleles correlated with the phenotype (combined concordance probability 0.003). Immunofluorescence (IF) experiments after dual co-transfection of WT and mutant podocin suggested altered co-localization of mutant homodimers. IF experiments after co-transfection of WT podocin and nephrin showed normal membrane localization, while both podocin variants interfered with normal trafficking, demonstrating perinuclear staining. Immunoprecipitation experiments showed stronger binding of mutant podocin to WT podocin or nephrin. <b><i>Conclusion:</i></b> The results support the hypothesis that certain hypomorphic podocin variants may act as adverse genetic modifiers when co-inherited with<i> COL4A3/A4</i> mutations, thus predisposing to FSGS and severe kidney function decline.