GENERATION OF A C. ELEGANS MODEL TO STUDY THE INTERALLELIC INTERACTIONS OF PODOCIN

Abstract Background and Aims NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. NPHS2 encodes podocin, a key component of the glomerular filtration barrier. Podocin is a 42 kDa integral membrane protein, which accumulates in lipid raft microdomains at the podocyte slit diaphragm and is known to form oligomers. The c.686G>A, p.R229Q is the most common non-silent variant of NPHS2. We formerly described that R229Q is pathogenic only when trans-associated to specific 3’ missense mutations on the other parental allele. These C-terminal podocin mutants exert a dominant negative effect on R229Q podocin and retain it in intracellular compartments. As such, R229Q is the first variant in human genetics with a mutation-dependent pathogenicity. Based on FRET analysis and structural modeling, we formerly showed that the dominant negative effect is mediated by an altered oligomerization. However, the pathogenicity of several [mutation];[R229Q] associations is still in question and the identification of other rare NPHS2 variants with a mutation-dependent pathogenicity require an in vivo model. We therefore aimed to generate a Caenorhabditis elegans model, deficient for the homologous gene of NPHS2 (mec-2) and coexpressing differently (GFP- and mCherry-) tagged human podocin pairs. MEC-2 shares 45% identity and 83% similarity over 275aa (72%) of podocin (383aa). Expressed in six neurons, it is responsible for gentle-touch mechanosensation. The mec-2 mutants are insensitive to gentle touch in the center part of the body. Method Vectors encoding C-terminal GFP- or mCherry-tagged MEC-2 or human podocin with C. elegans codon optimization under mec-2 promoter and a selection marker (unc-119) were generated. Double (mec-2 and unc-119) mutant strains were established. Mutant strains were transformed by microparticle bombardment. The gentle-touch mechanosensation was examined by cat’s whiskers in a blinded experiment. Results The expression pattern of GFP and mCherry under mec-2 promoter corresponded to the six neurons responsible for gentle-touch sensation, indicating the proper functioning of the promoter. Strains with extrachromosomal MEC-2 or podocin coding vectors were successfully established (GFP-tagged MEC-2: n= 55 strains, GFP-tagged podocin: n= 81, mCherry-tagged podocin: n= 18). However, we found no rescue of the gentle touch sensation in any of them in blinded experiments. We hypothesized that either the fluorescent tag or the lack of chromosomal integration prevents the rescue effect of MEC-2. We therefore aimed to achieve self-cleaving of MEC-2 and the fluorescent tag, and inserted a T2A self-cleaving peptide-encoding sequence between them. To achieve chromosomal integration, we are implementing the MosSCI (Mos1-mediated Single Copy Insertion) technique. Conclusion Once the rescue with MEC-2 is achieved, the rescue effect of wild type and next different human podocin variant(s) will be aimed to analyze. The generation of the first animal model to study human interallelic interactions is challenging.