Deciphering the pathomechanism of monogenic disorders associated with pathogenic variants in C20orf204, FBXW11 and WDHD1

• Applicant: Professorin Dr. Kerstin Kutsche
• Subject Area: Human Genetics
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 511275754

Next-generation sequencing technologies have accelerated the discovery of novel disease genes for monogenic disorders. To prove pathogenicity of genomic variants and improve our understanding of disease biology and pathophysiology, a variety of bio¬che¬mical and cell biological assays can be applied on patient-derived cells and/or well-established cell models. Functional studies are particularly important to decipher the function of uncharacterized open reading frames in which pathogenic variants cause disease.Within this project, we will perform functional studies for pathogenic variants in three genes. We identified the de novo FBXW11 missense variant c.1139C>T/p.(Ser380Phe) in a patient with craniosynostosis, microcephaly, cloverleaf scull, and facial dysmorphism. Seven de novo disease-associated FBXW11 missense variants in individuals with diverse phenotypes have been reported. FBXW11 encodes an F-box protein that functions as substrate adaptor for the S-phase kinase-associated protein (SKP)-cullin 1-F-box protein ubiquitin ligase complexes involved in proteasomal degradation of various substrates. To gain insight into the differential functional effects of FBXW11 variants, we will study the impact of p.(Ser380Phe) and four published variants on FBXW11 substrate binding by determining the set of binding partners of HA-tagged FBXW11 wildtype and mutants in a cellular system. Substrates with decreased or enhanced binding to FBXW11 mutants will be investigated further to determine their turnover rate and steady-state and ubiquitination levels. Possible further experiments will focus on specific pathways and/or cellular functions affected by p.(Ser380Phe) and/or other FBXW11 variants. We identified C20orf204, encoding a poorly characterized protein, as a novel autosomal recessive disease gene for a severe immunodeficiency. Our preliminary data revealed expression of C20orf204 in naïve T cells and a predicted function as a cytokine or cytokine inhibitor. To characterize the biological role of C20orf204 in T cells, we will collaborate with experts in the respective field. We will produce and purify recombinant C20orf204 protein in eukaryotic cells. We will then study the possible function of C20orf204 in controlling T cell homeostasis by analyzing if C20orf204 signals through the interleukin 7 receptor, if C20orf204 is a negative regulator of thymic stromal lymphopoietin and by investigating the role of C20orf204 in the survival and maintenance of naïve T cells. We identified biallelic WDHD1 pathogenic variants in individuals with microcephalic primordial dwarfism. WDHD1 integrates the CMG helicase complex and DNA polymerase α/primase complex to initiate DNA replication. By using patient-derived fibroblasts, we will study the impact of WDHD1 variants on cell cycle and cell proliferation, DNA damage response, and the DNA replication machinery. Our studies will advance our understanding of biological processes underlying human health and disease.

• DFG Programme: Research Grants
• Co-Investigators: Professorin Dr. Kerstin Borgmann; Professorin Dr. Eva Tolosa; Dr. Susanne Witt