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bioRxiv2026

Integrative proteomic analysis and molecular dynamics simulations of ANKLE2 reveal mechanisms of microcephaly

ANKLE2 is a scaffolding protein with crucial roles in neuroprogenitor cell division and embryonic brain development. Pathogenic variants in ANKLE2 cause primary microcephaly, a congenital disorder characterized by impaired neurodevelopment. Nonetheless, the underlying dysfunction of ANKLE2 is not understood. Here, we define the ANKLE2 protein interaction landscape, which includes cell division proteins and novel microcephaly candidates in the APC (anaphase-promoting complex). Analysis of six pathogenic variants reveals changes in this interactome that likely result in microcephaly. Using molecular simulations, we identify the structural consequences of five pathogenic substitutions, including disruption of important helical structures. Combined, these techniques suggest loss of interaction with the PP2A (protein phosphatase 2A) complex is a common mechanism for ANKLE2 pathogenesis. Phosphoproteomics reveals widespread alterations in ANKLE2- and PP2A-dependent phosphorylation of cell division proteins. Together, our integrative proteomics and simulation approach improves the molecular understanding of ANKLE2 function and its pathogenic changes in primary microcephaly.

systems biology↗

bioRxiv2024

Identification of CNTN2 as a genetic modifier of PIGA-CDG through pedigree analysis of a family with incomplete penetrance and functional testing in Drosophila

Loss of function mutations in the X-linked PIGA gene lead to PIGA-CDG, an ultra-rare congenital disorder of glycosylation (CDG), typically presenting with seizures, hypotonia, and neurodevelopmental delay. We identified two brothers (probands) with PIGA-CDG, presenting with epilepsy and mild developmental delay. Both probands carry PIGAS132C, an ultra-rare variant predicted to be damaging. Strikingly, the maternal grandfather and a great-uncle also carry PIGAS132C, but neither presents with symptoms associated with PIGA-CDG. We hypothesized genetic modifiers may contribute to this reduced penetrance. Using whole genome sequencing and pedigree analysis, we identified possible susceptibility variants found in the probands and not in carriers and possible protective variants found in the carriers and not in the probands. Candidate variants included heterozygous, damaging variants in three genes also involved directly in GPI-anchor biosynthesis and a few genes involved in other glycosylation pathways or encoding GPI-anchored proteins. We functionally tested the predicted modifiers using a Drosophila eye-based model of PIGA-CDG. We found that loss of CNTN2, a predicted protective modifier, rescues loss of PIGA in Drosophila eye-based model, like what we predict in the family. Further testing found that loss of CNTN2 also rescues patient-relevant phenotypes, including seizures and climbing defects in Drosophila neurological models of PIGA-CDG. By using pedigree information, genome sequencing, and in vivo testing, we identified CNTN2 as a strong candidate modifier that could explain the incomplete penetrance in this family. Identifying and studying rare disease modifier genes in human pedigrees may lead to pathways and targets that may be developed into therapies.

genetics↗