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The Investigation of The Effects of A Novel Missense Mutation In The Katanin-Like 2(katnal2) Gene In Patient Fibroblasts and Fibroblast-Derived Cells Using Functional Analysis Methods

Objective:KATNAL2(Katanin A-like2) is a microtubule-severing enzyme. Studies in cell cultures and lower organisms demostrated its role in neuron growth and cell division. In human, KATNAL2 is defined as a risk gene for autism spectrum disorder in a few reports. However, there is no functional studies. Our aim was to perform functional analyzes in cells obtained from a male patient with intellectual disability, epilepsy and autistic behavior carrying a pathogenic KATNAL2 variant(p.Ser392Pro)(Figure1). Methods:By conducting skin biopsy, fibroblast cultures were generated. Induced pluripotent stem cells(iPSCs) were derived from fibroblasts using episomal plasmid method. After performing the characterization of the iPSCs, they were transformed to neural progenitor cells(NPCs) using STEMdiff™-SMADi Neural Induction Kit(Figure2,3). Functional analyzes including Western blotting(WB), immunfluorescent(IF) antibody stainings, proteomics and RNA-Sequencing were performed in fibroblasts and NPCs. Results:KATNAL2 IF staining was weaker, and in tubulin-alpha IF staining, the mitotic spindles were irregular in patient's cells compared to control(Figure4). In fibroblasts, RNA-Seq enrichment analysis of the high/low expressed genes demonstrated an association with microtubule and spindle structures and their role in mitosis, cell division and DNA replication. Proteomics analysis demonstrated extracellular matrix proteins that interact with the cytoskeleton were organized differently in patient’s cells in enrichment/overrepresentation analyzes. WB revealed no significant change in protein amounts between the mutant and control cells. Conclusion:For the first time, we performed functional studies in KATNAL2-mutant human cells and demonstrated defects in microtubule-related functions. Our findings contributed to understand the disease mechanism and will form basis for further treatment studies. (This study is supported by TUBITAK-120S404)