Nörogelişimsel hastalıklarla ilişkilendirilen aday genlerin işlevsel validasyonu

This study aims to elucidate genetic diseases through the validation and characterization of novel mutations identified in a group of children born of consanguineous marriages. Three such pediatric patients were selected for further investigation. They were all clinically suspected of genetic diseases but were unable to be diagnosed with known genetic mutations. Clinical findings included microcephaly, motor-mental retardation, neuro-developmental disorders, and epilepsy. Whole exome sequencing identified previously unreported candidate mutations: nucleotide deletions in WDR62 and ATG9B genes resulting in frameshift and premature termination of translation, and a missense mutation in VARS1 gene resulting in single amino acid alteration. In the literature, WDR62 mutations were extensively reported as causative factor for microcephaly. Therefore, the WDR62 mutation was only validated by Sanger sequencing. The effects of the VARS1 mutation were studied by structural modeling and shown to have destabilizing effects on the VARS1 protein. The focus of this thesis was the ATG9B mutation. ATG9B has not been associated with any genetic disease and its function is poorly characterized in literature. The eleven-nucleotide deletion identified in the ATG9B gene causes a frameshift resulting in protein truncation. We characterized WT and mutant ATG9B by ectopic expression in cells and developed several mouse models for in vivo studies. We showed that truncated protein is not stable when expressed in cells. Using in vivo models, we demonstrated that Atg9b is expressed in the placenta and testis, but not essential for viability or fertility. We didn't observe any major histological differences between WT and mutant placenta. On the other hand, behavioral assays revealed reduced fear memory in the homozygous knockin animals. We additionally developed knockout and FLAG knockin mouse models of ATG9B, and generated antisera to study its functions in vivo and in vitro.