Identification of De Novo Germline Mutations and Causal Genes for Neurological Diseases Using Trio‐Based Whole‐Exome Sequencing in a Turkish Population

Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Gunel, Murat

Abstract

Recent progress in next-generation sequencing has greatly enhanced our comprehension of the genetic foundation of neurodevelopmental disorders. However, in populations with elevated consanguinity rates, particularly in cases of uncommon diseases, our understanding of pathogenic genetic variations is still restricted. Fifty-nine trios and two quads from clinical centers across Turkey were analyzed in our study, all with probands exhibiting neurological features of varying severity, including brain malformations. Our cohort was also enriched for consanguineous families. Whole-exome sequencing (WES) was performed, followed by bioinformatic analyses to detect potential pathogenic variants. Several modes of inheritance were assessed, including homozygous recessive, compound heterozygous, and de novo variants. We created a customized pathogenicity scoring system to prioritize likely causal mutations. Variants previously implicated in neurological disease such as CEP290, ATP1A3, and PAFAH1B1 were confirmed in our cohort. Additionally, novel variants were identified in candidate genes: TOX3, SOCS7, and PIK3R4, along with novel variants in known disease-associated genes UNC80, SPEN, and SURF1. Gene co-expression network analysis as well as module enrichment analysis were performed using transcriptomic datasets relevant to brain development and function. These analyses showed that TOX3, SOCS7, PIK3R4, UNC80, and SPEN are linked to modules enriched for neurological processes, with UNC80 further associated with epilepsy-related gene modules. Sanger sequencing confirmed the presence of our specific variants of interest in genes: TOX3, SOCS7, PIK3R4, UNC80, SPEN, and SURF1. Functional studies were then performed to assess the potential role of SOCS7 and TOX3 in neural development using Xenopus tropicalis embryos. CRISPR/Cas9 gene editing combined with optical coherence tomography imaging enabled early visualization of neural tube abnormalities as early as stage 36 (three days post-fertilization). By stage 46 (four days post-fertilization), structural changes in brain morphology were observed in the injected SOCS7 and TOX3 embryos compared to controls. Immunohistochemistry revealed disrupted neuronal organization in regions such as the diencephalon and telencephalon, with β-tubulin staining showing reduced and disorganized neuronal structures in the experimental group.

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