Nörogenetik Hastalıklarda Ekzom Dizileme Analizi ile Aday Genlerin Belirlenmesi ve Zebrafish Modellerinin Oluşturulması

Abstract

ABSTRACT

IDENTIFICATION OF CANDIDATE GENES IN NEUROGENETIC DISORDERS BY WHOLE EXOME SEQUENCING AND MODELING IN ZEBRAFISH

AYŞEGÜL OZANTÜRK

Doctor of Philosophy, Department of Biology, Molecular Biology Section

Supervisor: Prof. Dr. R. Köksal Özgül

September 2015, 171 pages

Application of whole exome and whole genome sequencing on affected individuals with unknown diseases has been using successfully to understand the cause of human inherited disorders. It became a diagnostic approach in medicine for undiagnosed families. By combining these high throughput-sequencing methods with functional studies that conducted on model organism accelerate the succes rate. The aim of this study is to identify candidate genes in patients with unknown conditions by applying whole exome sequencing and test pathogenicity of the determined alleles on zebrafish. This study includes six undiagnosed families with neurogenetic/neurocognitive clinical features. All families have two affected individuals with the same or similar neurological findings. The most common symptom of the affected individuals is intellectual disability. Three families have anatomical abnormalities; one family has cerebellar atrophy and two have craniofacial dysmorphology. First, all families, parents and affected individuals, were subjected to whole exome sequencing to list candidate genes that might be responsible for observed phenotypes. After defining the candidates, all selected alleles were tested on zebrafish to reveal their contribution to the disease phenotypes. Zebrafish is a relatively new organism to model human genetics disorders. There is a high amount of genomic sequence similarity, 70 %, between human and zebrafish.

iv Using these molecular approaches genetic causes of the disorders in three families were identified; Family 1, has two affected sibs, M-11-1496 and M-11-1497, who have intellectual disability, ambiguous genitale, and craniofacial dysmorphology, is a carrier of two CNVs which are located in 16q (13,5 Mb duplication) and 5p (7,5 Mb deletion). Clinical features of M-11-1472 and M-11-1473 in Family 2, failure to thrive, microcephaly, vision impairment and hearing loss, axial hypotony, coreic movement, were associated with Pontocerebellar hypoplasia type 2B which caused by a novel mutation, p.T364R, in TSEN2 gene. Family 3 has two affected sibs with dysmorphic features, bigger and low set ears, hypertelorism is associated with ATRX syndrome which was resulted in a novel mutation, p.D217G, in ATRX gene. Family 4 has two affected males with severe intellectual disability, autism spectrum disorders. Exome sequencing identified 5 candidate genes, ANKRD30A, TCEB3, PLIN2, VPS26B, METTL4 in this family. There was no association between these candidate genes and clinical features of affected males. Family 5 has two affected sibs, M-11-1500 and M-11-1501, with intellectual disability, autism spectrum disorders. 11 candidate genes identified by whole exome sequencing. This family was excluded from the functional study since there are too many candidate alleles need to tested on zebrafish. Family 6 has a clear candidate, CELSR3, in which functional study showed the severe phenotype that might be related neurological symptoms seen in patient M-11-1476 and M-11-1477. By combining genomics and functional aspects, we were able to define molecular causes of neurologic phenotypes in three families and a likely pathogenic variation in one family. Our results provided molecular diagnosis for these families. Therefore, these applied combined molecular approaches such as whole exome sequencing and zebrafish modeling are powerful methodologies to elucidate the molecular pathology of diseases and especially useful in identification of rare and unknown diseases.