טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDaniel-Farran Nada
SubjectDiscovering Mutations and Genes Causing Hearing Impairment
by Next Generation Sequencing
DepartmentDepartment of Medicine
Supervisor Professor Stavit Shalev
Full Thesis textFull thesis text - English Version


Abstract



Understanding the clinical implications of genetic variation in the context of genetic disorders is one of the important challenges in the field of human genetics. Hearing loss (HL) is the most prevalent sensory disorder, in which half of the cases are genetic in nature. HL is a heterogeneous disorder, with more than 80 genes that are known to cause deafness in human to-date.

This study aimed to decipher the genetic causes of deafness among a cohort of the Northern Israeli population. We hypothesized that families with multiple affected individuals, in which prevalent known pathogenic variants were not found, harbor novel alleles in known or novel deafness genes.

This research was empowered with the availability of next generation sequencing (NGS). This technology allows parallel sequencing of extended genomic regions. Our study focused on two strategies: first, the HEar-Seq, which targets 375 protein coding genes, known to cause deafness in humans and mice, and genes highly expressed in the auditory system, and the second was whole exome sequencing (WES). 

The study included 1 95 families, belonging to different ethnicities of Arabs and Jewish families living in northern Israel. The Arab families (169, 87% of the families) are characterized by high rates of consanguinity and fertility. Detailed pedigrees consistent with monogenic inheritance, and thorough medical data were obtained, followed by informed consent process. At first, the investigation included screening of known pathogenic variants in reference populations, including sequencing of the coding region of the GJB2 gene, and screening variants in TMC1, SLC26A4, and OTOF gene. These steps yielded diagnoses in 54 families (about 27%). A subset of 24 families further selected for NGS.

            Analysis of the 24 families led to the identification of variants in 16 of them (2/3), four of which are known rare pathogenic variants, and seven were novel variants in known deafness genes, including TBC1D24, LOXHD1, OTOG, TECTA and MYO15A, and a duplication disrupting theTJP2 gene. The variants found to successfully segregate in the families. Three additional variants were found in three families, involving the CLCNKA, COCH, and PCDH15 genes. These findings require further investigation for final conclusions. In additional family a variant in a candidate novel deafness gene SLC25A21 was found, led to extensive functional analysis including generation of Crisper/Cas9 transgenic mice. Presently, the results do not allow us to making final conclusions.

Finally, functional assays validated the abnormal gene product of the variants c.8340G>A and c.9083>A in MYO15A gene.

This study emphasize the heterogeneity of hearing loss in the population of north Israel, and manifest the advantage of NGS technology in such project. Still the results often require more investigation to make conclusion regarding the pathogenicity of the detected variants, complicated both by heterogeneity and pleiotropic nature of many genes. The value of genetic diagnosis of deafness to individuals and families is enormous, since it allows informed personal and medical decisions based on precise medical data. It is also highly valuable in broader medical aspects, as it might be the cornerstone of future diagnostic and even therapeutic measures.