טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTatour Yasmin
SubjectUsing advanced genetic analyses to identify the underlying
genetic basis for Hereditary Retinal Degeneration
DepartmentDepartment of Medicine
Supervisor Professor Tamar Ben-Yosef
Full Thesis textFull thesis text - English Version


Abstract

   The population of Northern Israel is a unique population because it includes multiple sub-ethnic groups, each of them has a unique genetic structure. Inherited Retinal Diseases (IRDs) are a heterogeneous group of blinding diseases, mainly affecting photoreceptor cells. To date, over 250 genes have been associated with different IRD phenotypes. IRDs are associated with marked clinical diversity, with the most common phenotypes being Retinitis Pigmentosa, Cone Dystrophy, and Leber Congenital Amaurosis.

To date there is no effective treatment for IRDs. Identifying the genetic cause of disease in IRD patients is highly important for two main reasons: first, it allows molecular diagnosis and genetic counseling. In addition, it is crucial for development of novel therapeutic approaches. The increasing list of known genes underlying IRD can be classified into several groups, based on their functional protein products. Therefore, both gene-based and non-gene based therapies will have to be tested on a set of patients with a known genetic diagnosis to prove their efficiency.

There are several available diagnostic approaches for IRD patients. These include: Sanger sequencing of specific IRD - causative genes; The Arrayed Primer Extension (APEX) technology; and next generation sequencing (NGS).

We took part in a study based in Radboud University in The Netherlands, named ERDC4000. This study is a collaborative effort involving 12 European, Canadian and Israeli research labs which are part of the European Retinal Degeneration Consortium (ERDC). In this study Molecular Inversion Probes (MIPs) - based sequence analysis of 108 genes associated with nonsyndromic IRDs was used to analyze ~4,000 probands, including 91 of our patients. Most of the patients sent for ERDC4000 study were isolated cases, that we were unable to solve using "conventional" methods.

For each DNA sample we sent we got the Bioinformatics results, which we analyzed using the following fixed criteria: variants located in exons or canonical splice sites; variants of the following variation types: Nonsense, in-frame deletions/insertions, frameshift, splice-site, missense. For missense variants we checked the pathogenicity with the following bioinformatics prediction tools: mutation taster, polyphen2, SIFT and CADD. Another criterion was the minor allele frequency in different populations, as it appears in the EXAC browser and the 1000 genomes browser. After the bioinformatic analysis, each putative mutation was validated by Sanger sequencing, and segregation in the family was tested (if possible). In addition, each mutation was cross- checked in unsolved patients with the same ethnic background. In total, the disease-causing mutation was identified in 45% of patients. A few unsolved patients were eventually solved by other strategies, including whole exome sequencing and Sanger sequencing of novel IRD genes which were not included in the original ERDC4000 panel.

The percent of solved cases using targeted NGS was similar to the percent obtained in our lab using whole exome sequencing, and therefore targeted NGS proved to be cost-effective. Our results are important since they contribute to molecular diagnosis, carrier screening and genetic counseling, in the relevant populations. In addition, they have a putative value for future development of novel therapeutic strategies for IRDs.