Effective Utilization of Molecular Genetic Screening of Patients with Sickle Cell Disease and Beta Thalassemia Major in Saudi Arabia

Hereditary blood diseases are prevalent in the Kingdom of Saudi Arabia. The majority of these blood disorders are sickle cell disease and β-thalassemia with variants located on the beta globin gene (HBB). Aim: To determine the profile of novel or previously reported causative mutations in more than 150 transfusion dependent individuals using TaqMan genotyping and next-generation DNA sequencing. In addition, I explored the genomic variation in a family with transfusion dependency but without a definitive genetic diagnosis related to HBB. I also attempted to detect unknown genetic variations in functionally related genes and applied in-silico analysis of the detected variants to propose candidate genes that may contribute to the severe etiology of thalassemia within a family. Methods: To identify HBB variants, I conducted Taqman genotyping tests using SCD, c.92+5G>C, c.92+1G>A, c.93-21G>A, c.27dupG, and c.118C>T as the most frequently identified HBB variants within the Saudi population. After that, targeted next generation sequencing was performed on samples with either negative or only heterozygous results for these variants. The use of different molecular techniques including MLPA alpha thalassemia, whole exome sequencing, cytoscan HD array, and whole genome sequencing was undertaken on samples that needed further investigation. Implementation of different data filtering approaches and several in-silico techniques were utilized to investigate the detected variants. Results: After Taqman genotyping of the 154 DNA samples, 100 samples were either homozygous or compound heterozygous for the most frequently known HBB variants. The rest of these samples were sequenced using targeted NGS and 20 different common and rare HBB variants were identified. Three out of the 154 samples did not have any apparent HBB mutation and further investigation was applied using additional molecular techniques. This led to the identification of two gene candidates, SMC5 and TALDO1, with possible novel associations in increasing the severity of clinical manifestation in transfusion-dependent patients with heterozygous pathogenic variant of beta thalassemia. Conclusion: Beta thalassemia is a heterogenous disease with a wide range of clinical severity and the steps towards identification of the underlying genetic cause of the phenotype is different from case to case and may require a combination of several molecular techniques. Therefore, the interaction of illness-causing variations with the rest of an individual's genome is crucial to gaining a complete understanding of the condition. Excellent detection rates in less time may be achieved with a specialized filtering technique and strategy, making this an option for primary laboratory workflow