Identification and functional investigation of genes in patients with inherited bleeding disorders

Abstract

Inherited bleeding disorders (IBDs) comprise a heterogeneous group of diseases that reflect abnormalities in blood vessels, coagulation proteins and platelets. The diagnosis of patients with IBDs is challenging and approximately 50-60% of patients recruited to the UK-GAPP study have no known cause of their bleeding despite a strongly indicative inherited component. Whole Exome Sequencing (WES) analysis using a bioinformatics pipeline workflow was carried out in 6 patients including 4 isolated individuals and two related individuals with bleeding episodes and low platelet counts. A stop gained variant within CD36: c.975T>G; p. Tyr325* was identified in related patients 5.1 and 5.2 and a number of other plausible candidate variants were also found for the other isolated individuals. Functional studies were conducted on stop gained variants within CD36 which showed the effect of this variant on the expression of CD36 protein using Western blot and flow cytometry techniques. This finding was confirmed through the activation of NFAT-luciferase reporter assay by the WT CD36, but not by the mutant constructs. In addition, a comprehensive bioinformatic analysis of 126 patients with suspected platelet disorders was carried out using the Congenica diagnostic software to identify both structural and sequence genetic variants. 28.2% of patients were noted with platelet function defects and 19.6% of patients represented thrombocytopenia only. A total of 135 variants in genes implicated in bleeding disorders were identified across all patients. 22 patients were classified as pathogenic and 26 patients as likely pathogenic, while 87 patients had uncertain pathogenicity. The Congenica software has also identified potential CNVs in 3 patients. In conclusion, this thesis has demonstrated that the application of combined phenotyping and genotyping coupled with WES technology and bioinformatic tools are efficient and effective approaches for refining and identifying new sequence variants in patients with suspected IBDs.