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MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / Factor X deficiency is a rare bleeding disorder with an incidence of one in a million in the general population. Patients with the severe form of factor X deficiency suffer from serious bleeds occurring mainly into the joints and the muscle. In the two factor X deficient families currently looked after at the Haemophilia Comprehensive Care Centre, there are definite differences in the bleeding tendencies between and within family members. We hypothesize the differences in genetic mutations and the influence of cytokines to be responsible for these bleeding variabilities. These factors were explored in our study. The study population included a total of fourteen members of the two families with factor X deficiency. Blood for factor X measurement, cytokine studies and genetic studies was collected in the Haemophilia Comprehensive Care Centre of the Charlotte Maxeke Johannesburg Academic Hospital. Each blood was processed according to the test to be performed. Factor X activity levels were measured using the factor X assay, and the information on each patient’s bleeding episodes was obtained from the Haemophiliac Clinic database. Cytokines were analyzed in all patients using the ELISA kits from Biosource. Factor X gene was amplified using PCR and sequenced with Spectrumedix SCE 2410. iv For cytokine studies, high levels of IL-1beta and TNF-alpha were observed in frequent bleeding patients compared to infrequent bleeders. These cytokines are known to be involved in acute inflammatory process leading to cellular infiltrate and joint swelling. This results in synovitis and the creation of massive joint bleeding. The low levels of IL-1beta and TNF-alpha detected in infrequent bleeding patients appear to be related to the high levels of IL-1Ra and IL-10. These anti-inflammatory cytokines are known to inhibit the inflammatory synovitis and lessen the severity of joint bleeding. For genetic studies, differences were observed between the amino acid sequence of the three frequent bleeding patients and the consensus. In addition, a novel mutation Cys350Phe was detected in two of these patients. This mutation is characterized by very low factor X levels which sometimes are not detectable in circulation. The substituted cystine is known to cause defect in the substrate binding, leading to the lost of enzyme activity. From these findings we have concluded that the origin of the heterogeneity of bleeding in factor X deficiency is multifactorial, cytokines and genetic mutations seems to have a role in determining the clinical manifestations of the factor X deficient patients.

von Willebrand Disease (VWD) is the most common inherited bleeding disorder in humans, resulting from quantitative or qualitative deficiencies of von Willebrand factor (VWF). Type 3 VWD is the rarest and most severe form of the disease. This thesis characterizes the phenotype-genotype correlations of a cohort of Canadian type 3 VWD patients and their family members. Three main findings are highlighted: 1) 50% of families showed evidence of co-dominant inheritance as opposed to recessive, 2) 42% of mutations identified were located in the VWF propeptide region (VWFpp), 3) index cases (IC) with mutations in the VWFpp had a more severe bleeding diatheses than IC with mutations elsewhere. We investigated two of the identified VWFpp mutations (ex4-5del and Cys633Arg) to elucidate their molecular mechanisms using two cellular models. Patient-derived blood outgrowth endothelial cells (BOEC) are ideal for studying the underlying molecular mechanism of VWF mutations as they represent the native vascular endothelium. BOEC were isolated from type 3 VWD IC and family members with the mutations of interest. A heterologous cellular system was also used to study the VWF mutations in vitro. The VWFpp mutations caused impaired VWF secretion, defective multimerization, qualitative and quantitative defects in Weibel-Palade body (WPB) formation, and resulted in VWF retention within the endoplasmic reticulum. We attempted to restore secretion and multimerization by co-transfecting each mutant with the wild-type VWF propeptide (VWFpp), which was unsuccessful. Additionally, we investigated a third mutation, c.8419_8422dupTCCC, which is unique to the Canadian VWD population and is found at a high frequency in a specific geographic population. While we hypothesized that this mutation would disrupt dimerization due to its location in the C-terminal cysteine knot (CK) domain of VWF we did not find this to be true. The results presented within this thesis provide new insight into the genetics and pathobiology of type 3 VWD, the functional contribution of the VWFpp to type 3 VWD and highlight the utility of BOEC as a cellular model for evaluating the pathogenic mechanisms of VWF mutations. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-10-17 21:15:37.685