| dc.description.abstract | INTRODUCTION AND AIM
Increased total and ‘ fibrinogen concentrations are associated with cardiovascular disease
(CVD) risk in part through their effects on fibrin clot properties. Interleukin-6 (IL-6) promotes the
expression of fibrinogen and is also independently associated with CVD. The fibrinogen
phenotype is heritable, although discrepancies exist between the outcome of heritability studies
and the ability of genome-wide association studies to identify contributing polymorphisms. This
dissertation aimed to address the ‗missing heritability‘ of fibrinogen by investigating an African
population known to have higher fibrinogen and IL-6 concentrations, together with greater
genetic variability and lower linkage disequilibrium (LD) than previously reported in Europeans.
Three approaches were followed; firstly, polymorphisms and haplotypes within the fibrinogen
gene cluster were investigated with the aim of identifying functional variants that had not been
identified due to high LD in the fibrinogen gene cluster in Europeans; secondly, as genetic
variation within the fibrinogen genes alters the magnitude of the IL-6-induced expression of
fibrinogen, the interactive effect of fibrinogen polymorphisms and IL-6 was investigated; and
lastly, the pleiotropic and polygenic co-regulation of fibrinogen was investigated in a candidate
gene analysis of multiple targeted genes, largely outside the fibrinogen genes. These
approaches were investigated in terms of total and ‘ fibrinogen, as well as their functional
effects through turbidity-derived indicators of clot formation, structure and lysis.
METHODS
Eighty-one single nucleotide polymorphisms (SNPs) were investigated in 2010 apparently
healthy Tswana individuals. Genotyping was performed through restriction fragment length
polymorphism techniques, TaqMan-based assays, the beadXpress® platform and competitive
allele-specific polymerase chain reaction methods. Fourteen SNPs were located in the
fibrinogen gene cluster and 67 spanned the APOB, APOE, CBS, CRP, F13A1, LDL-R, MTHFR,
MTR, PCSK-9 and SERPINE-1 genes. Total and ‘ fibrinogen concentrations were quantified
via the modified Clauss and enzyme-linked immunosorbent assay methods, respectively. IL-6
was quantified by means of automatic electrochemiluminescence and fibrin clot properties were
determined through turbidimetric analyses. Independent and IL-6-interactive associations of the
14 fibrinogen SNPs with the outcome phenotypes were determined. In addition, 78 candidate
SNPs were investigated in terms of their individual and accumulative associations (through
genetic risk score analyses) with the outcome phenotypes together with possible co-regulatory
processes as a result of the gain and loss of transcription factor binding sites (TFBS).
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RESULTS
Lower minor allele frequencies and higher recombination rates for the investigated SNPs were
observed in our study population compared to what has previously been reported for
Europeans. None of the common European fibrinogen haplotypes was present. Seven of the
fibrinogen SNPs were significantly associated with one or more of the outcome phenotypes.
The fibrinogen SNPs contributed 0.5% of the variance in total fibrinogen. Fibrinogen
significantly associated with IL-6, and thereby mediated associations with clot formation and
structure. Several significant interactions between the fibrinogen SNPs and IL-6 were observed
relating to total and ‘ fibrinogen and fibre diameter. These interactions were additive in their
association with total fibrinogen concentrations. FGB-rs7439160, -1420G/A and -148C/T were
acknowledged as SNPs possibly functional in the PURE population. The candidate gene
analysis revealed SNPs within and outside of the fibrinogen gene cluster to associate with
fibrinogen and clot-related phenotypes, including SNPs located in the F13A1, LDL-R, PCSK-9,
CBS and CRP genes, through the regulatory effects induced by the gain and loss of 75 TFBS.
An accumulative genetic risk was observed through genetic risk scores that were significantly
associated with all phenotypes apart from fibre diameter.
CONCLUSION
This dissertation highlights the distinctive African genome and stresses the importance of
conducting genetic research among Africans. Original contributions to the literature include the
investigation of three novel SNPs, a report of a lack of haplotypes in the fibrinogen genes and
an additive effect of risk alleles within the fibrinogen gene cluster of Africans, as well as
evidence of the involvement of PCSK-9 SNPs in the heritability of fibrinogen concentration.
Furthermore, evidence is given that polygenic transcriptional co-regulation of these SNPs
through their effects on TFBS forms the basis of their associations with the respective
phenotypes. The current study contributes to the investigation of fibrinogen‘s missing
heritability by widening the scope of involved genes from what has been discovered thus far, as
well as providing a new avenue for the exploration of transcriptional co-regulation of SNPs
outside of, and additive gene-environment contributions within the fibrinogen gene cluster. | en_US |
