A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree Master of Science.
Johannesburg 2015 / The controversy surrounding the findings that copy number variation (CNV), of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. In this study, we evaluated the standard method of quantitative Real-Time PCR (qPCR) and Droplet Digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR, with the addition of another 30 black individuals to the ddPCR cohort. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet Digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course. We further used the CCL4L copy number data to examine variation of these genes with
reference to measured stimulated CCL4 production by the same individuals. Although significant differences in the copy number range medians and patterns of distribution of the genes CCL4L1, CCL4L2 and combined CCL4L were observed between the two populations, on a whole, the two populations do not differ significantly with respect to CCL4 production. Caucasian females however had a higher level of protein production per copy of CCL4L than Black females. When stratifying production based on population specific copy number median, Black individuals showed a decreased level of protein production at a CCL4L copy number below the median, although this was not maintained when the CCL4L1 and CCL4L2 genes were analysed individually. CCL4 copy number and production data was compared to data generated for CCL3 in the same cohort. CCL4 chemokine levels were significantly higher in both the Black and Caucasian populations and Black individuals had a higher number of gene copies of the CCL3L genes giving rise to functional CCL3 protein than the CCL4L genes producing functional CCL4 protein. These results highlight genetic differences between divergent populations, differences in distribution of CCL4 and CCL3 encoding genes and protein production, and suggest an intricate regulation of the CCL4L encoding genes. In order to investigate the role of CCL4 in HIV-1 control, we next assessed variation in the numbers of CCL4L copies in relation to CCL4 production in a cohort of 14 long-term nonprogressors (LTNPs). While no associations between copy number and CCL4 production were observed, the LTNP cohort had significantly lower levels of CCL4 production than the HIV-1-uninfected cohort, and this was maintained when Black and Caucasian individuals were examined individually. When the LTNP cohort individuals were divided based on viral loads, individuals with viral loads <400 RNA copies/ml had significantly lower CCL4 production than those with viral loads >400 RNA copies/ml. This finding suggests a role for the amount of CCL4 produced in the reduced pace of HIV-1 progression observed in LTNP individuals. Since genetic variation other than copy number can also influence CCL4 protein production, we then proceeded with a thorough genetic characterization of the CCL4 gene of uninfected individuals and investigated any possible relationships with select variants and CCL4 production. Sequencing the complete gene
and flanking regions of 23 Black and 32 Caucasian revealed several intra as well as extragenic SNPs, with one newly identified SNP at position -1063. The Black population exhibited a higher degree of variability compared to the Caucasian population. We described four haplotypes in the Caucasian population, three haplotypes in the Black population, and a single haplotype shared between both population groups. Of the four indels that were identified, a three bp deletion (rs3216921) was the only indel present in the Caucasian population and was not identified in the Black population. Of all the indels, this indel had the highest allelic frequency (14%). Comparisons of haplotypes and prevalent SNPs with protein production in both population groups did not show any significant differences. Caucasian individuals harbouring the 3bp deletion however, had significantly higher levels of CCL4 production (p=0.024). These results form a good base from which to further investigate the impact of select genetic variants on CCL4 production and possibly HIV-1 control. This study has succeeded in optimising a ddPCR assay for the copy number determination of the CCL4 genes and has interrogated the relationship between CCL4L copy number and CCL4 production in both HIV-1-uninfected individuals as well as a subset of LTNPs. The results suggest a complex, intricate regulation of CCL4 that appears to play a role in HIV-1 control. In conclusion, this study forms the basis for future work to build on and to further explore the role of this CCR5 ligand in HIV-1 disease.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/19971 |
| Date | January 2015 |
| Creators | Bharuthram, Avani |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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