Modeling Human Genetic Variation in Non-Coding DNA in Humanized Mice

The increasing availability of sequencing data from genome-wide association studies and whole genome sequencing of the human genome has enabled rapid identification of genetic variations—mainly single nucleotide polymorphisms (SNPs)— in non-coding DNA of the human genome. However, it has been difficult to find the biological functions of the numerous SNPs in the genome. This gap in knowledge can be explained in part by our poor understanding of the function of non-coding DNA, and by the challenge of experimentally assigning function to SNPs that map to these non-coding regions. To clearly define the function of non-coding SNPs, we created genetically humanized mice to model human genetic variation in non-coding DNA in vivo. To generate the mice, we used a bacterial artificial chromosome (BAC) system harboring two genetically different human IL10 SNP haplotypes. The IL10 SNP haplotypes are “ATA” and “GCC,” which have been associated with differential IL-10 levels and disease susceptibility in humans. We found a robust allele-specific human IL-10 expression in both macrophages and CD4+ T cells. Specifically, GCC-hIL10BAC encodes for a high human IL-10 level relative to ATA-hIL10BAC in CD4+ T cells both in vitro and in vivo. The reverse was observed in macrophages. Accordingly, by complementing Il10 null mice with the GCC-hIL10BAC, namely Il10-/-/GCC-hIL10BAC mice, we were able to completely reverse disease outcome. The Il10-/-/GCC-hIL10BAC mice were susceptible to persistent leishmania infection as evidenced by a high parasite burden in the liver and spleen. In contrast, like Il10 null mice, the Il10-/-/ATA-hIL10BAC mice were refractory to disease. Therefore, our data demonstrate that human IL10 promoter SNP haplotypes alone can modulate IL-10 levels and disease risk. In the second part of this dissertation, we examined the regulation of IL-10 and its homolog, IL-24, as a means to indirectly demonstrate that we are not missing important regulatory elements within the hIL10BAC. We chose IL-24 from the remaining cytokines within the Il10 gene cluster because the gene encoding for IL-24 is localized at the extreme end of the Il10 locus in both mouse and man and also human IL24 gene is not included in the hIL10BAC. Thus, finding co-regulation of IL-10 and IL-24 expression would suggest that the two homologs share common regulatory elements. Interestingly, we found that IL-10 and IL-24 are regulated by distinct cell-type-specific regulatory pathways. Optimal IL-24 expression requires Stat6 and Stat4 in macrophages and NK cells; meanwhile, IL-10 expression is independent of Stat6 and dependent on Stat4 only in IL-12-treated NK cells. We also discovered an unexpected role for Type-I Interferons in mediating differential regulation of IL-10 and IL-24 expression in macrophages and NK cells. Thus, our results suggest that IL-24 and IL-10 are unlikely to share common regulatory elements within the Il10 locus. Altogether, our results undoubtedly demonstrate that we can model human genetic variation in non-coding DNA in vivo using genetically humanized hIL10BAC mice. In the future, the hIL10BAC approach can be extended to other human genes to accelerate rational development of safe and efficient personalized therapies, including vaccines

Expanding Research Capacity in Sub-Saharan Africa Through Informatics, Bioinformatics, and Data Science Training Programs in Mali

Bioinformatics and data science research have boundless potential across Africa due to its high levels of genetic diversity and disproportionate burden of infectious diseases, including malaria, tuberculosis, HIV and AIDS, Ebola virus disease, and Lassa fever. This work lays out an incremental approach for reaching underserved countries in bioinformatics and data science research through a progression of capacity building, training, and research efforts. Two global health informatics training programs sponsored by the Fogarty International Center (FIC) were carried out at the University of Sciences, Techniques and Technologies of Bamako, Mali (USTTB) between 1999 and 2011. Together with capacity building efforts through the West Africa International Centers of Excellence in Malaria Research (ICEMR), this progress laid the groundwork for a bioinformatics and data science training program launched at USTTB as part of the Human Heredity and Health in Africa (H3Africa) initiative. Prior to the global health informatics training, its trainees published first or second authorship and third or higher authorship manuscripts at rates of 0.40 and 0.10 per year, respectively. Following the training, these rates increased to 0.70 and 1.23 per year, respectively, which was a statistically significant increase (p < 0.001). The bioinformatics and data science training program at USTTB commenced in 2017 focusing on student, faculty, and curriculum tiers of enhancement. The program’s sustainable measures included institutional support for core elements, university tuition and fees, resource sharing and coordination with local research projects and companion training programs, increased student and faculty publication rates, and increased research proposal submissions. Challenges reliance of high-speed bandwidth availability on short-term funding, lack of a discounted software portal for basic software applications, protracted application processes for United States visas, lack of industry job positions, and low publication rates in the areas of bioinformatics and data science. Long-term, incremental processes are necessary for engaging historically underserved countries in bioinformatics and data science research. The multi-tiered enhancement approach laid out here provides a platform for generating bioinformatics and data science technicians, teachers, researchers, and program managers. Increased literature on bioinformatics and data science training approaches and progress is needed to provide a framework for establishing benchmarks on the topics

High SARS-CoV-2 Seroprevalence among Healthcare Workers in Bamako, Mali

In Mali, a country in West Africa, cumulative confirmed COVID-19 cases and deaths among healthcare workers (HCWs) remain enigmatically low, despite a series of waves, circulation of SARS-CoV-2 variants, the country’s weak healthcare system, and a general lack of adherence to public health mitigation measures. The goal of the study was to determine whether exposure is important by assessing the seroprevalence of anti-SARS-CoV-2 IgG antibodies in HCWs. The study was conducted between November 2020 and June 2021. HCWs in the major hospitals where COVID-19 cases were being cared for in the capital city, Bamako, Mali, were recruited. During the study period, vaccinations were not yet available. The ELISA of the IgG against the spike protein was optimized and quantitatively measured. A total of 240 HCWs were enrolled in the study, of which seropositivity was observed in 147 cases (61.8%). A continuous increase in the seropositivity was observed, over time, during the study period, from 50% at the beginning to 70% at the end of the study. HCWs who provided direct care to COVID-19 patients and were potentially highly exposed did not have the highest seropositivity rate. Vulnerable HCWs with comorbidities such as obesity, diabetes, and asthma had even higher seropositivity rates at 77.8%, 75.0%, and 66.7%, respectively. Overall, HCWs had high SARS-CoV-2 seroprevalence, likely reflecting a “herd” immunity level, which could be protective at some degrees. These data suggest that the low number of cases and deaths among HCWs in Mali is not due to a lack of occupational exposure to the virus but rather related to other factors that need to be investigated

Clinical risk factors associated with multidrug-resistant tuberculosis (MDR-TB) in Mali

Background: MDR-TB is a major threat to global TB control. In 2015, 580,000 were treated for MDR-TB worldwide. The worldwide roll-out of GeneXpert MTB/RIF® has improved diagnosis of MDR-TB; however, in many countries laboratories are unable to assess drug resistance and clinical predictors of MDR-TB could help target suspected patients. In this study, we aimed to determine the clinical factors associated with MDR-TB in Bamako, Mali. Methods: We performed a cross-sectional study of 214 patients with presumed MDR-TB admitted to University of Bamako Teaching Hospital, Point-G between 2007 and 2016. We calculated crude and adjusted odds ratios for MDR-TB disease diagnosis using SPSS. Results: We found that age ≤40 years (OR = 2.56. 95% CI: 1.44–4.55), two courses of prior TB treatment (OR = 3.25, 95% CI: 1.44–7.30), TB treatment failure (OR = 3.82, 95% CI 1.82–7.79), sputum microscopy with 3+ bacilli load (OR = 1.98, 95% CI: 1.13–3.48) and a history of contact with a TB patient (OR = 2.48, 95% CI: 1.11–5.50) were significantly associated with confirmation of MDR-TB disease. HIV was not a risk factor for MDR-TB (aOR = 0.88, 95% CI: 0.34–1.94). Conclusion: We identified several risk factors that could be used to identify MDR-TB suspects and prioritize them for laboratory confirmation. Prospective studies are needed to understand factors associated with TB incidence and clinical outcomes of TB treatment and disease. Keywords: Multi-Drug Resistant Tuberculosis, Risk factors, Mal