Mapping of DBLα Sequence Tags of Field Isolates from Two Malaria Endemic Sites in Kenya

Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) found on the surface of infected erythrocytes (IEs) mediate antigenic variation during P. falciparum infection enabling the parasite evade host immune responses and prolong infection. These molecules mediate binding of IEs to host endothelial cells and uninfected erythrocytes. Cytoadhesion of IE to host cells leads to sequestration in tissues and PfEMP1 is thought to play an important role in parasite virulence. Here we analysed 1725 sequence tags sampled from the DBLa region of PfEMP1 encoding “var” genes from 27 patients in two different geographical regions in Kenya, Mbita in Western Kenya and Twiga on the Kenyan coast. The objective of this study was to construct a network to assess the extent of shared position specific polymorphic blocks (PSPBs) in sequences isolated from genomic DNA of field isolates from the two malaria endemic sites in Kenya.. Sequences from Mbita study site and those from Tiwi largely clustered into separate giant networks with only a limited number of sequences from the two sites linking to each other. This observation suggests that the parasite populations from the two endemic sites could be genetically varied and that PfEMP1 sequencing could be a useful tool of understanding the genetics of parasite populations. Thus the network approach of studying relationships between DBLα sequences is a useful tool of uncovering the genetic structure of parasite populations circulating in different malaria endemic regions. Keywords: PfEMP1, Networks, Position Specific Polymorphic Groups, DBLα, Malaria, P. falciparu

PfEMP1 DBLalpha Sequence Tags in Genomic DNA of P. falciparum Field Isolates from Two Malaria Endemic Sites in Kenya

Background Malaria caused by Plasmodium falciparum remains a major cause of childhood morbidity and mortality in sub-Saharan Africa.  PfEMP1 protein, coded for by a family of about sixty variant var genes, is a parasite protein found on infected erythrocyte membrane. PfEMP1 protein mediates cytoadherence of infected erythrocytes on endothelial cells which may lead to severe symptoms of malaria. Although PCR amplification of the whole gene is difficult due to high variability, primers targeting the DBL? domain have been designed and used to study pfemp1 genes. This objective of this study was to establish the distribution of DBL? sequence tags in isolates of Plasmodium falciparum from two malaria endemic sites in Kenya. Methods DNA extracted from field isolates collected from Mbita (Western Kenya) and Tiwi (Coastal region) was used to isolate and amplify DBL? domain sequence tags of pfemp1 by PCR. PCR products were sequenced by 454 next generation sequencing. After assembly, the translated protein sequences (GenBank KP085750-KP087726) were then aligned in Mega 5.2 and classified into cys/PoLV groups based on the number of cysteine residues and the motifs at PoLV1 and PoLV2 within the sequence tag. Six sequence groups were found in sequences from both endemic sites. Group 4 sequences were the most prevalent (57.35% and 57.07% in isolates from Mbita and Tiwi respectively) in the isolates from both sites. Sequence tags from Tiwi had a higher proportion of cys2 (group 1 and 2) than sequences from Mbita although individual group 2 sequence tags were slightly higher in Mbita tags. Similarly the proportion of groups 5 and 6 sequence tags was higher in sequence tags from Tiwi than those from Mbita. Conclusion In conclusion, the frequency of the different cyc/PoLV groups of DBL? sequence tags at both endemic sites follow almost similar pattern with group four sequence tags being the majority among the sequence tags isolated from patient isolates from both study sites. However, in the absence of expression data, the impact of this genomic distribution pattern on malaria pathology remains unknown. Key Words: Malaria, PfEMP1, cys/PoLV, DBL?, var, Sequence tag

Genotyping for point mutations in selected codons of pfcrt and pfmdr-1 genes of Plasmodium falciparum among patients with uncomplicated malaria in Mbita district Kenya.

Background Malaria remains a leading cause of morbidity and mortality in Kenya, especially in young children and pregnant women. Due to widespread resistance of Plasmodium falciparum to drugs such as Chloroquine (CQ) and Sulfurdoxine-Pyrthamine (SP), Artemisisnin Combination Therapy (ACT) was adopted in Africa as a means of improving treatment efficacy and slowing the spread of resistance. The development of drug resistance by the parasites for the various malaria drug regimens that have been in use before has been attributed to point mutations within the parasite genome. Therefore this study investigated the prevalence of point mutations in selected codons of the pfcrt and pfmdr-1 genes of Plasmodium falciparum. It is however unclear whether ACT will be effective in preventing the selection of resistant parasites in Africa, where parasite transmission rates are generally much higher with parts of Asia and Africa already reporting a reduction in sensitivity to ACT. Methods The dot-blot/probe hybridization technique was used to identify point mutations in codons 74, 75 and 76 of the pfcrt gene and codons 1034, 1042 and 1246 of the pfmdr-1 gene in Mbita a malaria holoendemic site in Kenya. In the pfcrt gene, 76T mutation was found to be in 91 (79.83% CI 63.1-88.5) of 114 samples while the, the wild type allele 76K was present in 23 (20.17% CI 9.0-22.0) samples. Codons 1246 showed allelic variation with 1246D the wild type allele being 72.8% (CI 52.0-89.1). This was a significant increase in the 76K allele (p=0.001) in comparison to the year 2005 where prevalence of 76K was 6%.   Conclusion There’s an expansion of the wild-type allele 76K of the pfcrt gene and no significant difference in the 1246D allele of the pfmdr1 gene, moreover the prevalence of 76T allele is still high in Mbita hence it’s beneficial to continue using AL as treatment for uncomplicated malaria. Keywords: Malaria, Drug Resistance, Point Mutations

Breast cancer risk factors in relation to molecular subtypes in breast cancer patients from Kenya

Background Few studies have investigated risk factor heterogeneity by molecular subtypes in indigenous African populations where prevalence of traditional breast cancer (BC) risk factors, genetic background, and environmental exposures show marked differences compared to European ancestry populations. Methods We conducted a case-only analysis of 838 pathologically confirmed BC cases recruited from 5 groups of public, faith-based, and private institutions across Kenya between March 2012 to May 2015. Centralized pathology review and immunohistochemistry (IHC) for key markers (ER, PR, HER2, EGFR, CK5-6, and Ki67) was performed to define subtypes. Risk factor data was collected at time of diagnosis through a questionnaire. Multivariable polytomous logistic regression models were used to determine associations between BC risk factors and tumor molecular subtypes, adjusted for clinical characteristics and risk factors. Results The median age at menarche and first pregnancy were 14 and 21 years, median number of children was 3, and breastfeeding duration was 62 months per child. Distribution of molecular subtypes for luminal A, luminal B, HER2-enriched, and triple negative (TN) breast cancers was 34.8%, 35.8%, 10.7%, and 18.6%, respectively. After adjusting for covariates, compared to patients with ER-positive tumors, ER-negative patients were more likely to have higher parity (OR = 2.03, 95% CI = (1.11, 3.72), p = 0.021, comparing ≥ 5 to ≤ 2 children). Compared to patients with luminal A tumors, luminal B patients were more likely to have lower parity (OR = 0.45, 95% CI = 0.23, 0.87, p = 0.018, comparing ≥ 5 to ≤ 2 children); HER2-enriched patients were less likely to be obese (OR = 0.36, 95% CI = 0.16, 0.81, p = 0.013) or older age at menopause (OR = 0.38, 95% CI = 0.15, 0.997, p = 0.049). Body mass index (BMI), either overall or by menopausal status, did not vary significantly by ER status. Overall, cumulative or average breastfeeding duration did not vary significantly across subtypes. Conclusions In Kenya, we found associations between parity-related risk factors and ER status consistent with observations in European ancestry populations, but differing associations with BMI and breastfeeding. Inclusion of diverse populations in cancer etiology studies is needed to develop population and subtype-specific risk prediction/prevention strategies

Track D Social Science, Human Rights and Political Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138414/1/jia218442.pd

Investigations on Implications of Gas Flaring on Some Phytochemicals and Trace Metal Content of Bitter leaf (Vernonia amygdalina)

The effect of gas flaring on some phytochemicals and trace metals in Vernonia amygdalina in Obrikom, a gas flaring community (GF) and Rumualogu, a non gas flaring community (NGF) in Rivers state, Nigeria was investigated. There was significant increase (P>0.05) in the composition of alkaloid (NGF; 1.32±0.044 and GF; 3.10±0.001) and tannin (NGF; 0.03±0.001 and GF; 0.31±0.007) in the Vernonia amygdalina when the leaf samples from Rumualogu(NGF-Community) was compared with the Obrikom(GF-Community) samples. However, there was no significant difference (P<0.05) in the flavonoid (NGF; 0.79±0.012 and GF; 0.88±0.009) and saponin (NGF; 1.20±0.009 and GF; 1.27±0.018) compositions. There was significant increase (P>0.05) in the levels of Fe (2.21±0.01 to 2.96±0.01), Zn (0.86±0.01 to 1.10±0.01) and Pb (0.14±0.03 to 0.29±0.02) when the leaves grown in a non gas flaring site were compared with the gas flaring site samples.  There was no significant difference in Cr concentration; Cr (0.01±0.01 to 0.02±0.01). Cadmium level was below detection limit (BDL) in the vegetable from both sites. The implication of these findings is a possible change on the nutritional and medicinal values of Vernonia amygdalina. Keywords: Gas flaring, Obrikom, Phytochemicals, Rumualog

Adaptive immune receptor features related to breast cancer tissue in Kenyan patients: high immunoglobulin gene expression and high levels of gamma-delta T-cells

Background: Characterization of the breast cancer (BC) immune response may provide information for a point of intervention, such as application of immunotherapeutic treatments. In this study, we sought to recover and characterize the adaptive immune receptor (IR) recombination reads from genomics files representing Kenyan patients, to better understand the immune response specifically related to those patients. Methods: We used a previously applied algorithm and software to obtain productive IR recombination reads from cancer and adjacent normal tissue samples representing 22 Kenyan BC patients. Results: From both the RNAseq and exome files, there were significantly more T-cell receptor (TCR) recombination reads recovered from tumor samples compared to marginal tissue samples. Also, the immunoglobulin (IG) genes were expressed at a much higher level than the TCR genes (p-value = 0.0183) in the tumor samples. And, the tumor IG CDR3s consistently represented more positively charged amino acid R-groups, in comparison to the marginal tissue, IG CDR3s. Conclusion: For Kenyan patients, a high level of IG expression, representing specific CDR3 chemistries, was associated with BC. These results lay the foundation for studies that could support specific immunotherapeutic interventions for Kenyan BC patients

Breast cancer risk factors in relation to molecular subtypes in breast cancer patients from Kenya

Abstract: Background Few studies have investigated risk factor heterogeneity by molecular subtypes in indigenous African populations where prevalence of traditional breast cancer (BC) risk factors, genetic background, and environmental exposures show marked differences compared to European ancestry populations. Methods We conducted a case-only analysis of 838 pathologically confirmed BC cases recruited from 5 groups of public, faith-based, and private institutions across Kenya between March 2012 to May 2015. Centralized pathology review and immunohistochemistry (IHC) for key markers (ER, PR, HER2, EGFR, CK5-6, and Ki67) was performed to define subtypes. Risk factor data was collected at time of diagnosis through a questionnaire. Multivariable polytomous logistic regression models were used to determine associations between BC risk factors and tumor molecular subtypes, adjusted for clinical characteristics and risk factors. Results The median age at menarche and first pregnancy were 14 and 21 years, median number of children was 3, and breastfeeding duration was 62 months per child. Distribution of molecular subtypes for luminal A, luminal B, HER2-enriched, and triple negative (TN) breast cancers was 34.8%, 35.8%, 10.7%, and 18.6%, respectively. After adjusting for covariates, compared to patients with ER-positive tumors, ER-negative patients were more likely to have higher parity (OR = 2.03, 95% CI = (1.11, 3.72), p = 0.021, comparing ≥ 5 to ≤ 2 children). Compared to patients with luminal A tumors, luminal B patients were more likely to have lower parity (OR = 0.45, 95% CI = 0.23, 0.87, p = 0.018, comparing ≥ 5 to ≤ 2 children); HER2-enriched patients were less likely to be obese (OR = 0.36, 95% CI = 0.16, 0.81, p = 0.013) or older age at menopause (OR = 0.38, 95% CI = 0.15, 0.997, p = 0.049). Body mass index (BMI), either overall or by menopausal status, did not vary significantly by ER status. Overall, cumulative or average breastfeeding duration did not vary significantly across subtypes. Conclusions In Kenya, we found associations between parity-related risk factors and ER status consistent with observations in European ancestry populations, but differing associations with BMI and breastfeeding. Inclusion of diverse populations in cancer etiology studies is needed to develop population and subtype-specific risk prediction/prevention strategies