Role of MyD88-adaptor-like gene polymorphism rs8177374 in modulation of malaria severity in the Pakistani population

Abstract Introduction: The present study was designed to investigate the association between rs8177374 polymorphism and malaria symptoms due to exposure of Plasmodium vivax and Plasmodium falciparum. Materials and methods: A total of 454 samples were included in the study (228 malaria patients and 226 healthy individuals). Malaria patients, divided into P. vivax and P. falciparum groups on the basis of the causative species of Plasmodium, were categorized into mild and severe on the basis of clinical outcomes according to WHO criteria. Healthy individuals were used as controls. Allele specific PCR based strategy was used for the identification of rs8177374 SNP. Results: MyD88-adaptor-like gene polymorphism was associated with susceptibility to malaria (p < 0.001). C allele frequency (0.74) was higher in the population compared to T allele frequency (0.26). CT genotype increased the susceptibility of malaria (OR: 2.661; 95% CI: 1.722-4.113) and was positively associated with mild malaria (OR: 5.609; 95% CI: 3.479-9.044, p = 0.00). On the other hand, CC genotype was associated with severe malaria (OR: 3.116; 95% CI: 1.560-6.224, p = 0.00). P. vivax infection rate was higher in CT genotype carriers compared to other genotypes (OR: 3.616; 95% CI: 2.219-5.894, p < 0.001). Conclusion: MyD88-adaptor-like/TIR domain containing adaptor protein polymorphism for single nucleotide polymorphism rs8177374 is related with the susceptibility of malaria.

A meta-analysis of TLR4 and TLR9 SNPs implicated in severe malaria

Abstract Toll-like receptors (TLRs) are critical mediators of the inflammatory response to malarial infection, and gene polymorphisms affecting TLR function may be partially responsible for inter-individual variation in disease manifestation. However, there are inconsistencies in the associations of common genetic variants of TLR4 (D299G) and TLR9 (T-1237C and T-1486C) with malaria outcome. A comprehensive search was conducted to identify relevant and independent Plasmodium falciparum-infected case-control studies, and meta-analysis including six studies for each SNP was performed to obtain more precise estimates of the pooled effects of these variants. The results showed significant associations of the -1486C allele with the risk of severe malaria in allele contrast (T vs. C, p = 0.004, OR = 1.26) and homozygous (TT vs. CC, p = 0.03, OR = 1.51) genetic models. There was no association between the D299G or T-1237C variants and uncomplicated or severe malaria using any of the genetic models tested. However, in stratified analysis, -1237C was associated with the risk of severe malaria in Indian adults (TT vs. TC, p = 0.06, OR = 2.13; TT vs. TC+CC, p <0.00001, OR = 2.65), suggesting that our results must be considered preliminary. The robustness of -1486C as a risk factor warrants investigation into its functionality in malaria pathogenesis. Further, the lack of an association with the T-1237C variant was weak, and future studies examining more detailed individual data from different ethnic groups are essential for confirmation of its genetic contribution to malaria.

Evaluación del efecto genotóxico en pacientes con malaria de una región de Colombia / Evaluation of the genotoxic effect of malaria in a cohort of patients in Medellín and Quibdó

Introduction: During malaria infection, both parasite and host are under the effects of oxidative stress due to the increased production of reactive oxygen species, which can induce DNA damage by its genotoxic effects. Objective: To evaluate genotoxic effects in human lymphocytes in a cohort of patients with malaria from Medellin and Quibdó. Methods: We performed an observational cross sectional study in 100 individuals with malaria and 100 healthy controls. Patients infected with Plasmodium consulting the Institute Colombiano of Medicina Tropical of Medellin and the Hospital Ismael Roldán Valencia of Quibdó were included. Genotoxic effects (genetic damage) was analysed by electrophoresis using alkaline single cell gel (Commet assay). Results: The average of tail length of malaria samples (26.9 ± 9.8) was significantly higher than of controls (14.8 ± 3.2) (p < 0.01). Conclusion: In our study population, malaria infection was associated with increased genotoxicity, while other variables such as smoking, antimalarial treatment, and occupation were not.

Introducción: Durante la infección de la malaria, tanto el parásito como el hospedero están bajo los efectos de estrés oxidativo, dado que se aumenta la producción de especies reactivas del oxígeno, las cuales pueden inducir daños en el ADN debido a su gran efecto genotóxico. Objetivo: Evaluar el efecto genotóxico en linfocitos humanos en una cohorte de pacientes con malaria de Medellín y Quibdó. Métodos: Se realizó un estudio observacional transversal en 100 personas con malaria y 100 controles sanos. Se incluyeron pacientes infectados con Plasmodium, que consultaron en el Instituto Colombiano de Medicina Tropical de Medellín y el Hospital Ismael Roldán Valencia de Quibdó. Se realizó una valoración transversal del efecto (daño genético) mediante electro-foresis en gel de células individuales (ensayo Cometa). Resultados: El promedio de longitud de la cola de los pacientes (26,9 ± 9,8) fue significativamente mayor que la media de los controles sanos (14,8 ± 3,2) (p < 0,01). Conclusión: Se evidenció en la población de estudio que la infección por malaria generó genotoxicidad, no así variables como tabaquismo, tratamiento antimalárico y ocupación.

Estudo do sistema de reparo do DNA tipo“Mismatch Repair” em Plasmodium spp

Cepas de Plasmodium resistentes a diferentes drogas têm sido descritas ao redor do mundo. Embora os mecanismos de desenvolvimento de resistência não sejam bem conhecidos, sabe-se que defeitos nos sistemas de reparo do DNA podem estar envolvidos. Esses defeitos estão relacionados principalmente a mutações nas enzimas do sistema de reparo de mal pareamento do DNA ou mismatch repair(MMR) e já foram descritos em populações naturais de diversos organismos. Devido ao conhecimento limitado sobre o sistema MMR de Plasmodium, faz-se necessário um amplo estudo sobre os genes que codificam as proteínas envolvidas nesse sistema. Neste trabalho, foi realizado um estudo sobre as enzimas envolvidas no sistema de reparo do mal pareamento do DNA em Plasmodium: variabilidade intra e interespecífica em Plasmodium, principalmente nos domínios funcionais, e comparação entre níveis de expressão entre cepas/isolados de P. falciparum. Os parasitos foram também avaliados quanto ao número de cópias e expressão dos genes gch-1 e mdr1. Foram identificadas proteínas pertencentes às classes MSH2,MSH6, MLH1 e PMS1.

As sequências de proteínas mostraram-se muito conservadas, tanto entre o gênero Plasmodium, quanto em relação a outros organismos distantes evolutivamente. Foi encontrada um proteína homóloga a MutSque possui os domínios I e V, mas ainda não identificada quanto à sua classificação.O gene codificador desta proteína teve sua expressão confirmada neste e em outros trabalhos. Alguns SNPs foram encontrados em cepas/isolados depositados no PlasmoDB, no entanto, o sequenciamento da região que compreende os principais domínios funcionais apontou apenas 1 SNP na proteína PMS1. Os genes estudados, em sua maioria, apresentaram-se mais expressos entre 10 e 30 horas após a sincronização. W2 e 3D7 apresentam 2 cópias do genes gch-1 e mdr1. BHZ apresentou apenas 1 cópia do mdr1. Os resultados da análise de expressão desses genes ligados à resistência concordam com os resultados encontrados para o número de cópias gênicas. Este estudo fornece uma análise ampla das principais enzimas do MMR e será importante para estudos futuros do papel funcional destas enzimas e seu envolvimento no desenvolvimento de resistência às drogas

Estudo do sistema de reparo do DNA tipo“Mismatch Repair” em Plasmodium spp / Study of the DNA repair type

Cepas de Plasmodium resistentes a diferentes drogas têm sido descritas ao redor do mundo. Embora os mecanismos de desenvolvimento de resistência não sejam bem conhecidos, sabe-se que defeitos nos sistemas de reparo do DNA podem estar envolvidos. Esses defeitos estão relacionados principalmente a mutações nas enzimas do sistema de reparo de mal pareamento do DNA ou mismatch repair(MMR) e já foram descritos em populações naturais de diversos organismos. Devido ao conhecimento limitado sobre o sistema MMR de Plasmodium, faz-se necessário um amplo estudo sobre os genes que codificam as proteínas envolvidas nesse sistema. Neste trabalho, foi realizado um estudo sobre as enzimas envolvidas no sistema de reparo do mal pareamento do DNA em Plasmodium: variabilidade intra e interespecífica em Plasmodium, principalmente nos domínios funcionais, e comparação entre níveis de expressão entre cepas/isolados de P. falciparum. Os parasitos foram também avaliados quanto ao número de cópias e expressão dos genes gch-1 e mdr1. Foram identificadas proteínas pertencentes às classes MSH2,MSH6, MLH1 e PMS1. As sequências de proteínas mostraram-se muito conservadas, tanto entre o gênero Plasmodium, quanto em relação a outros organismos distantes evolutivamente. Foi encontrada um proteína homóloga a MutSque possui os domínios I e V, mas ainda não identificada quanto à sua classificação.O gene codificador desta proteína teve sua expressão confirmada neste e em outros trabalhos. Alguns SNPs foram encontrados em cepas/isolados depositados no PlasmoDB, no entanto, o sequenciamento da região que compreende os principais domínios funcionais apontou apenas 1 SNP na proteína PMS1. Os genes estudados, em sua maioria, apresentaram-se mais expressos entre 10 e 30 horas após a sincronização. W2 e 3D7 apresentam 2 cópias do genes gch-1 e mdr1. BHZ apresentou apenas 1 cópia do mdr1. Os resultados da análise de expressão desses genes ligados à resistência concordam com os resultados encontrados para o número de cópias gênicas. Este estudo fornece uma análise ampla das principais enzimas do MMR e será importante para estudos futuros do papel funcional destas enzimas e seu envolvimento no desenvolvimento de resistência às drogas.

Allelic diversity of merozoite surface protein 2 gene of P falciparum among children in Osogbo, Nigeria / Diversidad alélica del gen de la proteína de superficie del merozoíto 2 del P falciparum entre los niños de Osogobo, en Nigeria

The genetic diversity of Plasmodium falciparum (P falciparum) infections in humans is implicated in the pathogenesis of malaria. This study provides the first estimate of the genetic diversity and genotype multiplicity of Plasmodium falciparum infection in children with uncomplicated P falciparum malaria in Osogbo, Nigeria. One hundred and one isolates were used for analysis of parasite population polymorphism and genotyped by nested-PCR of merozoite surface protein 2 (MSP2) block 3. Amplicons were obtained for all the 101 genotyped samples in MSP2 PCR with 9 alleles varying in size between 300 and 800 base pair. Thirty-three (31.7%) samples had FC27 allele while 27 (26.7%) had 3D7 allele and 35 (34.7%) had mixed alleles (3D7+FC27). The Multiplicity of Infection (MOI) in the population was 1.6. Children in the age group of > 4-8 years had the highest number of different genotypes in their samples (1.8). The number of MSP2 bands per isolate was lower in the older age group (1.3) but the difference was not statistically significant. Children with parasite density range 5001-10 000 had the highest MOI of 2 while those with parasite density range 1000-5000 had the lowest of1.5. In conclusion, the present study shows that the field isolates are highly diverse in respect ofMSP2 and multiplicity of infection was neither age nor parasite density dependent in the study population.

La diversidad genética de las infecciones por Plasmodium falciparum en los humanos se halla implícita en la patogénesis de la malaria. Este estudio proporciona un primer estimado de la diversidad genética y multiplicidad del genotipo de la infección por Plasmodium falciparum en los niños con malaria por P falciparum malaria sin complicaciones en Osogbo, Nigeria. Ciento un aislados fueron usados para el análisis del polimorfismo de la población parasitaria, y genotipificados mediante reacción en cadena de la polimerasa (RCP) anidada de la proteína de superficie del merozoíto 2 (MSP2) bloque 3. Se obtuvieron amplicones para las 101 muestras genotipificadas con RCP de MSP2, con 9 alelos variando en tamaño entre 300 y 800 par de bases. Treinta y tres (31.7%) muestras tenían el alelo FC27 mientras 27 (26.7%) tenían el alelo 3D7 y 35 (34.7%) tenían alelos mezclado (3D7+FC27). La multiplicidad de infección (MOI) en la población fue 1.6. Los niños en el grupo etario de > 4-8 años tenían el número más alto de genotipos diferentes en sus muestras (1.8). El número de bandas de MSP2 por aislado era más bajo en el grupo etario de mayor edad (1.3) pero la diferencia no era estadísticamente significativa. Los niños con un rango de densidad parasitaria 5001-10 000 tenían el MOI más alto equivalente a 2, mientras aquéllos con rango de densidad parasitaria 1000-5000 tenían el MOI más bajo equivalente a 1.5. En conclusión, el presente estudio muestra que los aislados de campo son altamente diversos con respecto al MSP2, y que la multiplicidad de la infección no depende ni de la edad ni de la densidad parasitaria de la población en estudio.

Toll-like receptor (TLR) polymorphisms in African children: common TLR-4 variants predispose to severe malaria.

Genetic host factors play a substantial role in susceptibility to and severity of malaria, which continues to cause at least one million deaths per year. Recently, members of the toll-like receptor (TLR) family have been shown to be involved in recognition of the etiologic organism Plasmodium falciparum: The glycosylphosphatidylinisitol anchor induces signaling in host cells via TLR-2 and -4, while hemozoin-induced immune activation involves TLR-9. Binding of microbial ligands to the respective TLRs triggers the release of pro-inflammatory cytokines via the TLR/IL-1 receptor (TIR) domain and may contribute to the host response, including pro-inflammatory cytokine induction and malarial fever. In a case-control study among 870 Ghanaian children, we examined the influence of TLR-2, -4, and -9 polymorphisms in susceptibility to severe malaria. TLR-2 variants common in Caucasians and Asians were completely absent. However, we found a new, rare mutation (Leu658Pro), which impairs signaling via TLR-2. We failed to detect any polymorphisms within the TLR-9/interleukin-1 receptor domain. Two frequent TLR-9 promoter polymorphisms did not show a clear association with malaria severity. In contrast, the TLR-4-Asp299Gly variant occurred at a high rate of 17.6% in healthy controls, and was even more frequent in severe malaria patients (24.1%, p<0.05). Likewise, TLR-4-Thr399Ile was seen in 2.4% of healthy children and in 6.2% of patients (p=0.02). TLR-4-Asp299Gly and TLR-4-Thr399Ile conferred an 1.5- and 2.6-fold increased risk of severe malaria, respectively. These findings suggest TLR4-mediated responses to malaria in vivo and TLR-4 polymorphisms to be associated with disease manifestation. However some gray areas also suggest the scope for further improvements.

Genetic variants of beta-globin gene in Thai malaria patients.

Hemoglobin E (E26K variant of beta-globin gene) causing hemoglobinopathy is commonly observed in parts of Thailand, regardless of the hematologic disadvantage of the homozygotes. In order to detect further variants of the beta-globin gene, we performed variation screening for exon 1 of the beta-globin gene in 64 adult patients with P. falciparum malaria, living in northwest Thailand. We identified E26K and two novel variants, 59C>T and IVS+1G>T. IVS+1G>T lies on the splice donor site, and a substitution of A for G at the same site (IVS+1G>A) is known to be linked to beta-thalassemia. Thus, the biological significance of IVS+1G>T and its association with malarial infection should be clarified in future studies.

Lack of association between interleukin-10 gene promoter polymorphism, -1082G/A, and severe malaria in Thailand.

Interleukin-10 (IL-10) is an important cytokine in the down-regulation of inflammatory responses, and it has been reported that a low plasma concentration of IL-10 is associated with severe anemia and cerebral malaria in Plasmodium falciparum infections. The IL-10 gene is located on chromosome 1q31-32, and a promoter polymorphism (-1082G/A) is known to affect IL-10 protein production. In order to examine the possible association of the -1082G/A polymorphism with the severity of malaria, we studied 203 mild malaria, 164 non-cerebral severe malaria, and 109 cerebral malaria patients living in northwest Thailand. The genotyping was performed by a fluorescence resonance energy transfer (FRET) method. The frequencies of a major allele -1082A in mild malaria, in non-cerebral severe malaria, and in cerebral malaria patients were 92.6%, 92.1%, and 92.7% respectively. Our results showed no significant association of the -1082G/A polymorphism with the severity of malaria.

Polymorphisms of CD36 in Thai malaria patients.

The human protein CD36 is a major endothelial receptor for Plasmodium falciparum parasitized erythrocytes. Several polymorphisms causing CD36 deficiency have been identified to date: T1264G in Kenyan and Gambian patients, and C478T, 539delAC, and 1159insA in Japanese patients. The T1264G polymorphism is reportedly associated with protection from severe malaria in Kenyans, although there is a contradictory report suggesting the susceptibility of T1264G to severe malaria. The polymorphism of CD36 has not been thoroughly studied in Asian malaria patients. In this study, nucleotide sequence variations in exons 4, 5, 6, and 10 of CD36 were investigated in mild and cerebral malaria patients living in northwest Thailand. A novel synonymous substitution T1168C was detected in exon 10, whereas no variation was found in exons 4 and 6. The 539delAC allele in exon 5 was detected in Thai malaria patients, while T1264G, C478T, and 1159insA were not found. The 539delAC allele was observed in three cerebral malaria patients (3/107), but not in mild malaria patients (0/203). The frequency of 539delAC was significantly higher in cerebral malaria patients than in mild malaria patients (p = 0.040, Fisher's exact test). Although independent studies should be performed in order to confirm our findings, the 539delAC allele might be a high-risk variant for cerebral malaria in Thai.