Patrícia Machado

Malaria: looking for selection signatures in the human PKLR gene region

The genetic component of susceptibility to malaria is both complex and multigenic and the better‐known protective polymorphisms are those involving erythrocyte‐specific structural proteins and enzymes. In vivo and in vitro data have suggested that pyruvate kinase deficiency, which causes a nonspherocytic haemolytic anaemia, could be protective against malaria severity in humans, but this hypothesis remains to be tested. In the present study, we conducted a combined analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in the pyruvate kinase‐encoding gene (PKLR) and adjacent regions (chromosome 1q21) to look for malaria selective signatures in two sub‐Saharan African populations from Angola and Mozambique, in several groups with different malaria infection outcome. A European population from Portugal, including a control and a pyruvate kinase‐deficient group, was used for comparison. Data from STR and SNP loci spread along the PKLR gene region showed a considerably higher differentiation between African and Portuguese populations than that usually found for neutral markers. In addition, a wider region showing strong linkage disequilibrium was found in an uncomplicated malaria group, and a haplotype was found to be associated with this clinical group. Altogether, this data suggests that malaria selective pressure is acting in this genomic region.

Characterization and Expression Analysis of the Cytochrome bd Oxidase Operon from Desulfovibrio gigas

Although classified as anaerobic, Desulfovibrio gigas contains a functional canonical membrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In the present study, we report the identification of the operon cydAB encoding the two subunits of cytochrome bd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptional regulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity to cytochrome bd from other organisms, presenting the conserved residues typical from these proteins. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed the operon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in different media and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in the presence of 150 μM NO. However, in the presence of 10 μM NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 μM O2 or 10 μM NO.

Analysis of malaria associated genetic traits in Cabo Verde, a melting pot of European and sub Saharan settlers

Malaria has occurred in the Cabo Verde archipelago with epidemic characteristics since its colonization. Nowadays, it occurs in Santiago Island alone and though prophylaxis is not recommended by the World Health Organization, studies have highlight the prospect of malaria becoming a serious public health problem as a result of the presence of antimalarial drug resistance associated with mutations in the parasite populations and underscore the need for tighter surveillance. Despite the presumptive weak immune status of the population, severe symptoms of malaria are not observed and many people present a subclinical course of the disease. No data on the prevalence of sickle-cell trait and red cell glucose-6-phosphate dehydrogenase deficiency (two classical genetic factors associated with resistance to severe malaria) were available for the Cabo Verde archipelago and, therefore, we studied the low morbidity from malaria in relation to the particular genetic characteristics of the human host population. We also included the analysis of the pyruvate kinase deficiency associated gene, reported as putatively associated with resistance to the disease. Allelic frequencies of the polymorphisms examined are closer to European than to African populations and no malaria selection signatures were found. No association was found between the analyzed human factors and infection but one result is of high interest: a linkage disequilibrium test revealed an association of distant loci in the PKLR gene and adjacent regions, only in non-infected individuals. This could mean a more conserved gene region selected in association to protection against the infection and/or the disease.

Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria

Background Pyruvate kinase (PK) deficiency, causing hemolytic anemia, has been associated to malaria protection and its prevalence in sub-Saharan Africa is not known so far. This work shows the results of a study undertaken to determine PK deficiency occurrence in some sub-Saharan African countries, as well as finding a prevalent PK variant underlying this deficiency. Materials and Methods Blood samples of individuals from four malaria endemic countries (Mozambique, Angola, Equatorial Guinea and Sao Tome and Principe) were analyzed in order to determine PK deficiency occurrence and detect any possible high frequent PK variant mutation. The association between this mutation and malaria was ascertained through association studies involving sample groups from individuals showing different malaria infection and outcome status. Results The percentage of individuals showing a reduced PK activity in Maputo was 4.1% and the missense mutation G829A (Glu277Lys) in the PKLR gene (only identified in three individuals worldwide to date) was identified in a high frequency. Heterozygous carrier frequency was between 6.7% and 2.6%. A significant association was not detected between either PK reduced activity or allele 829A frequency and malaria infection and outcome, although the variant was more frequent among individuals with uncomplicated malaria. Conclusions This was the first study on the occurrence of PK deficiency in several areas of Africa. A common PKLR mutation G829A (Glu277Lys) was identified. A global geographical co-distribution between malaria and high frequency of PK deficiency seems to occur suggesting that malaria may be a selective force raising the frequency of this 277Lys variant.

La contribución de los polimorfismos humanos del eritrocito en la protección contra la malaria

The understanding of the complex life cycle of malaria has greatly improved in the last few years, however, despite decades of research and struggle against the disease, it continues to be a major public health problem, especially in the poorest areas of the world. Due to its long-term high prevalence in certain regions of the globe, malaria has exerted strong selective pressure on the human genome. The genetic component of malaria susceptibility is complex, with a variety of polymorphisms influencing both pathogenesis and host response. Evaluating these determinants of susceptibility and deciphering the mechanisms involved may lead to the discovery of new vaccines or targets for pharmacological agents. The most common and best characterized human genetic polymorphisms that confer protection against malaria involve specific structural erythrocyte proteins (such as haemoglobin S and C, thalassemias, the Duffy antigen, and blood group O) and enzymes (such as glucose-6-phosphate dehydrogenase deficiency and, more recently described, pyruvate kinase deficiency). This short review describes these genetic variants, reviews some of the controversial results that have been obtained, and discusses mechanisms that might explain the protection they provide. La contribución de los polimorfismos humanos del eritrocito en la protección contra la malaria

Analysis of TPI gene promoter variation in three sub-Saharan Africa population samples.

Population samples from Angola, Mozambique, and S. Tomé e Príncipe were screened for the TPI gene promoter variants ‐5A→G, ‐8G→A and ‐24T→G. Three haplotypes were identified in the three populations: the haplotype ‐5A‐8G‐24T (average frequency 65.3%) and two less common haplotypes ‐5G‐8G‐24T (average frequency 24.7%) and ‐5G‐8A‐24T (average frequency 10.0%). A population sample from Central Portugal showed the haplotype ‐5A‐8G‐24T in 139 chromosomes and one subject heterozygous for haplotype ‐5G‐8A‐24G. The exact test of sample differentiation among three groups of malaria‐infected individuals classified according to the severity of the disease showed no significant differences. We confirmed TPI gene diversity in sub‐Saharan Africa, but we could not detect any association between TPI promoter variation and a malarial protective effect. Larger scale epidemiological studies are thus required to clarify this putative mechanism of natural host defense against this worldwide public health problem. Am. J. Hum. Biol., 2009.

Triosephosphate isomerase gene promoter variation: -5G/A and -8G/A polymorphisms in clinical malaria groups in two African populations

TPI1 promoter polymorphisms occur in high prevalence in individuals from African origin. Malaria-patients from Angola and Mozambique were screened for the TPI1 gene promoter variants rs1800200A>G, (-5G>A), rs1800201G>A, (-8G>A), rs1800202T>G, (-24T>G), and for the intron 5 polymorphism rs2071069G>A, (2262G>A). -5G>A and -8G>A variants occur in 47% and 53% in Angola and Mozambique, respectively while -24T>G was monomorphic for the wild-type T allele. Six haplotypes were identified and -8A occurred in 45% of the individuals, especially associated with the GAG haplotype and more frequent in non-severe malaria groups, although not significantly. The arising and dispersion of -5G>A and -8G>A polymorphisms is controversial. Their age was estimated by analyses of two microsatellite loci, CD4 and ATN1, adjacent to TPI1 gene. The -5G>A is older than -8G>A, with an average estimate of approximately 35,000 years. The -8A variant arose in two different backgrounds, suggesting independent mutational events. The first, on the -5G background, may have occurred in East Africa around 20,800 years ago; the second, on the -5A background, may have occurred in West Africa some 7500 years ago. These estimates are within the period of spread of agriculture and the malaria mosquito vector in Africa, which could has been a possible reason for the selection of -8A polymorphism in malaria endemic countries.

Quantitative proteomics for the analysis of Plasmodium falciparum and its red blood cell host - a preliminary study

Background Malaria is a major cause of death and has been one of the strongest selective forces on the human genome selecting variants that influence pathogenesis, host response and may protect against disease severity [1, 2, 3]. Previous studies suggest the association between malaria and red blood cell (RBC) glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiencies in humans [4]. This study focuses on RBC-pathogen interactions and the effect of these two enzymatic deficiencies on parasite development. Proteomic information from Plasmodium infection is scarce, and proteomes of both G6PDand PKdeficient RBC and from parasites growing in these cells have not yet been characterized. We performed a proteomic study to detect the relative abundance of proteins from both G6PDand PK-deficient RBC, and also from Plasmodium infecting these cells. It will provide key information about malaria dynamics, but also about enzyme deficiencies causing important haemolytic anaemia. Furthermore, it will contribute to a better understanding of host-parasite interactions.

A contribuição dos polimorfismos humanos do eritrócito na proteção contra a malária

The understanding of the complex life cycle of malaria has greatly improved in the last few years, however, despite decades of research and struggle against the disease, it continues to be a major public health problem, especially in the poorest areas of the world. Due to its long-term high prevalence in certain regions of the globe, malaria has exerted strong selective pressure on the human genome. The genetic component of malaria susceptibility is complex, with a variety of polymorphisms influencing both pathogenesis and host response. Evaluating these determinants of susceptibility and deciphering the mechanisms involved may lead to the discovery of new vaccines or targets for pharmacological agents. The most common and best characterized human genetic polymorphisms that confer protection against malaria involve specific structural erythrocyte proteins (such as haemoglobin S and C, thalassemias, the Duffy antigen, and blood group O) and enzymes (such as glucose-6-phosphate dehydrogenase deficiency and, more recently described, pyruvate kinase deficiency). This short review describes these genetic variants, reviews some of the controversial results that have been obtained, and discusses mechanisms that might explain the protection they provide. La contribución de los polimorfismos humanos del eritrocito en la protección contra la malaria

The contribution of human erythrocyte polymorphisms in the protection against malaria

The understanding of the complex life cycle of malaria has greatly improved in the last few years, however, despite decades of research and struggle against the disease, it continues to be a major public health problem, especially in the poorest areas of the world. Due to its long-term high prevalence in certain regions of the globe, malaria has exerted strong selective pressure on the human genome. The genetic component of malaria susceptibility is complex, with a variety of polymorphisms influencing both pathogenesis and host response. Evaluating these determinants of susceptibility and deciphering the mechanisms involved may lead to the discovery of new vaccines or targets for pharmacological agents. The most common and best characterized human genetic polymorphisms that confer protection against malaria involve specific structural erythrocyte proteins (such as haemoglobin S and C, thalassemias, the Duffy antigen, and blood group O) and enzymes (such as glucose-6-phosphate dehydrogenase deficiency and, more recently described, pyruvate kinase deficiency). This short review describes these genetic variants, reviews some of the controversial results that have been obtained, and discusses mechanisms that might explain the protection they provide. La contribución de los polimorfismos humanos del eritrocito en la protección contra la malaria