Deficiência de piruvato quinase e risco de malária: estudo de base populacional na Amazônia Brasileira
Arquivos
Data
2024-09-06
Autores
Macedo, Evelyn Gonçalves 
Tipo
Trabalho de conclusão de curso
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Resumo
A suscetibilidade à infecção por estágios sanguíneos dos plasmódios e à malária clínica é modulada por características hereditárias dos hospedeiros que facilitam ou bloqueiam a invasão de glóbulos vermelhos (RBCs) e a multiplicação intracelular de parasitas. Um exemplo é a deficiência de piruvato quinase (PKD), um defeito hereditário de uma enzima eritrocitária decorrente de heterozigosidade composta ou homozigosidade para mutações no gene pklr. A PKD causa anemia hemolítica não esferocítica e limita o crescimento de Plasmodium falciparum nos eritrócitos humanos, mas pode aumentar o risco de malária vivax. Investigamos aqui a associação entre PKD e o risco de malária em nível populacional em uma coorte populacional exposta a P. falciparum e P. vivax. Em primeiro lugar, determinamos a frequência de três polimorfismos de nucleotídeo único (SNPs) representativos (tag SNPs) no gene pklr ou adjacentes – ou seja, rs1052176 (G>T), rs4971072 (A>G) e rs11264359 (A>G) – e o genótipos resultantes e testamos se esses polimorfismos estavam associados ao risco de infecção e doença por P. vivax e P. falciparum em mais de 2.000 participantes de uma coorte de base populacional em Mâncio Lima, Acre, o principal foco de malária urbana do Brasil. Ao todo, foram genotipados 2.043 participantes para rs1052176, 2.045 para rs4971072 e 2.044 para rs11264359. As frequências dos alelos mutantes variaram entre 0,46 e 0,55. Com o uso de modelos de regressão logística de efeitos mistos, testamos se havia associação entre alelos e genótipos de pklr e o risco de monoinfecção por P. vivax ou P. falciparum durante 7 inquéritos transversais realizados entre 2018 e 2021 (cerca de 9.000 observações), com resultados negativos. A heterozigosidade nos loci rs4971072 e rs11264359 pareceu conferir proteção contra a malária clínica (qualquer espécie) em modelos logísticos parcialmente ajustados, mas a significância estatística de tal associação desapareceu após o ajuste para outro polimorfismo de “resistência à malária”, a substituição T>C no motivo de ligação ao fator de transcrição GATA1 específico para eritrócitos, que elimina a expressão de DARC em hemácias, levando ao fenótipo Duffy-negativo. A seguir, investigamos a associação entre os três SNPs representativos e a atividade PK em hemácias. Para tanto, utilizamos hemácias recém-colhidas tratadas com EDTA em um ensaio colorimétrico padrão. A atividade enzimática de PK foi maior em hemácias de 77 participantes com a sequência de tipo selvagem em todos os SNPs (genótipo GG/AA/AA), com uma média de 7,45 U/g de hemoglobina, em comparação com 74 participantes com mutações em todos os SNPs (genótipo TT/GG/GG), com uma média de 6,58 U/g de hemoglobina. No entanto, encontramos taxas de prevalência semelhantes de PKD entre participantes com o genótipo de tipo selvagem GG/AA/AA (7 de 77, 9,1%) e aqueles com o genótipo mutado TT/GG/GG (7 de 74, 9,5%), sugerindo que os polimorfismos nos três loci analisados não eram capazes de predizer PKD nesta população. Concluímos que os SNPs representativos do gene pklr ou adjacentes são frequentemente encontrados nesta população exposta à malária, mas não estão associados à redução do risco de malária e nem são preditivos de PKD.
The susceptibility to blood-stage malarial infection and clinical malaria is modulated by inherited characteristics of the hosts that either favor or block red blood cell (RBC) invasion and intracellular multiplication of parasites. One example is pyruvate kinase deficiency (PKD), a hereditary RBC enzyme defect caused by compound heterozygosity or homozygosity for mutations in the pklr gene. PKD causes non-spherocytic hemolytic anemia and limits Plasmodium falciparum growth within human RBCs but has been reported to increase the risk of vivax malaria. Here, we investigate the association between PKD and malaria risk at the population level in a population-based cohort exposed to P. falciparum and P. vivax. We first determined the frequency of three pklr tag single nucleotide polymorphisms (SNPs) at or close to the pklr gene – namely, rs1052176 (G>T), rs4971072 (A>G), and rs11264359 (A>G) – and the resulting genotypes and tested whether these polymorphisms were associated with the risk of P. vivax and P. falciparum infection and disease in over 2,000 participants in a population based cohort in Mâncio Lima, Acre, the main urban malaria hotspot of Brazil. Overall, 2,043 participants were genotyped for rs1052176, 2,045 for rs4971072 and 2,044 for rs11264359. The frequencies of the mutant alleles ranged between 0.46 and 0.55. By using mixed-effects logistic regression models, we found no association between individual pklr alleles and genotypes the risk of single-species P. vivax infection or single-species P. falciparum infection during 7 cross sectional surveys between 2018 and 2021 (c. 9,000 observations). Heterozygosity at the rs4971072 and rs11264359 loci appeared to confer protection against clinical malarial (any species) in partially adjusted logistic models, but the statistical significance of such an association disappeared after adjusting for another putative “malaria resistance” polymorphism, the T>C substitution in the erythrocytic specific GATA1 transcription-factor binding motif that abolishes DARC expression on RBCs, leading to the Duffy-negative phenotype. We next investigated the association between the three tag SNPs and PK activity in RBCs. To this end, we used freshly collected RBCs treated with EDTA and a standard colorimetric assay. The PK activity in RBCs was higher 77 participants with the wild-type sequence in all tag SNPs (genotype GG/AA/AA), with a mean of 7.45 U/g hemoglobin, compared to 74 cohort participants with mutations in all tag SNPs (genotype TT/GG/GG), with a mean of 6.58 U/g hemoglobin. However, similar prevalence rates of PKD were found among participants with the wild-type GG/AA/AA genotype (7 of 77, 9.1%) and those with the mutated genotype TT/GG/GG (7 of 74, 9.5%), suggesting that the polymorphisms at the three tag loci analyzed were poor predictors of PKD in this population. We conclude that tag SNPs at or close to the pklr gene are frequently found in this malaria-exposed population but are neither associated with reduced malaria risk nor predictive of PKD.
The susceptibility to blood-stage malarial infection and clinical malaria is modulated by inherited characteristics of the hosts that either favor or block red blood cell (RBC) invasion and intracellular multiplication of parasites. One example is pyruvate kinase deficiency (PKD), a hereditary RBC enzyme defect caused by compound heterozygosity or homozygosity for mutations in the pklr gene. PKD causes non-spherocytic hemolytic anemia and limits Plasmodium falciparum growth within human RBCs but has been reported to increase the risk of vivax malaria. Here, we investigate the association between PKD and malaria risk at the population level in a population-based cohort exposed to P. falciparum and P. vivax. We first determined the frequency of three pklr tag single nucleotide polymorphisms (SNPs) at or close to the pklr gene – namely, rs1052176 (G>T), rs4971072 (A>G), and rs11264359 (A>G) – and the resulting genotypes and tested whether these polymorphisms were associated with the risk of P. vivax and P. falciparum infection and disease in over 2,000 participants in a population based cohort in Mâncio Lima, Acre, the main urban malaria hotspot of Brazil. Overall, 2,043 participants were genotyped for rs1052176, 2,045 for rs4971072 and 2,044 for rs11264359. The frequencies of the mutant alleles ranged between 0.46 and 0.55. By using mixed-effects logistic regression models, we found no association between individual pklr alleles and genotypes the risk of single-species P. vivax infection or single-species P. falciparum infection during 7 cross sectional surveys between 2018 and 2021 (c. 9,000 observations). Heterozygosity at the rs4971072 and rs11264359 loci appeared to confer protection against clinical malarial (any species) in partially adjusted logistic models, but the statistical significance of such an association disappeared after adjusting for another putative “malaria resistance” polymorphism, the T>C substitution in the erythrocytic specific GATA1 transcription-factor binding motif that abolishes DARC expression on RBCs, leading to the Duffy-negative phenotype. We next investigated the association between the three tag SNPs and PK activity in RBCs. To this end, we used freshly collected RBCs treated with EDTA and a standard colorimetric assay. The PK activity in RBCs was higher 77 participants with the wild-type sequence in all tag SNPs (genotype GG/AA/AA), with a mean of 7.45 U/g hemoglobin, compared to 74 cohort participants with mutations in all tag SNPs (genotype TT/GG/GG), with a mean of 6.58 U/g hemoglobin. However, similar prevalence rates of PKD were found among participants with the wild-type GG/AA/AA genotype (7 of 77, 9.1%) and those with the mutated genotype TT/GG/GG (7 of 74, 9.5%), suggesting that the polymorphisms at the three tag loci analyzed were poor predictors of PKD in this population. We conclude that tag SNPs at or close to the pklr gene are frequently found in this malaria-exposed population but are neither associated with reduced malaria risk nor predictive of PKD.