Eduardo José Melo dos Santos

Genetic Structure of Plasmodium falciparum Populations in the Brazilian Amazon Region

After a major increase in incidence between the 1970s and the 1990s, the Brazilian Amazon region now accounts for the most cases of Plasmodium falciparum malaria in the Americas. Polymorphism of 10 microsatellite loci in the P. falciparum genome was studied in 196 isolates obtained from 5 populations in the region. There was significant multilocus linkage disequilibrium, particularly within populations with lower proportions of mixed-genotype infections. However, most multilocus genotypes in different isolates were distinct, and there was no evidence of any recent epidemic expansion of particular clones. Genetic divergence between populations was very substantial but did not fit a simple model of isolation by distance. Thus, different foci of P. falciparum in Brazil are quite independent, with distinct population structures and minimal gene flow, a finding that has implications for strategies to control infection and to contain the spread of drug resistance at a regional level.

Origin of the hemoglobin S gene in a northern Brazilian population: the combined effects of slave trade and internal migrations

Com o objetivo de investigar a origem da mutacao bS na populacao da regiao norte do Brasil, foram analisados polimorfismos de DNA no complexo de genes b da hemoglobina em 30 pacientes com anemia falciforme na populacao de Belem, a capital do Estado do Para. Sessenta e sete por cento dos cromossomos bS analisados apresentaram o haplotipo Bantu, 30% o haplotipo Benin e 3% o haplotipo Senegal. A origem da mutacao bS na populacao de Belem, estimada de acordo com a distribuicao de haplotipos, nao esta de acordo com a esperada com base em dados historicos sobre o trafico de escravos para a regiao norte, os quais indicam uma reduzida contribuicao de escravos da regiao do Benin. Essas diferencas podem ser atribuidas ao trafico interno de escravos, bem como ao posterior fluxo de populacoes imigrantes, particularmente de nordestinos. A distribuicao de haplotipos em Belem nao difere significativamente da observada em outras regioes brasileiras, muito embora os dados historicos sugiram que a maioria dos escravos procedentes da regiao do Atlântico-Oeste africano, onde predomina o haplotipo Senegal, foi trazida para o norte do Brasil, enquanto que o nordeste (Bahia, Pernambuco e Maranhao) recebeu o maior contingente de escravos oriundos da regiao centro-oeste africana, onde o haplotipo Benin e o mais comum. Nos sugerimos que as diferencas regionais quanto a procedencia dos escravos africanos tambem foram modificadas pelo trafico de escravos estabelecido entre as diferentes regioes brasileiras e posteriormente pelos movimentos migratorios.

Amazonian Amerindians exhibit high variability of KIR profiles

Natural killer cell immunoglobulin-like receptors (KIRs) mediate cell lysis through the recognition of human leukocyte antigen class I complexes in target cells, playing an important role in innate immune response. In this context, disease-based selective pressures could be relevant, leaving signatures detected by population studies. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania, and Africa remain poorly studied. The aim of this study was to analyze the variability of KIR genes in Amerindian tribes from the Amazon region to infer about their evolutionary history. KIR profiles were estimated in 40 individuals from six Amazonian Amerindian tribes using single specific primer polymerase chain reaction. Twenty-five different profiles were identified, and surprisingly, the haplogroup A frequency was the lowest observed in human populations (16%). Results showed also that KIR variability was higher in this group in contrast to Venezuelan Amerindians. Principal components analysis evidenced that Amerindians formed a separated group from other worldwide populations and showed a higher intraethnic differentiation in comparison to other ethnic groups. Such pattern may reflect small effective size and intense genetic drift. However, because of the role of KIR in immune response, selective pressures cannot be entirely ruled out.

Genetical-demographic data from two amazonian populations composed of descendants of african slaves: Pacoval and Curiau

The Amazon region of Brazil includes communities founded by escaped slaves, some of which still remain relatively isolated. We studied two such Afro-Brazilian communities (Pacoval and Curiau), in the rural area of Alenquer, Para, and in the metropolitan region of Macapa, Amapa, respectively. Among 12 blood loci, alleles considered as markers of African ancestry, such as HBB*S, HBB*C, TF*D1, HP*2M, ABO*B, RH*D-, and CA2*2 were found at frequencies that are expected for populations with a predominantly African origin. Estimates of interethnic admixture indicated that the degree of the African component in Curiau (74%) is higher than that of Pacoval (44%); an Amerindian contribution was not detected in Curiau. Estimated values of African ancestry fit well with the degree of isolation and mobility of the communities. Pacoval exhibited a high proportion of immigrants among the parents and grandparents of the individuals studied, whereas persons living in Curiau exhibited a low level of mobility, despite its location in the metropolitan area of Macapa city, suggesting a relatively strong barrier against the interethnic admixture in this population. In addition, analysis of genetic data in a sub-sample consisting of individuals whose parents and grandparents were born in the study site, and that probably represents the populations two generations ago, indicated that gene flow from non-black people is not a recent event in both populations.

A database for curating the associations between killer cell immunoglobulin-like receptors and diseases in worldwide populations

The killer cell immunoglobulin-like receptors (KIR) play a fundamental role in the innate immune system, through their interactions with human leucocyte antigen (HLA) molecules, leading to the modulation of activity in natural killer (NK) cells, mainly related to killing pathogen-infected cells. KIR genes are hugely polymorphic both in the number of genes an individual carries and in the number of alleles identified. We have previously developed the Allele Frequency Net Database (AFND, http://www.allelefrequencies.net), which captures worldwide frequencies of alleles, genes and haplotypes for several immune genes, including KIR genes, in healthy populations, covering >4 million individuals. Here, we report the creation of a new database within AFND, named KIR and Diseases Database (KDDB), capturing a large quantity of data derived from publications in which KIR genes, alleles, genotypes and/or haplotypes have been associated with infectious diseases (e.g. hepatitis C, HIV, malaria), autoimmune disorders (e.g. type I diabetes, rheumatoid arthritis), cancer and pregnancy-related complications. KDDB has been created through an extensive manual curation effort, extracting data on more than a thousand KIR-disease records, comprising >50 000 individuals. KDDB thus provides a new community resource for understanding not only how KIR genes are associated with disease, but also, by working in tandem with the large data sets already present in AFND, where particular genes, genotypes or haplotypes are present in worldwide populations or different ethnic groups. We anticipate that KDDB will be an important resource for researchers working in immunogenetics. Database URL: http://www.allelefrequencies.net/diseases/

Expression of PLIN2 and PLIN3 during oocyte maturation and early embryo development in cattle.

In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.

Association of killer cell immunoglobulin-like receptor polymorphisms with chronic hepatitis C and responses to therapy in Brazil

Soroprevalence for Hepatitis C virus is reported as 2.12% in Northern Brazil, with about 50% of the patients exhibiting a sustained virological response (SVR). Aiming to associate polymorphisms in Killer Cell Immunoglobulin-like Receptors (KIR) with chronic hepatitis C and therapy responses we investigated 125 chronic patients and 345 controls. Additionally, 48 ancestry markers were genotyped to control for population stratification. The frequency of the KIR2DL2 and KIR2DL2+HLA-CAsp80 gene and ligand was higher in chronic infected patients than in controls (p < 0.0009, OR = 3.4; p = 0.001, OR = 3.45). In fact, KIR2DL3 is a weaker inhibitor of NK activity than KIR2DL2, which could explain the association of KIR2DL2 with chronic infection. Moreover, KIR2DS2 and KIR2DS2+HLA-CAsp80 (p < 0.0001, OR = 2.51; p = 0.0084, OR = 2.62) and KIR2DS3 (p < 0.0001; OR = 2.57) were associated with chronic infection, independently from KIR2DL2. No differences in ancestry composition were observed between control and patients, even with respect to therapy response groups. The allelic profile KIR2DL2/KIR2DS2/KIR2DS3 was associated with the chronic hepatitis C (p < 0.0001; OR = 3). Furthermore, the patients also showed a higher mean number of activating genes and a lower frequency of the homozygous AA profile, which is likely secondary to the association with non-AA and/or activating genes. In addition, the KIR2DS5 allele was associated with SVR (p = 0.0261; OR = 0.184).The ancestry analysis of samples ruled out any effects of population substructuring and did not evidence interethnic differences in therapy response, as suggested in previous studies.

Impact of population admixture on the distribution of immune response co-stimulatory genes polymorphisms in a Brazilian population.

Co-stimulatory molecules are essential in the orchestration of immune response and polymorphisms in their genes are associated with various diseases. However, in the case of variable allele frequencies among continental populations, this variation can lead to biases in genetic studies conducted in admixed populations such as those from Brazil. The aim of this study was to evaluate the influence of genomic ancestry on distributions of co-stimulatory genes polymorphisms in an admixed Brazilian population. A total of 273 individuals from the north of Brazil participated in this study. Nine single nucleotide polymorphisms in 7 genes (CD28, CTLA4, ICOS, CD86, CD40, CD40L and BLYS) were determined by polymerase chain reaction-restriction fragment length polymorphism. We also investigated 48 insertion/deletion ancestry markers to characterize individual African, European and Amerindian ancestry proportions in the samples. The analysis showed that the main contribution was European (43.9%) but also a significant contribution of African (31.6%) and Amerindian (24.5%) ancestry. ICOS, CD40L and CD86 polymorphisms were associated with genomic ancestry. However there were no significant differences in the proportions of ancestry for the other SNPs and haplotypes studied. Our findings reinforce the need to apply AIMs in genetic association studies involving these polymorphisms in the Brazilian population.

A web resource for mining HLA associations with adverse drug reactions: HLA-ADR

Abstract Human leukocyte antigens (HLA) are an important family of genes involved in the immune system. Their primary function is to allow the host immune system to be able to distinguish between self and non-self peptides—e.g. derived from invading pathogens. However, these genes have also been implicated in immune-mediated adverse drug reactions (ADRs), presenting a problem to patients, clinicians and pharmaceutical companies. We have previously developed the Allele Frequency Net Database (AFND) that captures the allelic and haplotype frequencies for these HLA genes across many healthy populations from around the world. Here, we report the development and release of the HLA-ADR database that captures data from publications where HLA alleles and haplotypes have been associated with ADRs (e.g. Stevens–Johnson Syndrome/toxic epidermal necrolysis and drug-induced liver injury). HLA-ADR was created by using data obtained through systematic review of the literature and semi-automated literature mining. The database also draws on data already present in AFND allowing users to compare and analyze allele frequencies in both ADR patients and healthy populations. The HLA-ADR database provides clinicians and researchers with a centralized resource from which to investigate immune-mediated ADRs. Database URL: http://www.allelefrequencies.net/hla-adr/.

Genetic relationships among native americans based on beta-globin gene cluster haplotype frequencies

The distribution of b-globin gene haplotypes was studied in 209 Amerindians from eight tribes of the Brazilian Amazon: Asurini from Xingu, Awa-Guaja, Parakana, Urubu-Kaapor, Zoe, Kayapo (Xikrin from the Bacaja village), Katuena, and Tiriyo. Nine different haplotypes were found, two of which (n. 11 and 13) had not been previously identified in Brazilian indigenous populations. Haplotype 2 (+ - - - -) was the most common in all groups studied, with frequencies varying from 70% to 100%, followed by haplotype 6 (- + + - +), with frequencies between 7% and 18%. The frequency distribution of the b-globin gene haplotypes in the eighteen Brazilian Amerindian populations studied to date is characterized by a reduced number of haplotypes (average of 3.5) and low levels of heterozygosity and intrapopulational differentiation, with a single clearly predominant haplotype in most tribes (haplotype 2). The Parakana, Urubu-Kaapor, Tiriyo and Xavante tribes constitute exceptions, presenting at least four haplotypes with relatively high frequencies. The closest genetic relationships were observed between the Brazilian and the Colombian Amerindians (Wayuu, Kamsa and Inga), and, to a lesser extent, with the Huichol of Mexico. North-American Amerindians are more differentiated and clearly separated from all other tribes, except the Xavante, from Brazil, and the Mapuche, from Argentina. A restricted pool of ancestral haplotypes may explain the low diversity observed among most present-day Brazilian and Colombian Amerindian groups, while interethnic admixture could be the most important factor to explain the high number of haplotypes and high levels of diversity observed in some South-American and most North-American tribes.