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Biomedical subjects

S D Pena

Publications and source records attributed to S D Pena.

At least 37 records · Page 2Linked to original sources
PubMed1999

The central Siberian origin for native American Y chromosomes.

Y chromosomal DNA polymorphisms were used to investigate Pleistocene male migrations to the American continent. In a worldwide sample of 306 men, we obtained 32 haplotypes constructed with the variation found in 30 distinct polymorphic sites. The major Y haplotype present in most Native Americans was traced back to recent ancestors common with Siberians, namely, the Kets and Altaians from the Yenissey River Basin and Altai Mountains, respectively. Going further back, the next common ancestor gave rise also to Caucasoid Y chromosomes, probably from the central Eurasian region. This study, therefore, suggests a predominantly central Siberian origin for Native American paternal lineages for those who could have migrated to the Americas during the Upper Pleistocene.

Genetic Variation↗
PubMed1999

Identification in Portugal and Brazil of a mtDNA lineage containing a 9-bp triplication of the intergenic COII/tRNALys region.

Although the deletion of one of the 9-bp repeats in region V of mitochondrial DNA is very common in Asians, Asian-derived populations and Africans, the triplication of the 9-bp segment was described only a few times, mostly on individuals from Asian origin. Here, we report for the first time the presence of the 9-bp triplication in Europeans. The triplication was initially found in one Brazilian individual. Sequencing of the hypervariable segments I (HVSI) and II (HVS2) of the control region and RFLP analysis of the coding region classified the mtDNA as belonging to the European haplogroup H. Since white Brazilians are predominantly of Portuguese descent, we screened 96 unrelated Northern Portuguese for the 9-bp triplication and found its presence in two of them (2.1%). One of these had an mtDNA haplotype identical to that of the Brazilian individual, while the other differed in a single base change in HVS2. The fact that the 9-bp triplication has reached polymorphic frequencies in Northern Portugal and that it has apparently differentiated into at least two lineages defined by the mutuation in HVS2 suggests that it probably occurred a long time ago.

Base Sequence↗
PubMed1999

The population structure of Trypanosoma cruzi: expanded analysis of 54 strains using eight polymorphic CA-repeat microsatellites.

Recently we cloned and sequenced the first eight Trypanosoma cruzi polymorphic microsatellite loci and studied 31 clones and strains to obtain valuable information about the population structure of the parasite. We have now studied 23 further strains, increasing from 11 to 31 the number of strains obtained from patients with chronic Chagas disease. This expanded set of 54 strains and clones analyzed with the eight microsatellites markers confirmed the previously observed diploidy, clonal population organization and very high polymorphism of T. cruzi. Moreover, this new study disclosed two new features of the population genetic structure of T. cruzi. The first was the discovery that, similarly to what we had previously shown for strains isolated from insect vectors, mammals and humans with acute disease, isolates from patients in the chronic phase of Chagas disease could also be multiclonal, albeit at a reduced proportion. Second, when we used parsimony to display the genetic relationship among the clonal lineages in an unrooted Wagner network we observed, like before, a good correlation of the tree topography with the classification in three clusters on the basis of single locus analysis of the ribosomal RNA genes. However, a significant new finding was that now the strains belonging to cluster 2 split in two distant sub-clusters. This observation suggests that the evolutionary history of T. cruzi may be more complex than we previously thought.

Animals↗
PubMed1999

Chagas' disease diagnosis: comparative analysis of parasitologic, molecular, and serologic methods.

During the course of chronic chagasic infection, low parasitemia levels prevent parasite detection by current techniques such as hemoculture and xenodiagnosis. Since serologic tests have sensitivity but lack specificity, molecular assays based on the polymerase chain reaction (PCR) have been proposed as alternative tools for parasite detection in individuals with chronic Chagas' disease. A variable degree of PCR efficiency has been reported in the literature and illustrates the need for further evaluation of large numbers of chagasic patients. In this study, we compared an optimized PCR technique with hemoculture and complement-mediated lysis (CoML) in 113 individuals from or living in endemic areas of Brazil who had conventional serologic results that were either positive, negative, or inconclusive. The PCR amplification yielded positive results in 83.5% (66 of 79) of individuals with positive serology, 47.6% (10 of 21) with negative serology, and 46.2% (6 of 13) with inconclusive serology. Of 10 patients with negative serology and positive PCR result, eight (80%) had positive CoML, indicating that they could have been chagasic but were not mounting immune responses. The PCR results were also positive for all individuals who had positive hemoculture, for 37 individuals with negative hemoculture and positive serology, and for two of six individuals with inconclusive serology and negative hemoculture. Thirteen individuals living in nonendemic areas who had negative serology were used as a negative control group: 100% had negative PCR results. Our results show that the optimized PCR protocol used here was very sensitive in detecting the presence of Trypanosoma cruzi in chronic chagasic patients. The PCR and CoML results were well correlated in all of the groups studied, which suggests that our PCR protocol may be effective in the evaluation of cure in patients who receive anti-parasite treatment.

Animals↗
PubMed1999

Multiple geographic sources of region V 9-bp deletion haplotypes in Brazilians.

We investigated 245 white Brazilians for the presence of the 9-bp deletion in the intergenic COII/tRNALys region of the mitochondrial DNA (mtDNA) and found the deletion in 21 individuals (8.6% of the sample). Because white Brazilians are believed to be predominantly of European descent and this marker is rare in Europe, we established the geographic origin of these 21 mtDNA sequences by sequencing the hypervariable segment I of the mtDNA control region and by performing an RFLP analysis. Only 1 European mtDNA lineage was identified. On the other hand, 16 of the individuals had matrilineages of Amerindian origin and 4 had African mtDNA haplotypes. These results demonstrate that in the formation of the present-day white Brazilian population there was a significant contribution of Amerindian and African matrilineages. Although these data initially appear surprising, they agree well with the historical records of Brazilian colonization.

Africa↗
PubMed1998

Characterization and mapping of four novel human expressed polymorphic trinucleotide microsatellites.

Recently, several important human genetic diseases have been shown to be due to pathological expansion of expressed trinucleotide microsatellites. Discovery of other such 'expansion diseases' will depend on characterization of more expressed loci containing trinucleotide repeats. We searched the expressed sequence tag database (dbEST) for repetitive trinucleotides and selected four loci for further studies (EST00586 EST05486, EST13299 and HHEA48B). For each locus, we have identified size polymorphism by PCR amplification and achieved chromosomal mapping using a somatic hybrid cell panel and the Stanford G3 radiation hybrid panel. Further searches of GenBank, dbEST and UniGene unraveled EST clusters for three of the loci, allowing construction of contigs and, in one instance, identification of a partial open reading frame. Three of the loci were linked to autosomal dominant human genetic diseases whose primary gene defect has not yet been established. Although it is a priori improbable that there is an etiological connection between the loci studied and the diseases, our results demonstrate that dbEST constitutes a useful starting point in the search for candidate loci for new expansion diseases.

Chromosome Mapping↗
PubMed1998

Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites.

We describe here the identification of eight polymorphic microsatellite loci with (CA)n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA)12 attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Salpha gene.

Animals↗
PubMed1998

Trypanosoma cruzi: optimization of polymerase chain reaction for detection in human blood.

We have optimized the conditions for DNA extraction and polymerase chain reaction (PCR) amplification to diagnose the presence of Trypanosoma cruzi DNA in the blood and serum of patients with chronic Chagas disease. The approximately 330-bp fragment of the kinetoplast minicircles was used as a target for amplification. The use of chemiluminescence on slot blots with a specific alkaline phosphatase-conjugated oligonucleotide probe detected specific product from as little as 0.1 fg of T. cruzi kDNA. An additional product of approximately 200 bp inadvertently amplified from the human genome was observed in human blood from T. cruzi-negative and -positive samples and served as an internal control of the amplification. Samples from other mammalian hosts were also assayed using the PCR protocol. The higher sensitivity of our PCR method observed in both acute and chronic phases of T.cruzi infections in mice and dog, respectively, could be useful in monitoring the course of infection during experimental drug tests in laboratory animals. Since this procedure showed a higher sensitivity than other protocols in the literature, it may be a suitable routine test in diagnosing Chagas disease, especially for patients presenting very low parasitemia levels.

Animals↗
PubMed1998

Polarity of mutations in tumor-associated microsatellite instability.

Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)3CAC(CAT)CAC(CAT)2CAC(CAT)CAC(CAT)15, thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms.

Adenocarcinoma↗
PubMed1998

Genetic relationships between Trypanosoma cruzi strains isolated from chronic chagasic patients in southern Brazil as revealed by RAPD and SSR-PCR analysis.

30 Trypanosoma cruzi strains isolated from chronic chagasic patients were studied at the genotype level by RAPD with arbitrary primers and SSR-PCR analysis. The genetic distance of strains was measured by the percentage of unshared bands. The results showed that the strains isolated from chronic patients from different Chagas' disease endemic areas of Brazil constituted a broad group significantly more correlated than reported in previous studies, presenting RAPD profiles with an average of 71% of shared bands and SSR-PCR patterns with a mean of 59% of shared bands. In contrast to other findings in the literature, these strains did not group on the basis of their geographic origin. These results suggest that a special adaptation of a parasite population in human hosts from mixed infective T. cruzi populations circulating in nature may have occurred.

Animals↗
PubMed1998

Genetic Variability of Trypanosoma cruzi:Implications for the Pathogenesis of Chagas Disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, has a variable clinical course, ranging from symptomless infection to severe chronic disease with cardiovascular or gastrointestinal involvement or even overwhelming acute episodes. The factors influencing this clinical variability have not been elucidated, but genetic variation of both the host and parasite is likely to be important. Here, Andréa M. Macedo and Sérgio D.J. Pena review the evidence showing a role for the genetic constitution of T. cruzi in determining the clinical characteristics of Chagas disease, and propose a ;clonal-histotropic model' for the pathogenesis of this disease.

Journal Article↗
PubMed1998

Characterization of a Schistosoma mansoni homologue of the gene encoding the breast basic conserved protein 1/L13 ribosomal protein.

The Schistosoma mansoni gene sequence encoding the breast basic conserved protein 1/ribosomal protein L13 has been isolated from an adult worm cDNA library using the Expressed Sequence Tag strategy. The cDNA codes for a putative protein of 184 amino acids which is approximately 55% identical to other eukaryotic L13 ribosomal proteins. A PCR amplified genomic fragment containing the coding region of the gene was seen to possess only a single large intron interrupting the open reading frame. Studies of gene expression by RT-PCR showed the transcript is expressed in distinct stages of the parasite life cycle. The cDNA was also hybridized with an ordered cosmid library of S. mansoni and the identified cosmids were mapped to chromosomes 3 and W by chromosomal in situ suppression hybridization.

Amino Acid Sequence↗
PubMed1998

Identification of genes encoding Schistosoma mansoni antigens using an antigenic sequence tag strategy.

Another approach for the identification of genes that code for antigenic products is described using an antigenic sequence tag (AST) strategy. A Schistosoma mansoni adult worm cDNA library was screened with affinity chromatography-purified immunoglobulins from infected human sera and a mild oxidation treatment with sodium periodate. From 1 or both ends of 30 cDNA clones, 30 ASTs were obtained. Of these, 22 were previously known Sm antigens. One clone had matches with entries for other organisms in the databases and 6 had homology with Sm-expressed sequence tags (EST) entries. These clones, together with another 1 that had no significant database matches, were considered new antigenic genes in S. mansoni. The strategy proved to be efficient for the identification of genes that could be used for immunological studies and evaluation as vaccine candidates.

Animals↗
PubMed1997

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein.

We have cloned and characterized a Schistosoma mansoni cDNA encoding a basic protein homologous to the human Y-box binding protein 1 (YB-1). The 1.3-kb S. mansoni YB-1 transcript, which was shown to be expressed in various stages of the parasite life cycle, codes for a protein of 217 amino acids containing, towards its N-terminus, a nucleic acid binding motif, known as the cold-shock domain (CSD). This domain is 64% identical to the cold-shock domain of other members of the Y-box binding protein family and 43% identical to the cold-shock protein CspA of Escherichia coli. In S. mansoni YB-1, the cold-shock domain possess some structural characteristics that permit dimer formation as occurs in the Bacillus subtilis cold-shock protein CspB. The C-terminal region of S. mansoni YB-1 differs from the other Y-box binding proteins because of the presence of tandem repeats of Arg and Gly, suggesting the formation of a fibroin-like beta-sandwich structure. This novel folding pattern for the C-terminus of S. mansoni YB-1 might suggest a distinct specific function for this protein in the parasite.

Amino Acid Sequence↗