Full-gene haplotypes refine CYP2D6 metabolizer phenotype inferences.

CYP2D6 is a critical pharmacogenetic target, and polymorphisms in the gene region are commonly used to infer enzyme activity score and predict resulting metabolizer phenotype: poor, intermediate, extensive/normal, or ultrarapid which can be useful in determining cause and/or manner of death in some autopsies. Current genotyping approaches are incapable of identifying novel and/or rare variants, so CYP2D6 star allele definitions are limited to polymorphisms known a priori. While useful for most predictions, recent studies using massively parallel sequencing data have identified additional polymorphisms in CYP2D6 that are predicted to alter enzyme function but are not considered in current star allele nomenclature. The 1000 Genomes Project data were used to produce full-gene haplotypes, describe their distribution in super-populations, and predict enzyme activity scores. Full-gene haplotypes generated lower activity scores than current approaches due to inclusion of additional damaging polymorphisms in the star allele. These findings are critical for clinical implementation of metabolizer phenotype prediction because a fraction of the population may be incorrectly considered normal metabolizers but actually may be poor or intermediate metabolizers.

Selection of highly informative SNP markers for population affiliation of major US populations.

Ancestry informative markers (AIMs) can be used to detect and adjust for population stratification and predict the ancestry of the source of an evidence sample. Autosomal single nucleotide polymorphisms (SNPs) are the best candidates for AIMs. It is essential to identify the most informative AIM SNPs across relevant populations. Several informativeness measures for ancestry estimation have been used for AIMs selection: absolute allele frequency differences (δ), F statistics (F ST), and informativeness for assignment measure (In). However, their efficacy has not been compared objectively, particularly for determining affiliations of major US populations. In this study, these three measures were directly compared for AIMs selection among four major US populations, i.e., African American, Caucasian, East Asian, and Hispanic American. The results showed that the F ST panel performed slightly better for population resolution based on principal component analysis (PCA) clustering than did the δ panel and both performed better than the In panel. Therefore, the 23 AIMs selected by the F ST measure were used to characterize the four major American populations. Genotype data of nine sample populations were used to evaluate the efficiency of the 23-AIMs panel. The results indicated that individuals could be correctly assigned to the major population categories. Our AIMs panel could contribute to the candidate pool of AIMs for potential forensic identification purposes.

STR data for 15 autosomal STR markers from Paraná (Southern Brazil).

Allelic frequencies for 15 STR autosomal loci, using AmpFℓSTR® Identifiler™, forensic, and statistical parameters were calculated. All loci reached the Hardy-Weinberg equilibrium. The combined power of discrimination and mean power of exclusion were 0.999999999999999999 and 0.9999993, respectively. The MDS plot and NJ tree analysis, generated by FST matrix, corroborated the notion of the origins of the Paraná population as mainly European-derived. The combination of these 15 STR loci represents a powerful strategy for individual identification and parentage analyses for the Paraná population.

Evaluation of Bcl-2, Bcl-x and cleaved caspase-3 in malignant peripheral nerve sheath tumors and neurofibromas.

AIMS: To study the expression of Bcl-2, Bcl-x, as well the presence of cleaved caspase-3 in neurofibromas and malignant peripheral nerve sheath tumors. The expression of Bcl-2 and Bcl-x and the presence of cleaved caspase 3 were compared to clinicopathological features of malignant peripheral nerve sheath tumors and their impact on survival rates were also investigated. MATERIALS AND METHODS: The evaluation of Bcl-2, Bcl-x and cleaved caspase-3 was performed by immunohistochemistry using tissue microarrays in 28 malignant peripheral nerve sheath tumors and 38 neurofibromas. Immunoquantification was performed by computerized digital image analysis. CONCLUSIONS: Apoptosis is altered in neurofibromas and mainly in malignant peripheral nerve sheath tumors. High levels of cleaved caspase-3 are more common in tumors with more aggressive histological features and it is associated with lower disease free survival of patients with malignant peripheral nerve sheath tumors.

Polymorphisms upstream of the melanocortin-1 receptor coding region are associated with human pigmentation variation in a Brazilian population.

OBJECTIVE: We describe an association of two SNPs, rs3212345:C>T and rs3212346:G>A, located approximately 2.5 kb upstream of the melanocortin-1 receptor (MC1R) translation initiation codon, with pigmentation phenotype variation in a Southeast Brazilian miscegenated population. METHODS: One hundred thirty-eight genetically unrelated subjects, with multicolor phenotype, were selected from the southeast region of Brazil. Skin, hair and eye color, and tanning ability were rated. Genotypes for each SNP (rs3212345:C>T and rs3212346:G>A) were determined. A logistic regression analysis was performed with the additive model to determine which of the polymorphisms contributed to a specific phenotype. RESULTS: We found that the rs3212345:C>T is associated with light skin, red hair, and poor tanning ability, while the rs3212346:G>A is associated with dark skin, black hair, and strong tanning ability. The presence of rs3212345-C and rs3212346-A alleles in human, chimpanzee, gorilla, orangutan, and marmoset genomes suggests that they are the ancestral alleles. CONCLUSION: These data suggest that the rs3212345-T and rs3212346-G alleles may have contributed to lighter pigmentation phenotypes in modern humans. Genotyping for these SNPs may prove useful to the fields of molecular anthropology and forensic genetics.

Genetic diversity of the melanocortin-1 receptor in an admixed population of Rio de Janeiro: Structural and functional impacts of Cys35Tyr variant.

The melanocortin-1 receptor (MC1R) is one of the key proteins involved in the regulation of melanin production and several polymorphisms have been associated with different phenotypes of skin and hair color in human and nonhuman species. Most of the knowledge is centered on more homogeneous populations and studies involving an admixed group of people should be encouraged due to the great importance of understanding the human color variation. This work evaluates the MC1R diversity and the possible impacts of MC1R variants in an admixed sample population of Rio de Janeiro, Brazil, which is a product of Native American, African, and European miscegenation. Sequencing of complete coding region and part of the 3´UTR of MC1R gene identified 31 variants including one insertion and three novel synonymous substitutions in sample population grouped according to skin, hair and eye pigmentation levels. In nonmetric multidimensional scaling analysis (NMDS), three main clusters were identified, in which the Brazilian dark skin group remained in the African cluster whereas the intermediate and the light skin color phenotype in the European one. None gathered with Asians since their immigration to Brazil was a recent event. In silico analyses demonstrated that Cys35Tyr, Ile155Thr and Pro256Ser, found in our population, have a negative effect on receptor function probably due to changes on the receptor structure. Notably, Cys35Tyr mutation could potentially impair agonist binding. Altogether, this work contributes to the understanding of the genetic background of color variation on an admixed population and gives insights into the damaging effects of MC1R variants.

Evaluation of 16S rRNA Hypervariable Regions for Bioweapon Species Detection by Massively Parallel Sequencing.

Molecular detection and classification of the bacterial groups in a sample are relevant in several areas, including medical research and forensics. Sanger sequencing of the 16S rRNA gene is considered the gold standard for microbial phylogenetic analysis. However, the development of massively parallel sequencing (MPS) offers enhanced sensitivity and specificity for microbiological analyses. In addition, 16S rRNA target amplification followed by MPS facilitates the combined use of multiple markers/regions, better discrimination of sample background, and higher sample throughput. We designed a novel set of 16S rRNA gene primers for detection of bacterial species associated with clinical, bioweapon, and biohazards microorganisms via alignment of 364 sequences representing 19 bacterial species and strains relevant to medical and forensics applications. In silico results indicated that the hypervariable regions (V1V2), (V4V5), and (V6V7V8) support the resolution of a selected group of bacteria. Interspecies and intraspecies comparisons showed 74.23%-85.51% and 94.48%-99.98% sequencing variation among species and strains, respectively. Sequence reads from a simulated scenario of bacterial species mapped to each of the three hypervariable regions of the respective species with different affinities. The minimum limit of detection was achieved using two different MPS platforms. This protocol can be used to detect or monitor as low as 2,000 genome equivalents of bacterial species associated with clinical, bioweapon, and biohazard microorganisms and potentially can distinguish natural outbreaks of pathogenic microorganisms from those occurring by intentional release.

Planktonic microbial profiling in water samples from a Brazilian Amazonian reservoir.

Our comprehension of the dynamics and diversity of freshwater planktonic bacterial communities is far from complete concerning the Brazilian Amazonian region. Therefore, reference studies are urgently needed. We mapped bacterial communities present in the planktonic communities of a freshwater artificial reservoir located in the western Amazonian basin. Two samples were obtained from rainy and dry seasons, the periods during which water quality and plankton diversity undergo the most significant changes. Hypervariable 16S rRNA and shotgun sequencing were performed to describe the first reference of a microbial community in an Amazonian lentic system. Microbial composition consisted mainly of Betaproteobacteria, Cyanobacteria, Alphaproteobacteria, and Actinobacteria in the dry period. The bacteria distribution in the rainy period was notably absent of Cyanobacteria. Microcystis was observed in the dry period in which the gene cluster for cyanotoxins was found. Iron acquisition gene group was higher in the sample from the rainy season. This work mapped the first inventory of the planktonic microbial community of a large water reservoir in the Amazon, providing a reference for future functional studies and determining other communities and how they interact.

DGGE analysis as a tool to identify point mutations, de novo mutations and carriers of the dystrophin gene.

Approximately 30% of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene and it is difficult to identify single nucleotide variations in genomic DNA using current diagnostic techniques. This represents a great obstacle in genetic analysis of these patients and genetic counselling of their families. In this work we performed denaturing gradient gel electrophoresis analysis to search for Duchenne muscular dystrophy mutations. We screened the whole dystrophin gene in 20 Brazilian Duchenne muscular dystrophy patients without a detectable deletion or duplication, and their mothers. The disease causing mutations, all of which have not been described before, were identified, and we could determine the carrier status of the mothers in all analyzed families. We concluded that denaturing gradient gel electrophoresis is very efficient in identifying small mutations and de novo mutations and in determining the carrier status of the mothers in these 30% of Duchenne muscular dystrophy patients. Denaturing gradient gel electrophoresis showed a high mutation detection rate (100%) for Duchenne muscular dystrophy and can be used as a current diagnostic procedure.

Genetic diversity and admixture data on 11 STRs (F13B, TPOX, CSF1PO, F13A01, D7S820, LPL, TH01, vWA, D13S317, FESFPS, and D16S539) in a sample of Rio de Janeiro European-descendants population, Brazil.

Allelic frequencies, forensic parameters and admixture values for eleven STR loci (F13B, TPOX, CSF1PO, F13A01, D7S820, LPL, TH01, vWA, D13S317, FESFPS, and D16S539) were determined in a sample of unrelated individuals, European descendants from Rio de Janeiro area, Brazil.

Y-chromosome variation in Rio de Janeiro, Brazil, population sample.

Allele and haplotype frequencies of seven Y-chromosome STR loci, DYS 19, DYS388, DYS389I, DYS389II, DYS390, DYS391 and DYS392, were determined from 109 unrelated males in Rio de Janeiro, Brazil, sample population.

Genetic composition of six miniSTR in a Brazilian Mulatto sample population.

The present study characterizes the genetic variability of Mulatto population based on the polymorphism of six miniSTR autosomal loci, known as Non Codis 01 and 02 (NC01 and NC02) and evaluate their applicability in forensic genetics. A sample of 102 unrelated Brazilian mulattoes were genotyped for miniSTR loci D1S1677, D2S441, D4S2364 (miniplex NC02) and 45 individuals for D10S1248, D14S1434, D22S1045 (miniplex NC01). No significant deviations from Hardy-Weinberg equilibrium expectations were detected. The combined power of discrimination (PD) and mean power of exclusion (PE) were 0.999996 and 0.98991, respectively. The results also support the effectiveness of the NC01and NC02 miniplexes for human identification.

Color and genomic ancestry in Brazilians: a study with forensic microsatellites.

The population of Brazil, formed by extensive admixture between Amerindians, Europeans and Africans, is one of the most variable in the world. We have recently published a study that used ancestry-informative markers to conclude that in Brazil, at an individual level, color, as determined by physical evaluation, was a poor predictor of genomic ancestry, estimated by molecular markers. To corroborate these findings we undertook the present investigation based on data from 12 commercially available forensic microsatellites that were utilized to estimate the personal genomic origin for each of 752 individuals from the city of São Paulo, belonging to different Brazilian color categories (275 Whites, 192 Intermediates and 285 Blacks). The genotypes permitted the calculation of a personal likelihood-ratio estimator of African or European ancestry. Although the 12 marker set proved capable of discriminating between European and African individuals, we observed very significant overlaps among the three color categories of Brazilians. This was confirmed quantitatively using a Bayesian analysis of population structure that did not demonstrate significant genetic differentiation between the three color groups. These results corroborate and validate our previous conclusions using ancestry-informative markers that in Brazil at the individual level there is significant dissociation of color and genomic ancestry.

Comparison of Rio de Janeiro DNA typing data with the FBI worldwide study

We analyzed 128 chromosomes for the D4S139 (pH30) locus and determined the allelic frequency of our sampling using the 31 fixed-bin key table. Using the frequency distribution of this locus in the different population database available, we compared our sample with eight distinct populations through scatter plot. Our result corroborates the notion that, in forensic science, there is no significant difference among polulations. We also typed 12 different individuals for four loci (D4S139, D1S7, D10S28, D2S44) and 3 profiles of three loci (D4S139, D1S7, D10S28). Individual genotype frequency was determined for each population. Whatever reference population database used, the result showed that each person has a very genotype frequency, ranging from 10E-11 to 10E-13, for four loci typing, and from 10E-07 to 10-E10, for three loci typing. As stated before, these results indicate that the statistical differences found among populations do not interfere in forensic science application and therefore, the use of a general population database will not produce a biased result.

Use of PCR for the determination of the frequency of the F508 mutation in Brasilian cistic fibrosis patients

The F508 mutation in the cystic fibrosis (CF) gene was studied in a population of 18 Brazilian CF patients and their 17 families by use of PCR and differential hybridization with oligonucleotides. In a total of 34 chromosomes considered, 12 (35%) carried the F508 deletion, a frequency much lower than that reported in most other populations. As a consequence, CF in Brazil would be predominantly caused by mutations different from the F508 deletion