Massively parallel sequencing, also referred to as next-generation sequencing, has positively changed DNA analysis, allowing further advances in genetics. Its capability of dealing with low ...quantity/damaged samples makes it an interesting instrument for forensics. The main advantage of MPS is the possibility of analyzing simultaneously thousands of genetic markers, generating high-resolution data. Its detailed sequence information allowed the discovery of variations in core forensic short tandem repeat loci, as well as the identification of previous unknown polymorphisms. Furthermore, different types of markers can be sequenced in a single run, enabling the emergence of DIP-STRs, SNP-STR haplotypes, and microhaplotypes, which can be very useful in mixture deconvolution cases. In addition, the multiplex analysis of different single nucleotide polymorphisms can provide valuable information about identity, biogeographic ancestry, paternity, or phenotype. DNA methylation patterns, mitochondrial DNA, mRNA, and microRNA profiling can also be analyzed for different purposes, such as age inference, maternal lineage analysis, body-fluid identification, and monozygotic twin discrimination. MPS technology also empowers the study of metagenomics, which analyzes genetic material from a microbial community to obtain information about individual identification, post-mortem interval estimation, geolocation inference, and substrate analysis. This review aims to discuss the main applications of MPS in forensic genetics.
Gene doping: Present and future Cantelmo, Rebeca Araujo; da Silva, Alessandra Pereira; Mendes-Junior, Celso Teixeira ...
European journal of sport science,
September 2020, Letnik:
20, Številka:
8
Journal Article
Recenzirano
Being an elite athlete is an extremely coveted position, which can lead an individual to use doping. As knowledge is extended, doping techniques have become increasingly sophisticated, and the newest ...method of doping is gene doping. This article aims to present an updated bibliographic survey that addresses gene doping between 1983 and 2018. Anti-doping agencies have not yet approved any detection technique for this type of doping. The possibility of eradicating such doping is almost zero mainly because gene therapy advances rapidly. In this scenario, the future of gene doping must be discussed and decided before irreversible limits are exceeded.
HLA‐B is among the most variable gene in the human genome. This gene encodes a key molecule for antigen presentation to CD8+ T lymphocytes and NK cell modulation. Despite the myriad of studies ...evaluating its coding region (with an emphasis on exons 2 and 3), few studies evaluated introns and regulatory sequences in real population samples. Thus, HLA‐B variability is probably underestimated. We applied a bioinformatics pipeline tailored for HLA genes on 5347 samples from 80 different populations, which includes more than 1000 admixed Brazilians, to evaluate the HLA‐B variability (SNPs, indels, MNPs, alleles, and haplotypes) in exons, introns, and regulatory regions. We observed 610 variable sites throughout HLA‐B; the most frequent variants are shared worldwide. However, the haplotype distribution is geographically structured. We detected 920 full‐length haplotypes (exons, introns, and untranslated regions) encoding 239 different protein sequences. HLA‐B gene diversity is higher in admixed populations and Europeans while lower in African ancestry individuals. Each HLA‐B allele group is associated with specific promoter sequences. This HLA‐B variation resource may improve HLA imputation accuracy and disease‐association studies and provide evolutionary insights regarding HLA‐B genetic diversity in human populations.
Achieving accurate STR genotyping by using next-generation sequencing data has been challenging. To provide the forensic genetics community with a reliable open-access STR database, we conducted a ...comprehensive genotyping analysis of a set of STRs of broad forensic interest obtained from 1000 Genome populations. We analyzed 22 STR markers using files of the high-coverage dataset of Phase 3 of the 1000 Genomes Project. We used HipSTR to call genotypes from 2504 samples obtained from 26 populations. We were not able to detect the D21S11 marker. The Hardy-Weinberg equilibrium analysis coupled with a comprehensive analysis of allele frequencies revealed that HipSTR was not able to identify longer alleles, which resulted in heterozygote deficiency. Nevertheless, AMOVA, a clustering analysis that uses STRUCTURE, and a Principal Coordinates Analysis showed a clear-cut separation between the four major ancestries sampled by the 1000 Genomes Consortium. Except for larger Penta D and Penta E alleles, and two very small Penta D alleles (2.2 and 3.2) usually observed in African populations, our analyses revealed that allele frequencies and genotypes offered as an open-access database are consistent and reliable.
Cytomegalovirus retinochoroiditis (CMV-R) is the primary cause of blindness among AIDS patients. Since HLA-G is associated with the modulation of the immune response, we hypothesized that variability ...at the 3' untraslated region (3'UTR) of the gene could be implicated on the predisposition to CMV-R. We evaluated whether HLA-G 3'UTR influences CMV-R development in Brazilian AIDS patients. Peripheral blood DNA was obtained from two groups of patients: (1) AIDS exhibiting CMV-R (n = 40) and (2) AIDS without CMV-R (n = 147). HLA-G 3'UTR typing was performed using sequencing analysis. Allele, genotype and haplotype frequencies were evaluated using Fisher's exact test accompanied by the calculation of the odds ratio and its 95% confidence interval (95% CI). The etiologic (EF) and preventive fractions were also estimated. Compared to AIDS patients without CMV-R, AIDS patients with CMV-R showed increased frequencies of the: (1) + 3001T allele, (2) the + 3001C/T genotype and (3) the UTR-17 (InsTTCCGTGACG) haplotype (EFs = 0.02-0.04). The UTR-3 (DelTCCGCGACG) haplotype was associated with protection against CMV-R development. Although the risk for developing CMR-V at the population level was relatively low (EF), the identification of HLA-G 3'UTR variation sites may help to further evaluate the role of post-transcriptional factors that may contribute to the existent immunosuppresion caused by HIV per se.
Leukocyte immunoglobulin (Ig)‐like receptors (LILR) LILRB1 and LILRB2 may play a pivotal role in maintaining self‐tolerance and modulating the immune response through interaction with classical and ...nonclassical HLA molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LILRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next‐generation sequencing in a population sample from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes, and 41 LILRB2 SNVs arranged into 11 haplotypes. Although we may not exclude as a possible effect of population structure, we found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have revealed that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Overall, our findings demonstrate that LILRB1 and LILRB2 are as polymorphic as HLA class Ib genes and provide strong evidence supporting the directional selection regime hypothesis.
HLA-G is a promiscuous immune checkpoint molecule. The HLA-G gene presents substantial nucleotide variability in its regulatory regions. However, it encodes a limited number of proteins compared to ...classical HLA class I genes. We characterized the HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe by using a state-of-the-art method to characterize polymorphisms and haplotypes from high-coverage next-generation sequencing data. We also provide insights regarding the HLA-G genetic diversity and a resource for future studies evaluating HLA-G polymorphisms in different populations and association studies. Despite the great haplotype variability, we demonstrated that: (1) most of the HLA-G polymorphisms are in introns and regulatory sequences, and these are the sites with evidence of balancing selection, (2) linkage disequilibrium is high throughout the gene, extending up to HLA-A, (3) there are few proteins frequently observed in worldwide populations, with lack of variation in residues associated with major HLA-G biological properties (dimer formation, interaction with leukocyte receptors). These observations corroborate the role of HLA-G as an immune checkpoint molecule rather than as an antigen-presenting molecule. Understanding HLA-G variability across populations is relevant for disease association and functional studies.
Vitiligo is the most frequent cause of depigmentation worldwide. Genetic association studies have discovered about 50 loci associated with disease, many with immunological functions. Among them is ...HLA-G, which modulates immunity by interacting with specific inhibitory receptors, mainly LILRB1 and LILRB2. Here we investigated the
and
association with vitiligo risk and evaluated the possible role of interactions between HLA-G and its receptors in this pathogenesis. We tested the association of the polymorphisms of
,
, and
with vitiligo using logistic regression along with adjustment by ancestry. Further, methods based on the multifactor dimensionality reduction (MDR) approach (MDR v.3.0.2, GMDR v.0.9, and MB-MDR) were used to detect potential epistatic interactions between polymorphisms from the three genes. An interaction involving rs9380142 and rs2114511 polymorphisms was identified by all methods used. The polymorphism rs9380142 is an
3'UTR variant (+3187) with a well-established role in mRNA stability. The polymorphism rs2114511 is located in the exonic region of
. Although no association involving this SNP has been reported, ChIP-Seq experiments have identified this position as an EBF1 binding site. These results highlight the role of an epistatic interaction between
and
in vitiligo pathogenesis.
The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new ...approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.
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