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1.
J Anim Breed Genet ; 130(4): 294-302, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23855631

RESUMO

We present here the first genome-wide characterization of linkage disequilibrium (LD) in the French Blonde d'Aquitaine (BLA) breed, a well-muscled breed renowned for producing high-yielding beef carcasses. To assess the pattern and extent of LD, we used a sample of 30 unrelated bulls and 36 923 single nucleotide polymorphisms (SNPs) covering all cattle autosomes. The squared correlation of the alleles at two loci (r(2) ) was used as a measure of LD. The analysis of adjacent marker pairs revealed that the level of LD decreases rapidly with physical distance between SNPs. Overall mean r(2) was 0.205 (±0.262). Strong LD (r(2)  > 0.8) and useful LD (measured as r(2 ) > 0.2) were observed within genomic regions of up to 720 and 724 kb, respectively. We analysed the genetic structure of the BLA population and found stratification. The observed genetic sub-structuring is consistent with the known recent demographic history that occurred during BLA breed formation. Our results indicate that LD mapping of phenotypic traits in the BLA population is feasible; however, because of this sub-structuring, special care is needed to reduce the likelihood of false-positive associations between marker loci and traits of interest.


Assuntos
Genômica , Desequilíbrio de Ligação , Animais , Cruzamento , Bovinos , Frequência do Gene , Polimorfismo de Nucleotídeo Único
2.
Anim Genet ; 43(4): 419-28, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22497629

RESUMO

Many studies in human genetics compare informativeness of single-nucleotide polymorphisms (SNPs) and microsatellites (single sequence repeats; SSR) in genome scans, but it is difficult to transfer the results directly to livestock because of different population structures. The aim of this study was to determine the number of SNPs needed to obtain the same differentiation power as with a given standard set of microsatellites. Eight chicken breeds were genotyped for 29 SSRs and 9216 SNPs. After filtering, only 2931 SNPs remained. The differentiation power was evaluated using two methods: partitioning of the Euclidean distance matrix based on a principal component analysis (PCA) and a Bayesian model-based clustering approach. Generally, with PCA-based partitioning, 70 SNPs provide a comparable resolution to 29 SSRs. In model-based clustering, the similarity coefficient showed significantly higher values between repeated runs for SNPs compared to SSRs. For the membership coefficients, reflecting the proportion to which a fraction segment of the genome belongs to the ith cluster, the highest values were obtained for 29 SSRs and 100 SNPs respectively. With a low number of loci (29 SSRs or ≤100 SNPs), neither marker types could detect the admixture in the Gödöllö Nhx population. Using more than 250 SNPs allowed a more detailed insight into the genetic architecture. Thus, the admixed population could be detected. It is concluded that breed differentiation studies will substantially gain power even with moderate numbers of SNPs.


Assuntos
Galinhas/genética , Cromossomos/genética , Genética Populacional , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Animais , Cruzamento , Análise por Conglomerados , Loci Gênicos , Marcadores Genéticos , Genótipo , Dinâmica Populacional , Análise de Componente Principal
3.
N Engl J Med ; 359(8): 789-99, 2008 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-18650507

RESUMO

BACKGROUND: Lowering low-density lipoprotein cholesterol with statin therapy results in substantial reductions in cardiovascular events, and larger reductions in cholesterol may produce larger benefits. In rare cases, myopathy occurs in association with statin therapy, especially when the statins are administered at higher doses and with certain other medications. METHODS: We carried out a genomewide association study using approximately 300,000 markers (and additional fine-mapping) in 85 subjects with definite or incipient myopathy and 90 controls, all of whom were taking 80 mg of simvastatin daily as part of a trial involving 12,000 participants. Replication was tested in a trial of 40 mg of simvastatin daily involving 20,000 participants. RESULTS: The genomewide scan yielded a single strong association of myopathy with the rs4363657 single-nucleotide polymorphism (SNP) located within SLCO1B1 on chromosome 12 (P=4x10(-9)). SLCO1B1 encodes the organic anion-transporting polypeptide OATP1B1, which has been shown to regulate the hepatic uptake of statins. The noncoding rs4363657 SNP was in nearly complete linkage disequilibrium with the nonsynonymous rs4149056 SNP (r(2)=0.97), which has been linked to statin metabolism. The prevalence of the rs4149056 C allele in the population was 15%. The odds ratio for myopathy was 4.5 (95% confidence interval [CI], 2.6 to 7.7) per copy of the C allele, and 16.9 (95% CI, 4.7 to 61.1) in CC as compared with TT homozygotes. More than 60% of these myopathy cases could be attributed to the C variant. The association of rs4149056 with myopathy was replicated in the trial of 40 mg of simvastatin daily, which also showed an association between rs4149056 and the cholesterol-lowering effects of simvastatin. No SNPs in any other region were clearly associated with myopathy. CONCLUSIONS: We have identified common variants in SLCO1B1 that are strongly associated with an increased risk of statin-induced myopathy. Genotyping these variants may help to achieve the benefits of statin therapy more safely and effectively. (Current Controlled Trials number, ISRCTN74348595.)


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Doenças Musculares/induzido quimicamente , Transportadores de Ânions Orgânicos/genética , Polimorfismo de Nucleotídeo Único , Sinvastatina/efeitos adversos , Idoso , Arteriopatias Oclusivas/tratamento farmacológico , Cromossomos Humanos Par 12 , Diabetes Mellitus/tratamento farmacológico , Feminino , Marcadores Genéticos , Genótipo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Pessoa de Meia-Idade , Doenças Musculares/genética , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/prevenção & controle , Risco , Sinvastatina/uso terapêutico
4.
Tissue Antigens ; 77(2): 100-6, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21214521

RESUMO

Polymorphisms in the central major histocompatibility complex (MHC) (particularly TNF and adjacent genes) associate with several immunopathological diseases and with susceptibility to pneumonia. The MHC is characterised by strong linkage disequilibrium (LD), so identification of loci affecting disease must be based on haplotypes. We have defined 31 tumour necrosis factor (TNF) block haplotypes (denoted FV1-31) in Caucasians, Asians and Australian Aboriginals. This study correlates the carriage of TNF block haplotypes with TNF and lymphotoxin alpha (LTA) protein production by peripheral blood mononuclear cells from 205 healthy Caucasian subjects, following in vitro stimulation with Streptococcus pneumoniae (S. pneumoniae; gram-positive bacteria), Escherichia coli (E. coli; gram-negative bacteria) or TNF over 4, 8 and 24 h. Fifteen haplotypes were present at >1%, accounting for 94.5% of the cohort. The haplotypes were grouped into five families based on common alleles. Following stimulation, cells from carriers of the FV10 haplotype (family 2) produced less LTA compared with non-FV10 carriers. Carriers of the FV18 haplotype (family 4) produced more LTA than other donors. Induction of TNF by S. pneumoniae following 24 h stimulation was also greater in donors with FV18. The FV18 haplotype associated with the 44.1 MHC ancestral haplotype (HLA-A2, -C5, -B44, -DRB1*0401 and -DQB1*0301) that has few disease associations. FV16 occurred in the 8.1 MHC haplotype (HLA-A2, B8, DR3) that is associated with multiple immunopathological diseases. FV16 did not affect TNF or LTA levels. The findings suggest that many genetic variations critical in vivo are not effectively modelled by short-term cultures.


Assuntos
Predisposição Genética para Doença , Haplótipos/genética , Linfotoxina-alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , População Branca/genética , Adulto , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Genótipo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Desequilíbrio de Ligação , Linfotoxina-alfa/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética
5.
Tissue Antigens ; 77(2): 126-30, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20887379

RESUMO

In human immunodeficiency virus (HIV) patients, neuropathy is a common adverse side effect to some antiretroviral treatments, particularly stavudine. As stavudine is cheap, it is widely used in Asia and Africa. We showed that increasing age and height moderately predict the development of neuropathy. This was improved by the inclusion of tumour necrosis factor (TNF)-1031 (rs1799964). To investigate this association, Malay (n = 64), Chinese (n = 74) and Caucasian patients (n = 37) exposed to stavudine were screened for neuropathy. DNA samples were genotyped for polymorphisms in the central major histocompatibility complex (MHC) near TNF, and haplotypes were derived. The haplotype group FVa6,7,8 (incorporating TNF-1031) was found to be associated with neuropathy in Chinese patients in bivariate analyses (P = 0.03), and in Malays and Chinese in a multivariate analysis correcting for age and height (P = 0.02, P = 0.03, respectively). This trend was also confirmed in Caucasians.


Assuntos
Fármacos Anti-HIV/efeitos adversos , Haplótipos/genética , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/genética , Polimorfismo Genético/genética , Estavudina/efeitos adversos , Fatores de Necrose Tumoral/genética , Antropometria , Povo Asiático/genética , Estatura , Genótipo , HIV/patogenicidade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Reação em Cadeia da Polimerase , Medição de Risco , Fatores de Risco , População Branca/genética
6.
Sci Rep ; 11(1): 17109, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429448

RESUMO

The evolution of large vultures linked to mountainous habitats was accompanied by extreme physiological and behavioral specializations for energetically efficient flights. However, little is known on the genetic traits associated with the evolution of these obligate soaring scavengers. Mitochondrial DNA plays a vital role in regulating oxidative stress and energy production, and hence may be an important target of selection for flight performance. Herein, we characterized the first mitogenomes of the Andean and California condors, the world's heaviest flying birds and the only living representatives of the Vultur and Gymnogyps genus. We reconstructed the phylogenetic relationships and evaluated possible footprints of convergent evolution associated to the life-history traits and distributional range of vultures. Our phylogenomic analyses supported the independent evolution of vultures, with the origin of Cathartidae in the early Paleogene (~ 61 Mya), and estimated the radiation of extant condors during the late Miocene (~ 11 Mya). Selection analyses indicated that vultures exhibit signals of relaxation of purifying selection relative to other accipitrimorph raptors, possibly indicating the degeneration of flapping flight ability. Overall, our results suggest that the extreme specialization of vultures for efficient soaring flight has compensated the evolution of large body sizes mitigating the selection pressure on mtDNA.


Assuntos
Aves/genética , Evolução Molecular , Genoma Mitocondrial , Animais , Aves/classificação , Espécies em Perigo de Extinção , Filogenia , Seleção Genética
7.
Genome Med ; 12(1): 18, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075696

RESUMO

The European Union (EU) initiative on the Digital Transformation of Health and Care (Digicare) aims to provide the conditions necessary for building a secure, flexible, and decentralized digital health infrastructure. Creating a European Health Research and Innovation Cloud (HRIC) within this environment should enable data sharing and analysis for health research across the EU, in compliance with data protection legislation while preserving the full trust of the participants. Such a HRIC should learn from and build on existing data infrastructures, integrate best practices, and focus on the concrete needs of the community in terms of technologies, governance, management, regulation, and ethics requirements. Here, we describe the vision and expected benefits of digital data sharing in health research activities and present a roadmap that fosters the opportunities while answering the challenges of implementing a HRIC. For this, we put forward five specific recommendations and action points to ensure that a European HRIC: i) is built on established standards and guidelines, providing cloud technologies through an open and decentralized infrastructure; ii) is developed and certified to the highest standards of interoperability and data security that can be trusted by all stakeholders; iii) is supported by a robust ethical and legal framework that is compliant with the EU General Data Protection Regulation (GDPR); iv) establishes a proper environment for the training of new generations of data and medical scientists; and v) stimulates research and innovation in transnational collaborations through public and private initiatives and partnerships funded by the EU through Horizon 2020 and Horizon Europe.


Assuntos
Pesquisa Biomédica/organização & administração , Computação em Nuvem , Difusão de Inovações , Guias de Prática Clínica como Assunto , Pesquisa Biomédica/métodos , União Europeia , Disseminação de Informação/legislação & jurisprudência , Disseminação de Informação/métodos
8.
Genes Immun ; 10(7): 607-15, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19536152

RESUMO

The region spanning the tumour necrosis factor (TNF) cluster in the human major histocompatibility complex is implicated in susceptibility to immunopathological disease, but ethnic differences and linkage disequilibrium have hampered identification of critical polymorphisms. Here, we investigate Europeans, Asians (Bidayuh, Chinese, Indian, Jehai, Malay, Temuan) and Australian Aborigines to provide a framework for disease-association studies. DNA from 999 unrelated healthy donors was genotyped at 38 loci, primarily in coding and promoter regions over a 60-kb region spanning seven genes near TNF. The PHASE algorithm was used to statistically infer TNF block haplotypes and estimate their frequencies in each population. The TNF block is carried as 31 haplotypes in all populations combined, with <19 in any single population. Only six haplotypes have a unique tag single nucleotide polymorphism (SNP) valid for all populations, but seven haplotypes could be tagged with individual SNPs in selected populations. Four to eight TNF block haplotypes exist across all ethnicities, and hence must pre-date the divergence of these populations from a common ancestor >160,000 years ago. Some haplotypes are unique to isolated populations, but they do not contain unique SNP. Hence, they reflect restricted migration and/or extinction of some families rather than de novo mutation.


Assuntos
Povo Asiático/genética , Frequência do Gene/genética , Haplótipos/genética , Havaiano Nativo ou Outro Ilhéu do Pacífico/genética , Fatores de Necrose Tumoral/genética , População Branca/genética , Alelos , Cromossomos Humanos Par 6/genética , Evolução Molecular , Variação Genética , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas
9.
Tissue Antigens ; 74(1): 57-61, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19392789

RESUMO

Associations between major histocompatibility complex (MHC) ancestral haplotypes (AHs) and immunopathological diseases are traditionally ascribed to human leukocyte antigen (HLA) class I or class II alleles. However, polymorphisms in TNF and nearby genes in the central MHC can influence risk. We have defined TNF block haplotypes in Asian, European and Australian Aboriginal donors and shown conservation of TNF block haplotypes in geographically distinct populations, consistent with a common evolutionary origin. Here we show that most TNF block haplotypes do not align with a single MHC AH and associations often vary with ethnicity. This suggests more recent recombination events between the TNF block and the HLA alleles.


Assuntos
Frequência do Gene/genética , Complexo Principal de Histocompatibilidade/genética , Fator de Necrose Tumoral alfa/genética , Alelos , Povo Asiático/genética , Sequência Conservada , Genótipo , Haplótipos/genética , Humanos , Havaiano Nativo ou Outro Ilhéu do Pacífico/genética , Polimorfismo de Nucleotídeo Único , População Branca/genética
10.
Arthritis Rheum ; 58(9): 2670-4, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18759306

RESUMO

OBJECTIVE: We recently showed, using a candidate gene approach in a case-control association study, that a 65-kb block encompassing tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 is strongly associated with rheumatoid arthritis (RA). Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure and are not likely to be hampered by variation in population allele frequencies, as is seen for many genetic polymorphisms, including the TRAF1/C5 locus. The aim of this study was to confirm this association in populations of European origin by using a family-based approach. METHODS: A total of 1,356 western European white individuals from 452 "trio" families were genotyped for the rs10818488 polymorphism, using the TaqMan allelic discrimination assay. RESULTS: We observed evidence for association, demonstrating departure from Mendel's law, with an overtransmission of the rs10818488 A allele (A = 55%; P = 0.036). By taking into consideration parental phenotypes, we also observed an increased A allele frequency in affected versus unaffected parents (A = 64%; combined P = 0.015). Individuals carrying the A allele had a 1.2-fold increased risk of developing RA (allelic odds ratio 1.24, 95% confidence interval 1.04-1.50). CONCLUSION: Using a family-based study that is robust against population stratification, we provide evidence for the association of the TRAF1/C5 rs10818488 A allele and RA in populations of European descent, further substantiating our previous findings. Future functional studies should yield insight into the biologic relevance of this locus to the pathways involved in RA.


Assuntos
Artrite Reumatoide/genética , Complemento C5/genética , Polimorfismo Genético/genética , Fator 1 Associado a Receptor de TNF/genética , Alelos , Estudos de Casos e Controles , Família , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , População Branca/genética
11.
Cytogenet Genome Res ; 117(1-4): 22-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17675841

RESUMO

A comprehensive linkage map for chicken chromosome Z was constructed as the result of a large-scale screening of single nucleotide polymorphisms (SNPs). A total of 308 SNPs were assigned to Z based on the genotype distribution among 182 birds representing several populations. A linkage map comprising 210 markers and spanning 200.9 cM was established by analyzing a small Red junglefowl/White Leghorn intercross. There was excellent agreement between the linkage map for Z and a recently released assembly of the chicken genome (May 2006). Almost all SNPs assigned to chromosome Z in the present study are on Z in the new genome assembly. The remaining 12 loci are all found on unassigned contigs that can now be assigned to Z. The average recombination rate was estimated at 2.7 cM/Mb but there was a very uneven distribution of recombination events with both cold and hot spots of recombination. The existence of one of the major hot spots of recombination, located around position 39.4 Mb, was supported by the observed pattern of linkage disequilibrium. Thirteen markers from unassigned contigs were shown to be located on chromosome W. Three of these contigs included genes that have homologues on chromosome Z. The preliminary assignment of three more genes to the gene-poor W chromosome may be important for studies on the mechanism of sex determination and dosage compensation in birds.


Assuntos
Galinhas/genética , Mapeamento Físico do Cromossomo/métodos , Recombinação Genética/genética , Cromossomos Sexuais/genética , Animais , Feminino , Marcadores Genéticos , Genótipo , Masculino , Polimorfismo Genético/genética , Polimorfismo de Nucleotídeo Único/genética
12.
Oncogene ; 36(40): 5648-5657, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28581523

RESUMO

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.


Assuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Neoplasias/genética , Animais , Pareamento de Bases , Elementos Facilitadores Genéticos , Genoma Humano , Humanos , Regiões Promotoras Genéticas
13.
Endocr Relat Cancer ; 13(4): 1223-36, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17158767

RESUMO

Mutations of the MEN1 gene lead to the occurrence of multiple endocrine neoplasia type 1 (MEN1). To gain insights into the mechanisms of the tumorigenesis related to MEN1 inactivation, we have used mice in which the Men1 gene was specifically disrupted in pancreatic beta-cells. In these mice, we observed full penetrance of insulinoma with defined histological characteristics of tumorigenesis. To identify the genetic factors taking part in the tumour development, we performed gene expression profiling analysis of these insulinomas at different stages. Here, we show that in late stage insulinomas, 56 genes are up-regulated and 194 are down-regulated more than fourfold compared with normal pancreatic islets. Clustering analysis reveals the deregulation of Hox gene family and the genes involved in cell proliferation and cell cycle control. The altered expression of Igf2, Igfbp3 and Igfbp6 as well as cyclin A2, B2 and D2 are confirmed by quantitative RT-PCR, with the overexpression of all the three cyclins found in early stage insulinomas. Moreover, an increased proportion of cyclin A2- and D2-expressing cells and the overexpression of insulin-like growth factor 2 (IGF2) protein are detected in mouse Men1 insulinomas by immunostaining. Interestingly, the analysis of DNA methylation patterns by quantitative serial pyrosequencing reveals that four specific CpGs in the intragenic differentially methylated region 2 (DMR2) region of the Igf2 gene known to augment transcription through methylation are significantly hypermethylated in insulinomas of Men1 beta-cell mutant mice at 6 and 10 months of age, even before IGF2 overexpression can be detected. Thus, our data indicate the involvement of both genetic and epigenetic mechanisms in early tumorigenesis of beta-cells related to MEN1 inactivation.


Assuntos
Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasias Pancreáticas/genética , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Células Secretoras de Insulina/patologia , Insulinoma/metabolismo , Insulinoma/patologia , Integrases/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Nucleic Acids Res ; 28(23): E100, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11095696

RESUMO

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of ss-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the ss-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.


Assuntos
Técnicas Genéticas , Polimorfismo de Nucleotídeo Único/genética , Animais , Bovinos , DNA/genética , Genótipo , Humanos , Receptores Adrenérgicos beta 2/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
Nucleic Acids Res ; 28(5): E13, 2000 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-10666474

RESUMO

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.


Assuntos
Precursor de Proteína beta-Amiloide/genética , Técnicas Genéticas , Fator Estimulador de Colônias de Granulócitos/genética , Polimorfismo de Nucleotídeo Único/genética , Códon , Análise Mutacional de DNA , Genótipo , Humanos , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Nat Commun ; 7: 13507, 2016 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-27886173

RESUMO

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression.


Assuntos
Colite Ulcerativa/genética , Doença de Crohn/genética , Metilação de DNA/genética , Predisposição Genética para Doença , Locos de Características Quantitativas/genética , Adulto , Estudos de Casos e Controles , Estudos de Coortes , Colite Ulcerativa/sangue , Doença de Crohn/sangue , Epigênese Genética , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Interação Gene-Ambiente , Genótipo , Humanos , Desequilíbrio de Ligação , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/genética
17.
J Mol Biol ; 255(1): 1-13, 1996 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-8568858

RESUMO

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.


Assuntos
Evolução Molecular , Genes MHC da Classe II/genética , Sequência de Aminoácidos , Animais , Composição de Bases , Sequência de Bases/genética , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Pseudogenes/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Especificidade da Espécie
18.
Hum Mutat ; 17(6): 475-92, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11385706

RESUMO

Automation for genotyping of single nucleotide polymorphisms (SNPs) can be split into the automation of the sample preparation and the automation of the analysis technology. SNP genotyping methods are reviewed and solutions for their automation discussed. A panacea for SNP genotyping does not exist. Different scientific questions require adapted solutions. The choice of a technology for SNP genotyping depends on whether few different SNPs are to be genotyped in many individuals, or many different SNPs are to be genotyped in few individuals. The requirements of throughput and the ease of establishing an SNP genotyping operation are important, as well as the degree of integration. The potential and state-of-the-art of different solutions are outlined.


Assuntos
Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA/métodos , Genótipo , Humanos
19.
Pharmacogenetics ; 10(9): 781-8, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11191882

RESUMO

Polymerase chain reaction-restriction fragment length polymorphism based genotyping assays were used to determine the frequency of polymorphisms in CYP1A1 (3'-flanking region), CYP2E1 (5'-flanking region and intron 6), EPHX (exon 3 and exon 4), GSTM1 (deletion), GSTP1 (exon 5) and GSTT1 (deletion) in a group of 416 Czech individuals. A comprehensive overview of the methodology is also presented. We have found the following frequencies of mutated alleles: CYP1A1-m2, 0.097; CYP2E1-C, 0.077; CYP2E1-c2, 0.023; EPHX(exon 3)-His, 0.381; EPHX(exon 4)-Arg, 0.198; GSTM1-null, 0.51; GSTP1-Val, 0.3; GSTT1-null, 0.164. These values are similar to those presented in the majority of studies on European Caucasians, although a few cases of significant differences in the distribution of genotypes were found. These differences were most probably caused by methodological variations or statistical bias in the analyses of low numbers of samples in the control groups of some authors. Based on the results of EPHX genotyping, the activity of its protein product was deduced and the Czech population was divided into three subgroups with low, medium and high EPHX activity. We found that 43% of the Czech population would fall into the low, 44% into the medium and 13% into the high EPHX activity group. The data obtained may prove to be very useful for epidemiological studies on the influence of genetic polymorphisms of biotransformation enzymes on carcinogenesis or other environment-related diseases.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Epóxido Hidrolases/genética , Frequência do Gene , Glutationa Transferase/genética , Polimorfismo Genético , Alelos , Biotransformação/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2E1/genética , República Tcheca , DNA Intergênico , Etnicidade , Éxons , Genótipo , Íntrons
20.
Biochem Pharmacol ; 47(12): 2233-42, 1994 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8031317

RESUMO

Exposure to benzene was reported to lower the cytochrome P450 (CYP; EC 1.14.14.1) content in phenobarbital-pretreated (PB) rats in vivo (Gut I, Zbl Pharm 122: 1139-1161, 1983). In this paper we followed the ability of benzene and its metabolites, phenol, catechol, hydroquinone and benzoquinone to destroy CYP in liver microsomes from PB rats in vitro. The spectrophotometric determinations of the total CYP content, 7-pentoxyresorufin O-depentylase and aniline hydroxylase activities, electrophoresis and western blot analysis after incubation of PB-microsomes with benzene or its metabolites revealed that: (1) benzene is metabolically activated to intermediates causing CYP destruction; phenol is not responsible for this effect. (2) Quinonic metabolites of benzene cause CYP destruction with different potency (30% CYP was destroyed by 3 mM catechol, 0.3 mM hydroquinone and 0.03 mM benzoquinone). (3) Low concentrations of quinones are capable of protecting CYP against reactive oxygen species produced in the CYP futile cycle. (4) Ascorbate effectively protects CYP against quinones, apparently by maintaining them in the reduced state. (5) Quinones attack both heme and protein of CYP. (6) CYP activities differ in the sensitivity to quinone-mediated destruction. In conclusion, we suggest that quinones may be responsible for CYP destruction by benzene in vivo.


Assuntos
Benzeno/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Sequestradores de Radicais Livres , Microssomos Hepáticos/efeitos dos fármacos , Quinonas/metabolismo , Animais , Benzeno/metabolismo , Citocromo P-450 CYP2B1 , Sistema Enzimático do Citocromo P-450/química , Masculino , Microssomos Hepáticos/enzimologia , NADP/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Fenobarbital , Ratos , Ratos Wistar , Fatores de Tempo
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