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1.
Hum Immunol ; 76(12): 910-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26037172

RESUMO

Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.


Assuntos
Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade , Alelos , Linhagem Celular , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Software , Fluxo de Trabalho
2.
Tissue Antigens ; 83(1): 32-40, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24355006

RESUMO

The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence.


Assuntos
Artefatos , DNA/análise , Antígenos HLA-DR/genética , Teste de Histocompatibilidade , Reação em Cadeia da Polimerase/métodos , Alelos , Troca Genética/genética , Primers do DNA , Bases de Dados de Ácidos Nucleicos , Erros de Diagnóstico/prevenção & controle , Éxons , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase/tendências , Análise de Sequência de DNA
3.
Tissue Antigens ; 81(3): 141-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23398507

RESUMO

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.


Assuntos
Biblioteca Gênica , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Microfluídica/métodos , Análise de Sequência de DNA/métodos , Linhagem Celular , Primers do DNA/metabolismo , Humanos , Reação em Cadeia da Polimerase , Software
4.
Int J Immunogenet ; 40(1): 72-6, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23302098

RESUMO

Human leucocyte antigen (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA-based genotyping methods, including Sanger sequence-based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high-resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next-generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to (i) template preparation including short- and long-range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele-calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.


Assuntos
DNA/genética , Antígenos HLA , Sequenciamento de Nucleotídeos em Larga Escala , Transplante de Órgãos , Alelos , Genótipo , Antígenos HLA/classificação , Antígenos HLA/genética , Antígenos HLA/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade , Humanos , Polimorfismo Genético , Análise de Sequência de DNA , Software
5.
Diabetologia ; 55(9): 2394-401, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22706720

RESUMO

AIMS/HYPOTHESIS: The study aimed to assess, in multiple populations, the role of HLA alleles on early and late age at onset of type 1 diabetes. METHODS: Stepwise linear regression models were used to determine which HLA class I and class II risk alleles to include. High-resolution genotyping data for patients from the Type 1 Diabetes Genetics Consortium (T1DGC) collection (n = 2,278) and four independent cohorts from Denmark, Sardinia and the USA (Human Biological Data Interchange [HBDI] and Joslin Diabetes Center) (n = 1,324) (total n = 3,602) were used to assess the role of HLA variation on age of onset and predict early onset (age ≤ 5 years) and late onset (age ≥ 15 years) of type 1 diabetes. RESULTS: In addition to carriage of HLA class I alleles A*24:02, B*39:06, B*44:03 and B*18:01, HLA class II DRB1-DQB1 loci significantly contributed to age at onset, explaining 3.4% of its variance in the combined data. HLA genotypes, together with sex, were able to predict late onset in all cohorts studied, with AUC values ranging from 0.58 to 0.63. Similar AUC values (0.59-0.70) were obtained for early onset for most cohorts, except in the Sardinian study, in which none of the models tested had significant predictive power. CONCLUSIONS/INTERPRETATION: HLA associations with age of onset are consistent across most white populations and HLA information can predict some of the risk of early and late onset of type 1 diabetes. Considerable heterogeneity was observed between Sardinian and other populations, particularly with regard to early age of onset.


Assuntos
Diabetes Mellitus Tipo 1/epidemiologia , Nefropatias Diabéticas/epidemiologia , Retinopatia Diabética/epidemiologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Falência Renal Crônica/epidemiologia , Idade de Início , Criança , Estudos de Coortes , Dinamarca/epidemiologia , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/genética , Retinopatia Diabética/genética , Feminino , Predisposição Genética para Doença , Variação Genética , Genótipo , Humanos , Itália/epidemiologia , Falência Renal Crônica/genética , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estados Unidos/epidemiologia
6.
Tissue Antigens ; 79(4): 279-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22296171

RESUMO

Follicular lymphoma (FL) is an indolent, sometimes, fatal disease characterized by recurrence at progressively shorter intervals and is frequently refractive to therapy. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region on chromosome 6p21.32-33 that are statistically significantly associated with FL risk. Low to medium resolution typing of single or multiple HLA genes has provided an incomplete picture of the total genetic risk imparted by this highly variable region. To gain further insight into the role of HLA alleles in lymphomagenesis and to investigate the independence of validated SNPs and HLA alleles with FL risk, high-resolution HLA typing was conducted using next-generation sequencing in 222 non-Hispanic White FL cases and 220 matched controls from a larger San Francisco Bay Area population-based case-control study of lymphoma. A novel protective association was found between the DPB1*03:01 allele and FL risk [odds ratio (OR) = 0.39, 95% confidence interval (CI) = 0.21-0.68]. Extended haplotypes DRB1*01:01-DQA1*01:01-DQB1*05:01 (OR = 2.01, 95% CI = 1.22-3.38) and DRB1*15-DQA1*01-DQB1*06 (OR = 0.55, 95% CI = 0.36-0.82) also influenced FL risk. Moreover, DRB1*15-DQA1*01-DQB1*06 was highly correlated with an established FL risk locus, rs2647012. These results provide further insight into the critical roles of HLA alleles and SNPs in FL pathogenesis that involve multi-locus effects across the HLA region.


Assuntos
Alelos , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Linfoma Folicular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade
7.
Diabetologia ; 55(4): 996-1000, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22278338

RESUMO

AIMS/HYPOTHESIS: Over 50 regions of the genome have been associated with type 1 diabetes risk, mainly using large case/control collections. In a recent genome-wide association (GWA) study, 18 novel susceptibility loci were identified and replicated, including replication evidence from 2,319 families. Here, we, the Type 1 Diabetes Genetics Consortium (T1DGC), aimed to exclude the possibility that any of the 18 loci were false-positives due to population stratification by significantly increasing the statistical power of our family study. METHODS: We genotyped the most disease-predicting single-nucleotide polymorphisms at the 18 susceptibility loci in 3,108 families and used existing genotype data for 2,319 families from the original study, providing 7,013 parent-child trios for analysis. We tested for association using the transmission disequilibrium test. RESULTS: Seventeen of the 18 susceptibility loci reached nominal levels of significance (p < 0.05) in the expanded family collection, with 14q24.1 just falling short (p = 0.055). When we allowed for multiple testing, ten of the 17 nominally significant loci reached the required level of significance (p < 2.8 × 10(-3)). All susceptibility loci had consistent direction of effects with the original study. CONCLUSIONS/INTERPRETATION: The results for the novel GWA study-identified loci are genuine and not due to population stratification. The next step, namely correlation of the most disease-associated genotypes with phenotypes, such as RNA and protein expression analyses for the candidate genes within or near each of the susceptibility regions, can now proceed.


Assuntos
Diabetes Mellitus Tipo 1/genética , Grupo com Ancestrais do Continente Europeu/genética , Loci Gênicos , Predisposição Genética para Doença , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único
8.
Genes Immun ; 12(8): 605-14, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21716314

RESUMO

We have conducted a pathway-based analysis of genome-wide single-nucleotide polymorphism (SNP) data in order to identify genetic susceptibility factors for cervical cancer in situ. Genotypes derived from Affymetrix 500k or 5.0 arrays for 1076 cases and 1426 controls were analyzed for association, and pathways with enriched signals were identified using the SNP ratio test. The most strongly associated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were Asthma (empirical P=0.03), Folate biosynthesis (empirical P=0.04) and Graft-versus-host disease (empirical P=0.05). Among the 11 top-ranking pathways were 6 related to the immune response with the common denominator being genes in the major histocompatibility complex (MHC) region on chromosome 6. Further investigation of the MHC revealed a clear effect of HLA-DPB1 polymorphism on disease susceptibility. At a functional level, DPB1 alleles associated with risk and protection differ in key amino-acid residues affecting peptide-binding motifs in the extracellular domains. The results illustrate the value of pathway-based analysis to mine genome-wide data, and point to the importance of the MHC region and specifically the HLA-DPB1 locus for susceptibility to cervical cancer.


Assuntos
Carcinoma in Situ/genética , Predisposição Genética para Doença , Cadeias beta de HLA-DP/genética , Neoplasias do Colo do Útero/genética , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Neoplasia Intraepitelial Cervical/genética , Éxons , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Risco , Transdução de Sinais , Suécia
9.
Tissue Antigens ; 77(3): 206-17, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21299525

RESUMO

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.


Assuntos
Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/tendências , Alelos , Sequência de Bases , Método Duplo-Cego , Características da Família , Genótipo , Antígenos HLA/análise , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Estudos Multicêntricos como Assunto , Análise de Sequência de DNA/métodos , Software
11.
Bone Marrow Transplant ; 45(5): 846-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20348972

RESUMO

The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.


Assuntos
Antígenos HLA , Terminologia como Assunto , Organização Mundial da Saúde , Humanos
12.
Genes Immun ; 10 Suppl 1: S33-41, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19956098

RESUMO

The Type I Diabetes Genetics Consortium (T1DGC) has collected thousands of multiplex and simplex families with type I diabetes (T1D) with the goal of identifying genes involved in T1D susceptibility. These families have all been genotyped for the HLA class I and class II loci and a subset of samples has been typed for an major histocompatibility complex (MHC) single-nucleotide polymorphism (SNP) panel. In addition, the T1DGC has genotyped SNPs in candidate genes to evaluate earlier reported T1D associations. Individual SNPs and SNP haplotypes in IL4R, which encodes the alpha-chain of the IL4 and IL13 receptors, have been associated with T1D in some reports, but not in others. In this study, 38 SNPs in IL4R were genotyped using the Sequenom iPLEX Gold MassARRAY technology in 2042 multiplex families from nine cohorts. Association analyses (transmission-disequilibrium test and parental-disequilibrium test) were performed on individual SNPs and on three-SNP haplotypes. Analyses were also stratified on the high-risk HLA DR3/DR4-DQB1*0302 genotype. A modest T1D association in HBDI families (n=282) was confirmed in this larger collection of HBDI families (n=424). The variant alleles at the non-synonymous SNPs (rs1805011 (E400A), rs1805012 (C431R), and rs1801275 (Q576R)), which are in strong linkage disequilibrium, were negatively associated with T1D risk. These SNPs were more associated with T1D among non-DR3/DR4-DQB1*0302 genotypes than DR3/DR4-DQB1*0302 genotypes. This association was stronger, both in terms of odds ratio and P-values, than the initial report of the smaller collection of HBDI families. However, the IL4R SNPs and the three-SNP haplotype containing the variant alleles were not associated with T1D in the total data. Thus, in the overall families, these results do not show evidence for an association of SNPs in IL4R with T1D.


Assuntos
Diabetes Mellitus Tipo 1/genética , Loci Gênicos , Predisposição Genética para Doença , Subunidade alfa de Receptor de Interleucina-4/análise , Polimorfismo de Nucleotídeo Único , Alelos , Diabetes Mellitus Tipo 1/imunologia , Genótipo , Humanos , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/imunologia , Fatores de Risco
13.
Genes Immun ; 10 Suppl 1: S54-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19956102

RESUMO

The Type I Diabetes Genetics Consortium (T1DGC) has collected thousands of multiplex and simplex families with type I diabetes (T1D) with the goal of identifying genes involved in T1D susceptibility. These families have been genotyped for the HLA class I and class II loci and, recently, for a genome-wide panel of single-nucleotide polymorphisms (SNPs). In addition, multiple SNPs in specific candidate genes have been genotyped in these families in an attempt to evaluate previously reported T1D associations, including the C883A (Pro-Thr) polymorphism in exon 2 of TCF7, a T-cell transcription factor. The TCF7 883A allele was associated with T1D in subjects with T1D not carrying the high-risk HLA genotype DR3/DR4. A panel of 11 SNPs in TCF7 was genotyped in 2092 families from 9 cohorts of the T1DGC. SNPs at two positions in TCF7 were associated with T1D. One associated SNP, C883A (rs5742913), was reported earlier to have a T1D association. A second SNP, rs17653687, represents a novel T1D susceptibility allele in TCF7. After stratification on the high T1D risk DR3/DR4 genotype, the variant (A) allele of C883A was significantly associated with T1D among non-DR3/DR4 cases (transmission=55.8%, P=0.004; OR=1.26) but was not significantly associated in the DR3/DR4 patient subgroup, replicating the earlier report. The reference A allele of intronic SNP rs17653687 was modestly associated with T1D in both DR3/DR4 strata (transmission=54.4% in DR3/DR4; P=0.03; transmission=52.9% in non-DR3/DR4; P=0.03). These results support the previously reported association of the non-synonymous Pro-Thr SNP in TCF7 with T1D, and suggest that other alleles at this locus may also confer risk.


Assuntos
Diabetes Mellitus Tipo 1/genética , Loci Gênicos , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Fator 1 de Transcrição de Linfócitos T/genética , Alelos , Diabetes Mellitus Tipo 1/imunologia , Genótipo , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/imunologia , Antígeno HLA-DR4/genética , Antígeno HLA-DR4/imunologia , Humanos , Fatores de Risco , Fator 1 de Transcrição de Linfócitos T/imunologia
14.
Tissue Antigens ; 74(5): 393-403, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19845894

RESUMO

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.


Assuntos
Características da Família , Antígenos HLA/genética , Ensaios de Triagem em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Alelos , Sequência de Bases , Feminino , Frequência do Gene , Genótipo , Antígenos HLA/análise , Teste de Histocompatibilidade/métodos , Humanos , Masculino , Pais , Polimorfismo Genético , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia
15.
Tissue Antigens ; 74(3): 268-70, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19691641

RESUMO

The analysis of families collected by the T1DGC and typed at high resolution for the HLA class I and class II loci provided an opportunity for identifying new alleles and rare recombination events. In one American Caucasian family, a novel allele (HLA-DPB1*1302), detected as an unusual pattern of probe binding, was identified in the mother and in one child. Amplicons from both individuals were sequenced and a new variant of DPB1*1301 with an A->T mutation [TAC to TTC in codon 64, (amino acid 35); Y to F] was confirmed. In another American Caucasian family, one child inherited an unusual haplotype, DRB1*1501-DQA1*0102-DQB1*0609-DPA1*0103-DPB1*0601 resulting from a recombination between the DRB1 loci on the maternal chromosomes DRB1*1501-DQA1*0102-DQB1*0602-DPA1*0103-DPB1*0401 and DRB1*1302-DQA1*0102-DQB1*0609-DPA1*0103-DPB1*0601.


Assuntos
Alelos , Antígenos HLA-DP/genética , Antígenos de Histocompatibilidade Classe II/genética , Núcleo Familiar , Recombinação Genética , Sequência de Bases , Criança , Mapeamento Cromossômico , Códon , Sequência Consenso , Primers do DNA/genética , Sondas de DNA/metabolismo , Diabetes Mellitus Tipo 1/genética , Grupo com Ancestrais do Continente Europeu/genética , Éxons , Frequência do Gene , Variação Genética , Genótipo , Cadeias beta de HLA-DP , Haplótipos , Humanos , Dados de Sequência Molecular , Mães , Mutação Puntual , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , Homologia de Sequência do Ácido Nucleico , Terminologia como Assunto , Estados Unidos
16.
Diabetes Obes Metab ; 11 Suppl 1: 25-30, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19143812

RESUMO

AIM: The goal of this study was to develop and implement methodology that would aid in the analysis of extended high-density single nucleotide polymorphism (SNP) major histocompatibility complex (MHC) haplotypes combined with human leucocyte antigen (HLA) alleles in relation to type 1 diabetes risk. METHODS: High-density SNP genotype data (2918 SNPs) across the MHC from the Type 1 Diabetes Genetics Consortium (1240 families), in addition to HLA data, were processed into haplotypes using PedCheck and Merlin, and extended DR3 haplotypes were analysed. RESULTS: With this large dense set of SNPs, the conservation of DR3-B8-A1 (8.1) haplotypes spanned the MHC (>/=99% SNP identity). Forty-seven individuals homozygous for the 8.1 haplotype also shared the same homozygous genotype at four 'sentinel' SNPs (rs2157678 'T', rs3130380 'A', rs3094628 'C' and rs3130352 'T'). Conservation extended from HLA-DQB1 to the telomeric end of the SNP panels (3.4 Mb total). In addition, we found that the 8.1 haplotype is associated with lower risk than other DR3 haplotypes by both haplotypic and genotypic analyses [haplotype: p = 0.009, odds ratio (OR) = 0.65; genotype: p = 6.3 x 10(-5), OR = 0.27]. The 8.1 haplotype (from genotypic analyses) is associated with lower risk than the high-risk DR3-B18-A30 haplotype (p = 0.01, OR = 0.23), but the DR3-B18-A30 haplotype did not differ from other non-8.1 DR3 haplotypes relative to diabetes association. CONCLUSION: The 8.1 haplotype demonstrates extreme conservation (>3.4 Mb) and is associated with significantly lower risk for type 1 diabetes than other DR3 haplotypes.


Assuntos
Diabetes Mellitus Tipo 1/genética , Antígeno HLA-DR3/genética , Polimorfismo de Nucleotídeo Único/genética , Sequência Conservada , Predisposição Genética para Doença/genética , Genótipo , Haplótipos , Heterozigoto , Homozigoto , Humanos , Linhagem , Fatores de Risco
17.
Genes Immun ; 9(7): 613-23, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18650831

RESUMO

Cervical cancer has been associated with specific human leukocyte antigen (HLA) haplotypes/alleles and with polymorphisms at the nearby non-HLA loci TNF, LTA, TAP1 and TAP2. Distinguishing effects of individual loci in the major histocompatibility complex (MHC) region are difficult due to the complex linkage disequilibrium (LD) pattern characterized by high LD, punctuated by recombination hot spots. We have evaluated the association of polymorphism at HLA class II DQB1 and the TNF, LTA, TAP1 and TAP2 genes with cervical cancer risk, using 1306 familial cases and 288 controls. DQB1 was strongly associated; alleles *0301, *0402 and (*)0602 increased cancer susceptibility, whereas *0501 and *0603 decreased susceptibility. Among the non-HLA loci, association was only detected for the TAP2 665 polymorphism, and interallelic disequilibrium analysis indicated that this could be due to LD with DQB1. As the TAP2 665 association was seen predominantly in non-carriers of DQB1 susceptibility alleles, we hypothesized that TAP2 665 may have an effect not attributable to LD with DQB1. However, a logistic regression analysis suggested that TAP2 665 was strongly influenced by LD with DQB1. Our results emphasize the importance of large sample sizes and underscore the necessity of examining both HLA and non-HLA loci in the MHC to assign association to the correct locus.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Linfotoxina-alfa/genética , Fator de Necrose Tumoral alfa/genética , Neoplasias do Colo do Útero/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Antígenos HLA-DQ/fisiologia , Cadeias beta de HLA-DQ , Humanos , Desequilíbrio de Ligação/genética , Linfotoxina-alfa/fisiologia , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Fator de Necrose Tumoral alfa/fisiologia
18.
Genes Immun ; 8(8): 628-33, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17728790

RESUMO

Autoimmune diabetes shows extreme variation in age of onset and clinical presentation, although most studies have been done in children with the most severe subtype. Disease risk is strongly associated with HLA-DRB1*0301-DQA1*0501-DQB1*0201 (DR3-DQ2), but it has not been possible to separate the effects of the DR and DQ alleles. We have identified a large Bedouin kindred in which a high prevalence of islet autoimmunity is associated with two different DR3 haplotypes, one carrying the usual DQ2 and the other carrying DQA1*0102-DQB1*0502 (DQ5). Results of prospective follow-up studies indicate that DR3 is associated with the initial activation of islet autoimmunity whereas DQ2 is associated with early-onset and severe clinical disease. The association signals map to a 350-kb interval, thus implicating primary effects for DR3 and DQ2. Overall, our results emphasize the importance of prospective genetic studies that examine the full range of variation in the initiation, progression and expression of autoimmune disease.


Assuntos
Autoimunidade/genética , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Ilhotas Pancreáticas/imunologia , Adolescente , Adulto , Idade de Início , Idoso , Árabes/genética , Criança , Pré-Escolar , Feminino , Frequência do Gene , Cadeias alfa de HLA-DQ , Cadeias beta de HLA-DQ , Cadeias HLA-DRB1 , Haplótipos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade
19.
Tissue Antigens ; 70(2): 110-27, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17610416

RESUMO

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.


Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Haplótipos , Europa (Continente) , Genótipo , Cadeias beta de HLA-DQ , Cadeias HLA-DRB1 , Humanos
20.
Tissue Antigens ; 69 Suppl 1: 185-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17445197

RESUMO

The Biostatistics Component of the 13th International Histocompatibility Workshop (IHWS) developed the PyPop (Python for Population Genomics) software framework for high-throughput analysis and quality control (QC) assessments of highly polymorphic genotype data. Since its initial release, the software has had several new analysis modules added to it. These additions, combined with improved data filtering and QC modules, facilitate analyses of data at different levels (allele, haplotype, amino acid sequence, and nucleotide sequence). Since the 13th IHWS, much of the human leukocyte antigen (HLA) data from the workshop, QCed via PyPop and other methods, have been made publicly available through the Major Histocompatibility Complex database web site at the National Center for Biotechnology Information (http://ncbi.nih.gov/mhc/). The Anthropology/Human Genetic Diversity component (AHGDC) data have been used in a variety of studies. Prugnolle et al. used this data to corroborate a model of pathogen-driven selection as a factor related to high levels of diversity at HLA loci. Using a comparative genomics approach contrasting results for HLA and non-HLA markers, Meyer et al. analyzed a subset of the 13th IHWS AHGDC data and showed that HLA loci show detectable signs of both natural selection and the demographic history of populations.


Assuntos
Bases de Dados como Assunto , Antígenos HLA/genética , Teste de Histocompatibilidade/métodos , Imunogenética , Complexo Principal de Histocompatibilidade/imunologia , Coleta de Dados , Genética Populacional , Antígenos HLA/imunologia , Humanos , Internet
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