RESUMO
BACKGROUND: The risk for type 1 diabetes mellitus (T1DM) and celiac disease (CD) is related to human leukocyte antigen (HLA) DQA1, DQB1 and DRB1 loci. Unfortunately, HLA typing has been too difficult and costly for frequent use. Automated genotyping focused on risk alleles could provide access to HLA typing in diagnostic evaluations, epidemiological screening and contribute to preventive strategies. METHODS: A sequence specific primer amplification method requiring a total of four PCR reactions, one restriction enzyme digestion and a single electrophoretic step provides low to medium resolution typing of DQA1, DQB1 and DRB1 using Applied Biosystems 3730 DNA analyzer. The method was validated using 261 samples with genotypes determined using a reference method. RESULTS: Specific fluorescent DQA1, DQB1 and DRB1 amplicons were of expected size. Concordance with the reference method was 100% for DQA1 and DQB1 alleles and 99.8% for DRB1 alleles. CONCLUSIONS: We have developed a high throughput HLA typing method that accurately distinguishes risk alleles for T1DM and CD. This method allows screening of several thousand samples per week, consuming 32 ng of DNA template, low reagent volumes and minimal time for data review.
Assuntos
Alelos , Automação , Doença Celíaca/genética , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Sequência de Bases , Primers do DNA , Genótipo , Humanos , Reação em Cadeia da PolimeraseRESUMO
The risk for celiac disease (CD) is clearly related to specific HLA DQA1 and DQB1 alleles, but HLA -typing is often considered too costly for frequent use.Here we present a method using sequence-specific primed PCR (PCR-SSP) for HLA-DR-DQ genotyping optimized for capillary electrophoresis on Applied Biosystems 3130xl Genetic Analyzer. Requiring a total of three PCR reactions and a single electrophoretic step, this method reduces the reagent expenses and technical time for directed HLA typing to distinguish risk alleles for CD, with a sufficient throughput for large-scale screening projects.
Assuntos
Doença Celíaca/genética , Doença Celíaca/imunologia , Eletroforese Capilar/métodos , Predisposição Genética para Doença , Técnicas de Genotipagem/métodos , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Homozigoto , Humanos , Reação em Cadeia da Polimerase , Medição de Risco , Estatística como AssuntoRESUMO
BACKGROUND: Susceptibility to celiac disease is essentially restricted to carriers of specific HLA DQA1 and DQB1 alleles. We have developed a semi-automated sequence specific primer (SSP) PCR method for clinical HLA typing and compared the test results with those from a commercial method. METHODS: Primers for each DQA1 and DQB1 allele group were included in our PCR-SSP reaction to allow differentiation of homozygous from heterozygous carriers of risk alleles. Primers detecting the tightly linked DRB1*04, *03, *07 and *09 alleles were included to resolve potentially ambiguous results. Fluorescently labeled PCR products of 119 clinical samples were analyzed by capillary electrophoresis, and results were compared to those previously obtained from the DELFIA® Type 1 Diabetes Genetic Predisposition assay. RESULTS: The risk assessment derived from the two methods was 100% concordant. One previously unreported haplotype was detected and haplotype assignments in two of the 119 samples were improved from previous reports. CONCLUSIONS: The use of three PCR reactions and a single electrophoretic step for DQA1, DQB1 and DRB1 typing provides distinction of celiac disease associated alleles and their homo- or heterozygous status. This multiplex analysis reduces reagent costs, personnel and instrument time, while enabling improved allelic assignment through HLA-DR-DQ haplotype association.