Multiplex HLA-DR-DQ genotyping : for genetic epidemiology and clinical risk assessment

The human leukocyte antigens (HLA) are highly polymorphic cell surface proteins encoded in the major histocompatibility complex (MHC) region on chromosome 6. The HLA system has been well known as transplantation antigens but the primary biological role of the HLA molecules is regulation of immune response by presenting peptide fragments to T-lymphocytes. As regulators of immune responses the HLA molecules are also of importance for susceptibility to several autoimmune and inflammatory diseases. Genotyping of these loci is therefore significant in research targeting the mechanisms of HLA associated diseases, in exploring new epidemiological associations between HLA and specific disease, and as a clinical tool for risk assessment for diseases with well defined associations. Although several commercial HLA genotyping methods are available, many require multiple steps, have low throughput and high cost. The aim of the work within this dissertation was to develop a robust, costeffective method for HLA-DRB1, -DQA1 and -DQB1 genotyping suitable for use in an epidemiological context and clinical investigation. The method was optimized with specific focus on risk alleles for type 1 diabetes mellitus and celiac disease, two autoimmune disorders with significant impact on public health. By combining PCR with sequence specific primers (PCR-SSP), product separation by capillary gel electrophoresis and fluorescence detection in the developed method, all three loci could be analyzed in a single step, resulting in low reagent cost and fast turnaround time. This in combination with the low total consumption of DNA template allows the method to be used in epidemiological studies. 10 A simplified version of the developed method is currently used for clinical risk assessment for celiac disease when histological and/or serologic results are ambiguous in investigated subjects or when a gluten-free diet has been initiated before diagnostic tests have been performed. The low cost of this newly developed method has enabled HLA typing as a tool in screening programs for high-risk groups, such as individuals with Down syndrome or type 1 diabetes, to preclude the risk for celiac disease and thus avoid periodic screening for auto-antibodies. This method is also used to analyze samples from children all over Sweden with newly diagnosed diabetes in the Better Diabetes Diagnosis project. The developed method was also used in two explorative association studies not related to type 1 diabetes or celiac disease. In one study the association between HLA-DRB1, -DQA1 and -DQB1 and acute myocardial infarction was investigated showing only weak associations. In the second study the HLA-DR-DQ haplotype effect on developing chronic pain after inguinal hernia surgery was explored demonstrating an HLA dependent risk of developing pai

HLA DR-DQ Genotyping by Capillary Electrophoresis for Risk Assessment for Celiac Disease.

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

A new automated human leukocyte antigen genotyping strategy to identify DR-DQ risk alleles for celiac disease and type 1 diabetes mellitus

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. Clin Chem Lab Med 2009;47:1489–95.Peer Reviewe

A new PCR-SSP method for HLA DR-DQ risk assessment for celiac disease.

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

Decrease by 50% of plasma IgA tissue transglutaminase antibody concentrations within 2 months after start of gluten-free diet in children with celiac disease used as a confirming diagnostic test

Background Histological examination of small bowel biopsies is normally the gold standard for the diagnosis of celiac disease (CD). The objective of this study was to investigate whether the rate of decreases in elevated plasma IgA tissue transglutaminase antibody (IgA-tTG) and/or IgG deamidated gliadin peptides antibody (IgG - DGP) concentrations could be used as a confirming test for CD in children on a gluten-free diet (GFD) when biopsy was omitted in the diagnostic process. Methods In this retrospective study we compared children (≤18 years old) with a CD-confirming biopsy (n = 16) to children without a biopsy (n = 18). After initiation of GFD the antibody half-life (the time (T½) when the antibody concentration is 50% decreased) was determined in all children. Results Children with a biopsy (IgA-tTG, T½ = 1.9 months; IgG - DGP, T½ = 2.2 months) and children without a biopsy (IgA-tTG, T½ = 1.6 months; IgG - DGP, T½ = 2.7 months) had comparable T½ (mean) results (p < 0.05) supporting that all children had the CD diagnosis. Conclusions When biopsy was omitted a rapid rate of decrease in CD antibody concentrations confirmed the CD diagnosis in children on GFD. The half-lives (T½) of IgA-tTG were less than 2 months in CD children

Multiple Loci in the HLA Complex Are Associated with Addison's Disease.

Context: A strong association between autoimmune Addison's disease (AAD) and major histocompatibility complex class II-encoded HLA-DRB1-DQA1-DQB1 haplotypes is well known. Recent evidence from other autoimmune diseases has suggested that class I-encoded HLA-A and HLA-B gene variants confer HLA-DRB1-DQA1-DQB1-independent effects on disease. Objective: We aimed to explore AAD predisposing effects of HLA-A and -B and further investigate the role of MICA and HLA-DRB1-DQA1-DQB1 in a much larger material than has previously been studied. Design: HLA-A, -B, -DRB1, and -DQB1 and a microsatellite in MICA were genotyped in 414 AAD patients and 684 controls of Norwegian origin. Results: The strongest association was observed for the DRB1 locus, in which the DRB1*03:01 and DRB1*04:04 conferred increased risk of AAD, particularly in a heterozygous combination [odds ratio 22.13; 95% confidence interval (11.39-43.98); P = 6 × 10(-20)]. After conditioning on DRB1, association with AAD was still present for HLA-B and MICA, suggesting the presence of additional risk factors. Conclusions: The major histocompatibility complex harbors multiple risk loci for AAD, in which DRB1 appears to represent the main risk factor