Next Generation Sequencing Reveals the Association of DRB3*02:02 With Type 1 Diabetes

Abstract

The primary associations of the HLA class II genes, HLA-DRB1 and HLA-DQB1, and the class I genes, HLA-A and HLA-B, with type 1 diabetes (T1D) are well established. However, the role of polymorphism at the HLA-DRB3, HLA-DRB4, and HLA-DRB5 loci remains unclear. In two separate studies, one of 500 subjects and 500 control subjects and one of 366 DRB1*03:01–positive samples from selected multiplex T1D families, we used Roche 454 sequencing with Conexio Genomics ASSIGN ATF 454 HLA genotyping software analysis to analyze sequence variation at these three HLA-DRB loci. Association analyses were performed on the two HLA-DRB loci haplotypes (DRB1-DRB3, -DRB4, or -DRB5). Three common HLA-DRB3 alleles (*01:01, *02:02, *03:01) were observed. DRB1*03:01 haplotypes carrying DRB3*02:02 conferred a higher T1D risk than did DRB1*03:01 haplotypes carrying DRB3*01:01 in DRB1*03:01/*03:01 homozygotes with two DRB3*01:01 alleles (odds ratio [OR] 3.4 [95% CI 1.46–8.09]), compared with those carrying one or two DRB3*02:02 alleles (OR 25.5 [3.43–189.2]) (P = 0.033). For DRB1*03:01/*04:01 heterozygotes, however, the HLA-DRB3 allele did not significantly modify the T1D risk of the DRB1*03:01 haplotype (OR 7.7 for *02:02; 6.8 for *01:01). These observations were confirmed by sequence analysis of HLA-DRB3 exon 2 in a targeted replication study of 281 informative T1D family members and 86 affected family-based association control (AFBAC) haplotypes. The frequency of DRB3*02:02 was 42.9% in the DRB1*03:01/*03:01 patients and 27.6% in the DRB1*03:01/*04 (P = 0.005) compared with 22.6% in AFBAC DRB1*03:01 chromosomes (P = 0.001). Analysis of T1D-associated alleles at other HLA loci (HLA-A, HLA-B, and HLA-DPB1) on DRB1*03:01 haplotypes suggests that DRB3*02:02 on the DRB1*03:01 haplotype can contribute to T1D risk.

HLA-DRB1*07:01 is associated with a higher risk of asparaginase allergies

“…49,50 Genetic variants within HLA genes have been associated with several diseases including celiac disease, 51 rheumatoid arthritis, 52,53 type I diabetes, 54,55 and multiple sclerosis. 56 Several HLA-B alleles have been associated with small molecule drug allergy, including abacavir, 57,58 and adverse reactions to carbamazepine 59 and allopurinol.…”

DNA Storage under High Temperature Conditions Does Not Affect Performance in Human Leukocyte Antigen Genotyping via Next-Generation Sequencing (DNA Integrity Maintained in Extreme Conditions)

“…Roche 454 fusion primers consist of a 10-base multiplex ID (MID) tag, flanked by a locus-specific primer on the 3¢ end, and an ''A'' or ''B'' 454-specific primer adaptor sequence on the 5¢ end. 6,7 The MID tag serves as a barcode for sample identification by data analysis programs. 7,8 PCRs were performed in a 25 mL volume.…”

“…6,7 The MID tag serves as a barcode for sample identification by data analysis programs. 7,8 PCRs were performed in a 25 mL volume. Each reaction contained 20 ng of genomic DNA, 2 units of AmpliTaq Gold Polymerase (Life Technologies, Carlsbad, CA), 1X AmpliTaq PCR Gold Buffer (Life Technologies, Carlsbad, CA), 1.5 mM AmpliTaq Gold MgCl 2 Solution (Life Technologies, Carlsbad, CA), 0.3 mM of PCR Grade Nucleotide Mix (Roche Applied Sciences, Indianapolis, IN), and 10% Amresco brand glycerol (Amresco, Solan, OH).…”

“…6,7 DNA bound beads were enriched and deposited onto a single region 454 Titanium PicoTiter plate (Roche 454, Branford, CT). 6,7 Molecules were pyrosequenced on the Roche 454 GS Junior system (Roche 454, Branford, CT). 6,7 Data analysis and statistics FASTA formatted sequencing reads were filtered based on alignment to an International Immunogenetics Information System (IMGT) HLA reference database using the Roche 454 Amplicon Variant Analyzer (Roche 454, Branford, CT).…”

“…6,7 Molecules were pyrosequenced on the Roche 454 GS Junior system (Roche 454, Branford, CT). 6,7 Data analysis and statistics FASTA formatted sequencing reads were filtered based on alignment to an International Immunogenetics Information System (IMGT) HLA reference database using the Roche 454 Amplicon Variant Analyzer (Roche 454, Branford, CT). 9 Sequencing reads from a subsequent FASTA file were analyzed with SCORE TM software and Conexio Assign TM ATF HLA genotyping softwares.…”