16(th) IHIW : review of HLA typing by NGS.

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.

Age-dependent prevalence of vascular disease-associated polymorphisms among 2689 volunteer blood donors.

BACKGROUND: The development of vascular disease involves the interaction of genetic and environmental factors. Because vascular disease is a major contributor to mortality in Western societies, we hypothesized that deleterious polymorphisms associated with hemostasis decrease in frequency among a healthy population as a function of age. METHODS: The frequencies of factor V G1691A Leiden (FVL), factor II (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, glycoprotein Ia (GPIa) C807T, glycoprotein IIIa (Pl(A1)/Pl(A2)) T1565C, and angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) alleles were determined among 2689 healthy Caucasian whole-blood donors. For analysis, participants were divided into three age groups: 17-39 years (n = 979; 505 males and 474 females), 40-59 years (n = 900; 526 males and 374 females), and 60-85 years (n = 810; 530 males and 280 females). RESULTS: The Pl(A2) allele frequency decreased from 17.5% to 15.7% and 14.1% in the 17-39 years, 40-59 years, and 60-85 years age groups, respectively (n = 5094 alleles; P = 0.025). Among ACE DD males, the Pl(A2) allele frequency decreased from 20.8% to 16.1% and 9.1% in the same groups, respectively (n = 810 alleles; P = 0.001). No statistically significant decrease in genotype or allele frequency was observed among carriers of FVL, FII 20210A, MTHFR 677T, GPIa 807T, or ACE D. CONCLUSIONS: These data suggest that Pl(A2) carriers, especially those who are ACE DD, are statistically less prevalent among older healthy blood donors compared with their younger counterparts. These observations suggest an important, deleterious, time-dependent impact of the Pl(A2) allele, as well as the ACE DD/Pl(A2) allelic combination, on overall health and longevity.

Sequence-based typing of HLA class II DQB1.

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.

Allelic distribution of the glycoprotein Ia (alpha2-integrin) C807T/G873A dimorphisms among caucasian venous thrombosis patients and six racial groups.

Two linked silent dimorphisms, 807 C --> T (Phe224) and 873 G --> A (Thr246) within the glycoprotein Ia (GPIa) gene have been correlated with low and high platelet receptor density, respectively, and associated with vascular disease. A multiplexed allele-specific PCR assay was used to determine the GPIa 807T/873A allele frequency among 331 Caucasian venous thrombosis patients and 3571 unrelated individuals belonging to six different racial groups. The 807T/873A allele frequencies were 54%, 51%, 39%, 39%, 38%, 34% and 30% among Native Americans, Hispanics, Caucasians, Caucasian venous thrombosis patients, Asian Indians, African-Americans, and Koreans, respectively. Significant differences in the GPIa allele frequency among racial groups were revealed which emphasized the need for appropriate controls in studies evaluating the association of GPIa genotype to vascular disease.

Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers.

The cooperative effects of the GPIa 807TT, MTHFR 677TT and prothrombin 20210GA genotypes with the FV Leiden 1691GA (FVL) genotype were evaluated by comparing these genotype frequencies in 77 asymptomatic and 156 symptomatic heterozygous FVL carriers. The GPIa 807TT and MTHFR 677TT genotypes did not segregate within the symptomatic FVL carrier group and did not contribute to venous thrombotic risk in this patient cohort. There was no difference in the prothrombin 20210GA genotype frequency between asymptomatic FVL carriers and a random Caucasian control group; however, the prothrombin 20210GA genotype was nearly 5 times as prevalent (19/156 v 2/77; P < 0.02) in the symptomatic FVL carriers (odds ratio 5.21; 95% confidence interval 1.20-47.62), demonstrating that this important prothrombotic risk factor acts synergistically with FVL.

DRB1*1316: evolutionary and functional implications of a novel polymorphism at codon 86.

HLA-DRB1 molecules contain extensive polymorphism localized to specific functional regions of the antigen binding site (ABS). Position 86 of HLA-DRB1 molecules modulates a hydrophobic pocket in the ABS which acts as a peptide anchoring site [1]. We report the nucleotide sequence of HLA-DRB1*1316 which is identical to HLA-DRB1*1301 and *1302 except at codon 86. The novel allele encodes aspartate rather than valine or glycine at position 86. Val86 and Gly86 have been exclusively observed in thousands of oligotyped specimens; thus Asp86 is a rare polymorphism which is significant with respect to hypotheses concerning evolution and structure-function relationships of HLA-DRB1 molecules.