RESUMO
HLA class I assignments were obtained at single genotype, G-level resolution from 98 855 volunteers for an unrelated donor registry in the United States. In spite of the diverse ancestry of the volunteers, over 99% of the assignments at each locus are common. Within this population, 52 novel alleles differing in exons 2 and 3 are identified and characterized. Previously reported alleles with incomplete sequences in the IPD-IMGT/HLA database (n = 519) were selected for full gene sequencing and, from this sampling, another 27 novel alleles are described.
Assuntos
Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Antígenos de Histocompatibilidade Classe I/genética , Teste de Histocompatibilidade/métodos , Sistema de Registros/estatística & dados numéricos , Análise de Sequência de DNA/métodos , Alelos , Genótipo , Voluntários Saudáveis , Humanos , Estados UnidosRESUMO
BACKGROUND: Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. MATERIALS AND METHODS: Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. RESULTS: Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons. CONCLUSION: Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories.
Assuntos
Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Antígenos de Histocompatibilidade Classe I/genética , Teste de Histocompatibilidade/métodos , Hibridização de Ácido Nucleico/métodos , Sistema de Registros , Alelos , Sequência de Aminoácidos , Éxons , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Sondas de Oligonucleotídeos/química , Doadores não RelacionadosRESUMO
High-resolution DNA sequencing was used to identify the human leukocyte antigen (HLA) -A, -B, -C, and -DRB1 alleles found in 552 individuals from the United States indicating Southern European (Italian or Spanish) heritage. A total of 46 HLA-A, 80 HLA-B, 32 HLA-C, and 50 DRB1 alleles were identified. Frequent alleles included A*02:01:01G (allele frequency = 0.26 in Italian Americans and 0.22 in Spanish Americans); B*07:02:01G (Italian Americans allele frequency = 0.11); B*44:03 (Spanish Americans allele frequency = 0.07); C*04:01:01G and C*07:01:01G (allele frequency = 0.13 and 0.16, respectively, in Italian Americans; 0.15 and 0.12, respectively, in Spanish Americans); and DRB1*07:01:01 (allele frequency = 0.12 in each population). The action of balancing selection was inferred at the HLA-B and -C loci in both populations. The A*01:01:01G-C*07:01:01G-B*08:01:01G-DRB1*03:01:01 haplotype was the most frequent A-C-B-DRB1 haplotype in Italian Americans (haplotype frequency = 0.049), and was the second most frequent haplotype in Spanish Americans (haplotype frequency = 0.021). A*29:02:01-C*16:01:01-B*44:03-DRB1*07:01:01 was the most frequent A-C-B-DRB1 in Spanish Americans (haplotype frequency = 0.023), and was observed at a frequency of 0.015 in Italian Americans. Pairwise F'(st) values measuring the degree of differentiation between these Southern European American populations as well as European and European American populations suggest that Spanish Americans constitute a distinct subset of the European American population, most similar to Mexican Americans, whereas Italian Americans cannot be distinguished from the larger European American population.
