High Resolution Human Leukocyte Antigen Class I Allele Frequencies and HIV-1 Infection Associations in Chinese Han and Uyghur Cohorts

Background

Host immunogenetic factors such as HLA class I polymorphism are important to HIV-1 infection risk and AIDS progression. Previous studies using high-resolution HLA class I profile data of Chinese populations appeared insufficient to provide information for HIV-1 vaccine development and clinical trial design. Here we reported HLA class I association with HIV-1 susceptibility in a Chinese Han and a Chinese Uyghur cohort.

Methodology/Principal Findings

Our cohort included 327 Han and 161 Uyghur ethnic individuals. Each cohort included HIV-1 seropositive and HIV-1 seronegative subjects. Four-digit HLA class I typing was performed by sequencing-based typing and high-resolution PCR-sequence specific primer. We compared the HLA class I allele and inferred haplotype frequencies between HIV-1 seropositive and seronegative groups. A neighbor-joining tree between our cohorts and other populations was constructed based on allele frequencies of HLA-A and HLA-B loci. We identified 58 HLA-A, 75 HLA-B, and 32 HLA-Cw distinct alleles from our cohort and no novel alleles. The frequency of HLA-B*5201 and A*0301 was significantly higher in the Han HIV-1 negative group. The frequency of HLA-B*5101 was significantly higher in the Uyghur HIV-1 negative group. We observed statistically significant increases in expectation-maximization (EM) algorithm predicted haplotype frequencies of HLA-A*0201-B*5101 in the Uyghur HIV-1 negative group, and of Cw*0304-B*4001 in the Han HIV-1 negative group. The B62s supertype frequency was found to be significantly higher in the Han HIV-1 negative group than in the Han HIV-1 positive group.

Conclusions

At the four-digit level, several HLA class I alleles and haplotypes were associated with lower HIV-1 susceptibility. Homogeneity of HLA class I and Bw4/Bw6 heterozygosity were not associated with HIV-1 susceptibility in our cohort. These observations contribute to the Chinese HLA database and could prove useful in the development of HIV-1 vaccine candidates.     

Measuring Ambiguity in HLA Typing Methods

In hematopoietic stem cell transplantation, donor selection is based primarily on matching donor and patient HLA genes. These genes are highly polymorphic and their typing can result in exact allele assignment at each gene (the resolution at which patients and donors are matched), but it can also result in a set of ambiguous assignments, depending on the typing methodology used. To facilitate rapid identification of matched donors, registries employ statistical algorithms to infer HLA alleles from ambiguous genotypes. Linkage disequilibrium information encapsulated in haplotype frequencies is used to facilitate prediction of the most likely haplotype assignment. An HLA typing with less ambiguity produces fewer high-probability haplotypes and a more reliable prediction. We estimated ambiguity for several HLA typing methods across four continental populations using an information theory-based measure, Shannon's entropy. We used allele and haplotype frequencies to calculate entropy for different sets of 1,000 subjects with simulated HLA typing. Using allele frequencies we calculated an average entropy in Caucasians of 1.65 for serology, 1.06 for allele family level, 0.49 for a 2002-era SSO kit, and 0.076 for single-pass SBT. When using haplotype frequencies in entropy calculations, we found average entropies of 0.72 for serology, 0.73 for allele family level, 0.05 for SSO, and 0.002 for single-pass SBT. Application of haplotype frequencies further reduces HLA typing ambiguity. We also estimated expected confirmatory typing mismatch rates for simulated subjects. In a hypothetical registry with all donors typed using the same method, the entropy values based on haplotype frequencies correspond to confirmatory typing mismatch rates of 1.31% for SSO versus only 0.08% for SBT. Intermediate-resolution single-pass SBT contains the least ambiguity of the methods we evaluated and therefore the most certainty in allele prediction. The presented measure objectively evaluates HLA typing methods and can help define acceptable HLA typing for donor recruitment.     

HLA Typing for the Next Generation

Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.     

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.     

Allele and haplotype frequencies of human leukocyte antigen-A, -B, -C,-DRB1, and -DQB1 in Chinese patients with hematological diseases

The human leukocyte antigen (HLA) system plays a central role in the immune response to pathogens, as well as in organ and allogenic hematopoietic stem cell transplantation (HSCT). Finding a five-locus (i.e., HLA-A, -B, -C, -DRB1, and -DQB1) matched unrelated donor for a patient awaiting HSCT is a major clinical challenge, due to the lack of HLA-identical sibling donors and the high polymorphism of HLA. To date, most studies providing HLA allele frequencies (AF) and haplotype frequencies (HF) in Chinese populations have focused on donors instead of the recipients and have provided data for three loci (HLA-A, -B, and -DR); however, data from five-locus HLA typing in a large sample of patients, especially those with hematological diseases, remains unavailable. Therefore, this study was designed to determine HLA AF and two-, three-, four- and five-locus HF in a large cohort of Chinese Han patients with hematological diseases. The AF and the HF were determined using high-resolution HLA typing data from 2,878 patients. The total number of HLA-A, -B, -C, -DRB1, and -DQB1 alleles was determined to be 48, 92, 49, 52, and 24, respectively. Hardy-Weinberg equilibrium (HWE) analyses indicated significant deviations from HWE for HLA-A, -C, -DRB1, and -DQB1 AF, but not for HLA-B locus. The three most common alleles at each locus were A*11:01, A*24:02, A*02:01; B*46:01, B*40:01, B*13:02; C*01:02, C*07:02, C*06:02; DRB1*09:01, DRB1*15:01, DRB1*07:01; DQB1*03:01, DQB1*03:03, and DQB1*06:01. Our data may help to determine whether the current bone marrow registry contains sufficient diversity to meet the demand.     

High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging

Background

Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.

Results

Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.

Conclusions

The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-864) contains supplementary material, which is available to authorized users.     

Simulation of Y-chromosomal haplotype data

The non-recombining nature of the Y-chromosome determines the non-independence of alleles between loci. The evolution of short tandem repeat (STR) loci in the Y-chromosome is the result of different factors such as differential mutation rates, mutation modes, gene conversion, selection and demographic processes. The degree of correlation between loci is dependent on the magnitude of these processes. The simulation of data is a routine tool used for testing hypotheses in population and evolutionary studies. The most basic parameters hitherto used in lineage haplotype simulations are the allele frequency distributions and mutation rates, assuming either full independence or linkage between loci. In this study we introduce use of the Spearman correlation coefficient to estimate the degree of dependence between non-recombining loci. Then, both the interdependence between loci and the allele frequency distributions at multi-allelic loci are incorporated in an algorithm for simulating haplotypes. We illustrate the method using published and unpublished Y-chromosome STR data.     

DOES FREQUENCY‐DEPENDENT SELECTION WITH COMPLEX DOMINANCE INTERACTIONS ACCURATELY PREDICT ALLELIC FREQUENCIES AT THE SELF‐INCOMPATIBILITY LOCUS IN ARABIDOPSIS HALLERI?

Frequency-dependent selection is a major force determining the evolutionary dynamics of alleles at the self-incompatibility locus (S-locus) in flowering plants. We introduce a general method using numerical simulations to test several alternative models of frequency-dependent selection on S-locus data from sporophytic systems, taking into account both genetic drift and observed patterns of dominance interactions among S-locus haplotypes (S-haplotypes). Using a molecular typing method, we estimated S-haplotype frequencies in a sample of 322 adult plants and of 245 offspring obtained from seeds sampled on 22 maternal plants, collected in a single population of Arabidopsis halleri (Brassicaceae). We found eight different S-haplotypes and characterized their dominance interactions by controlled pollinations. We then compared the likelihood of different models of frequency-dependent selection: we found that the observed haplotype frequencies and observed frequency changes in one generation best fitted a model with (1) the observed dominance interactions and (2) no pollen limitation. Overall, our population genetic models of frequency-dependent selection, including patterns of dominance interactions among S-haplotypes and genetic drift, can reliably predict polymorphism at the S-locus. We discuss how these approaches allow detecting additional processes influencing the evolutionary dynamics of the S-locus, such as purifying selection on linked loci.     

Development of a high-resolution NGS-based HLA-typing and analysis pipeline

The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in transplantation medicine and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. High-quality NGS-based methods are in proprietary use and not publicly available. Here, we introduce the first highly automated open-kit/open-source HLA-typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm allows for highly confident allele-calling to three-field resolution (cDNA nucleotide variants). The method was validated on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, our method provides the flexibility to add further enrichment target regions.     

汉族人群中22个HLA-Cw等位基因全长序列单核苷酸多态性分析

为探讨HLA-Cw (Human leukocyte antigen-Cw)基因全长序列分子遗传多态性, 文章对28个HLA-Cw基因型已知的汉族个体样本, 采用长距离PCR技术和高保真性的Pfu酶, 扩增HLA-Cw基因全长序列4.5 kb, 进行分子克隆和单倍体测序。采用群体遗传学研究方法分析了HLA-Cw等位基因全长序列中各亚区的单核苷酸多态性。结果表明: 在28个样本中共检测出22种等位基因, 序列均已提交GenBank和国际IMGT/HLA数据库并获得了认可; 其中Cw*0706、Cw*030301、Cw*140201的全长序列为首次报道, 尤其是Cw*0706内含子序列的获得, 能够重新设计对该等位基因测序分型的引物, 避免测序分型中可能对这一等位基因的漏检。将28个样本的56条单倍体序列用Clustal软件进行序列排比, 输入到DnaSP4.0进行多态性分析, 共发现244个SNPs, 10处插入/缺失多态性。对HLA-Cw等位基因各亚区多态性的分析, 发现第4内含子及以前并没有受到关注的第5外显子受到平衡选择的作用, 在进化中受到了选择压力, 预示着它们在免疫系统的进化过程中可能扮演着重要的角色。     

Significance of regional population HLA immunogenetic datasets in the efficacy of umbilical cord blood banks and marrow donor registries: a study of Cretan HLA genetic diversity

Background aimsThe high genetic diversity of HLA across populations significantly confines the effectiveness of a donor or umbilical cord blood search for allogeneic hematopoietic stem cell transplantation (HSCT). This study aims to probe the HLA immunogenetic profile of the population of Crete, a Greek region with specific geographic and historical characteristics, and to investigate potential patterns in HLA distribution following comparison with the Deutsche Knochenmarkspenderdatei (DKMS) donor registry. It also aims to highlight the importance of regional public cord blood banks (PCBBs) in fulfilling HSCT needs, especially in countries with significant genetic diversity.MethodsA cohort of 1835 samples representative of the Cretan population was typed for HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1, HLA-DPB1) loci by high-resolution second field next-generation sequencing. Data were compared with the respective HLA profiles of 12 DKMS populations (n = 20 032). Advanced statistical and bioinformatics methods were employed to assess specific intra- and inter-population genetic indexes associated with the regional and geographic distribution of HLA alleles and haplotypes.ResultsA considerable HLA allelic and haplotypic diversity was identified among the Cretan samples and between the latter and the pooled DKMS cohort. Even though the HLA allele and haplotype frequency distribution was similar to regions of close geographic proximity to Crete, a clinal distribution pattern from the northern to southern regions was identified. Significant differences were also observed between Crete and the Greek population of DKMS.ConclusionsThis study provides an in-depth characterization of the HLA immunogenetic profile in Crete and reveals the importance of demographic history in HLA heterogeneity and donor selection. The novel HLA allele and haplotype frequency comparative data between the Cretan and other European populations signify the importance of regional PCBBs in prioritizing HLA diversity to efficiently promote the HSCT program at the national level and beyond.     

On estimating HLA/disease association with application to a study of aplastic anemia

A multiplicative model is described relating HLA typing information to disease incidence. A likelihood-based method for estimating parameters in this model is proposed for use with data sets in which HLA haplotype information is available on a series of cases and their parents. This approach is extended to incorporate information from a matched control series for the purpose of estimating HLA and environmental risk factor effects simultaneously. The method is applied to data from aplastic anemia patients treated by bone marrow transplantation and the results are compared to unmatched case-control analyses using the same case series and several different control series.     

Common HLA alleles associated with health, but not with facial attractiveness

Three adaptive hypotheses have been proposed to explain the link between the human leucocyte antigen (hla) genes, health measures and facial attractiveness: inbreeding avoidance, heterozygote advantage and frequency-dependent selection. This paper reports findings that support a new hypothesis relating HLA to health. We suggest a new method to quantify the level of heterozygosity. HLA heterozygosity did not significantly predict health measures in women, but allele frequency did. Women with more common HLA alleles reported fewer cold and flu bouts per year, fewer illnesses in the previous year and rated themselves healthier than women with rare alleles. To our knowledge, this is the first study to show a positive correlation between HLA allele frequency and general health measures. We propose that certain common HLA alleles confer resistance to prevalent pathogens. Nevertheless, neither HLA heterozygosity nor allele frequency significantly predicted how healthy or attractive men rated the female volunteers. Three non-mutually exclusive explanations are put forward to explain this finding.     

Autoimmune diabetes mellitus, Hodgkin's disease and Graves' ophthalmopathy in a patient with 8.1 ancestral haplotype.

In this report, we describe the case of a 43-year-old woman affected by type 1 diabetes mellitus diagnosed 8 years before, who developed Graves' disease 2 years after chemotherapy and mantle radiotherapy treatment for Hodgkin's disease. Bilateral Graves' ophthalmopathy appeared four months before our observations. Intravenous methyl-prednisolone therapy was started, but was interrupted due to severe metabolic failure. Autoantibodies (anti-islet cells, anti-thyroid, thyroid-stimulating, non-organ-specific) were positive. Since the clinical picture suggested a genetic immunological ground predisposing to autoimmunity, we evaluated her HLA haplotype. Genomic typing of the patient permitted identification of the 8.1 ancestral haplotype, a Caucasoid haplotype unique in its association with many immunopathological diseases. Moreover, we also observed a haplotype unusual in Caucasians, trans DRB1*1101, DQA1*0103, DQB1*0603. To our knowledge, HLA-related genetic risk of developing thyroid autoimmunity after neck irradiation has never been studied. Although we cannot confirm a direct association between the 8.1 ancestral haplotype or DRB1*1101, DQA1*0103, DQB1*0603 and the diseases described, we suggest considering immunological parameters and HLA typing in candidate patients for mantle radiation therapy for Hodgkin's disease or other tumors. HLA haplotype determination could be useful in identifying the patients at raised risk of developing autoimmune diseases after irradiation, thus permitting a more appropriate follow-up schedule.     

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20^th, 31^st, and 81^sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68^th, 79^th, and 95^th haplotypes), one was predominant in the South and Southwest China regions (the 18^th haplotype), one was relatively common in the Northeast and North China regions (the 94^th haplotype), and one was common in the Northeast, North and Northwest China (the 40^th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.     

A method for detecting recent selection in the human genome from allele age estimates

Mutations that have recently increased in frequency by positive natural selection are an important component of naturally occurring variation that affects fitness. To identify such variants, we developed a method to test for recent selection by estimating the age of an allele from the extent of haplotype sharing at linked sites. Neutral coalescent simulations are then used to determine the likelihood of this age given the allele's observed frequency. We applied this method to a common disease allele, the hemochromatosis-associated HFE C282Y mutation. Our results allow us to reject neutral models incorporating plausible human demographic histories for HFE C282Y and one other young but common allele, indicating positive selection at HFE or a linked locus. This method will be useful for scanning the human genome for alleles under selection using the haplotype map now being constructed.