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1.  Phase-defined complete sequencing of the HLA genes by next-generation sequencing 
BMC Genomics  2013;14:355.
Background
The human leukocyte antigen (HLA) region, the 3.8-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, mostly autoimmune diseases. Due to the complex nature of HLA genes, there are difficulties in elucidating complete HLA gene sequences especially HLA gene haplotype structures by the conventional sequencing method. We propose a novel, accurate, and cost-effective method for generating phase-defined complete sequencing of HLA genes by using indexed multiplex next generation sequencing.
Results
A total of 33 HLA homozygous samples, 11 HLA heterozygous samples, and 3 parents-child families were subjected to phase-defined HLA gene sequencing. We applied long-range PCR to amplify six HLA genes (HLA-A, -C, -B, DRB1, -DQB1, and –DPB1) followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. Paired-end reads (2 × 250 bp) derived from the sequencer were aligned to the six HLA gene segments of UCSC hg19 allowing at most 80 bases mismatch. For HLA homozygous samples, the six amplicons of an individual were pooled and simultaneously sequenced and mapped as an individual-tagging method. The paired-end reads were aligned to corresponding genes of UCSC hg19 and unambiguous, continuous sequences were obtained. For HLA heterozygous samples, each amplicon was separately sequenced and mapped as a gene-tagging method. After alignments, we detected informative paired-end reads harboring SNVs on both forward and reverse reads that are used to separate two chromosomes and to generate two phase-defined sequences in an individual. Consequently, we were able to determine the phase-defined HLA gene sequences from promoter to 3′-UTR and assign up to 8-digit HLA allele numbers, regardless of whether the alleles are rare or novel. Parent–child trio-based sequencing validated our sequencing and phasing methods.
Conclusions
Our protocol generated phased-defined sequences of the entire HLA genes, resulting in high resolution HLA typing and new allele detection.
doi:10.1186/1471-2164-14-355
PMCID: PMC3671147  PMID: 23714642
HLA; Next generation sequencer
2.  Accurate HLA type inference using a weighted similarity graph 
BMC Bioinformatics  2010;11(Suppl 11):S10.
Background
The human leukocyte antigen system (HLA) contains many highly variable genes. HLA genes play an important role in the human immune system, and HLA gene matching is crucial for the success of human organ transplantations. Numerous studies have demonstrated that variation in HLA genes is associated with many autoimmune, inflammatory and infectious diseases. However, typing HLA genes by serology or PCR is time consuming and expensive, which limits large-scale studies involving HLA genes. Since it is much easier and cheaper to obtain single nucleotide polymorphism (SNP) genotype data, accurate computational algorithms to infer HLA gene types from SNP genotype data are in need. To infer HLA types from SNP genotypes, the first step is to infer SNP haplotypes from genotypes. However, for the same SNP genotype data set, the haplotype configurations inferred by different methods are usually inconsistent, and it is often difficult to decide which one is true.
Results
In this paper, we design an accurate HLA gene type inference algorithm by utilizing SNP genotype data from pedigrees, known HLA gene types of some individuals and the relationship between inferred SNP haplotypes and HLA gene types. Given a set of haplotypes inferred from the genotypes of a population consisting of many pedigrees, the algorithm first constructs a weighted similarity graph based on a new haplotype similarity measure and derives constraint edges from known HLA gene types. Based on the principle that different HLA gene alleles should have different background haplotypes, the algorithm searches for an optimal labeling of all the haplotypes with unknown HLA gene types such that the total weight among the same HLA gene types is maximized. To deal with ambiguous haplotype solutions, we use a genetic algorithm to select haplotype configurations that tend to maximize the same optimization criterion. Our experiments on a previously typed subset of the HapMap data show that the algorithm is highly accurate, achieving an accuracy of 96% for gene HLA-A, 95% for HLA-B, 97% for HLA-C, 84% for HLA-DRB1, 98% for HLA-DQA1 and 97% for HLA-DQB1 in a leave-one-out test.
Conclusions
Our algorithm can infer HLA gene types from neighboring SNP genotype data accurately. Compared with a recent approach on the same input data, our algorithm achieved a higher accuracy. The code of our algorithm is available to the public for free upon request to the corresponding authors.
doi:10.1186/1471-2105-11-S11-S10
PMCID: PMC3024871  PMID: 21172045
3.  The genetics of scleroderma: looking into the postgenomic era 
Current opinion in rheumatology  2012;24(6):677-684.
Purpose of review
The last decade has seen enormous progress in understanding genetic associations of systemic sclerosis to explain the observed heritability. This review highlights the most recent findings and places them in the context of proposed functional roles.
Recent findings
Over 30 genes and gene regions have now been identified as scleroderma susceptibility loci. These include both human leukocyte antigen (HLA) and non-HLA genes, most of which involve immune-related pathways and modifiers of immune function. Many of these associations have also been reported in other systemic autoimmune diseases and suggest that there are multiple autoimmunity genes resulting in disease occurrence.
Summary
In spite of these advances, only a small proportion of the heritability of systemic sclerosis has been explained. Ongoing studies include fine mapping and sequencing studies to identify causal variants, whereas other studies focus on functional consequences of these variants in order to identify the link between these genetic variants and disease susceptibility. Such knowledge should lead to more targeted and effective treatment in this disease.
doi:10.1097/BOR.0b013e328358575b
PMCID: PMC3678376  PMID: 23026857
genetic variants; genome-wide association studies; scleroderma; single nucleotide polymorphisms; systemic sclerosis
4.  The Relationship of HLA Class I and II Alleles and Haplotypes with Autism: A Case Control Study 
Autism Research and Treatment  2014;2014:242048.
Earlier reports showed the relationship between autism and immune genes located in the human leukocyte antigen (HLA). In this current study, we compared the HLA class I and class II alleles and haplotypes in 35 autistic children with 100 control subjects from Saudi Arabia, using PCR-SSP method and Luminex technology. In class I the HLA-A*01 (P = 0.03, OR 2.68), A*02 (P = 0.001, OR 3.02) and HLA-B*07 (P = 0.01, OR 3.27), were significantly associated with autism. Also, the haplotype A*02-B*07 was significantly higher in autistic patients than in controls (P = 0.007, OR 5.83). In class II, DRB1*1104 was significantly higher in patients than in controls (P = 0.001, OR 8.75). The DQB1*0202 (P = 0.001, OR 0.24), DQB1*0302 (P = 0.001, OR 0.14), and DQB1*0501 (P = 0.012, OR 0.25), were negatively associated with disease. While the four-loci genotype study showed that A*01-B*07-DRB1*0701-DQB1*0602 (P = 0.001, OR 41.9) and the A*31-B*51-DRB1*0103-DQB1*0302 (P = 0.012, OR 4.8) are positively associated with autism among Saudi patients. This is the first report on a foreseeable risk of association of HLA-B*07 allele with autism. Thus, HLA-B*07 allele and the closely linked haplotype A*01 B*07 DRB1*0701 DQB1*0602 may serve as a marker for genetic susceptibility to autism in Saudis.
doi:10.1155/2014/242048
PMCID: PMC3929985  PMID: 24672722
5.  Evolutionary Analysis of Classical HLA Class I and II Genes Suggests That Recent Positive Selection Acted on DPB1*04∶01 in Japanese Population 
PLoS ONE  2012;7(10):e46806.
The human leukocyte antigen (HLA) genes exhibit the highest degree of polymorphism in the human genome. This high degree of variation at classical HLA class I and class II loci has been maintained by balancing selection for a long evolutionary time. However, little is known about recent positive selection acting on specific HLA alleles in a local population. To detect the signature of recent positive selection, we genotyped six HLA loci, HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 in 418 Japanese subjects, and then assessed the haplotype homozygosity (HH) of each HLA allele. There were 120 HLA alleles across the six loci. Among the 80 HLA alleles with frequencies of more than 1%, DPB1*04∶01, which had a frequency of 6.1%, showed exceptionally high HH (0.53). This finding raises the possibility that recent positive selection has acted on DPB1*04∶01. The DPB1*04∶01 allele, which was present in the most common 6-locus HLA haplotype (4.4%), A*33∶03-C*14∶03-B*44∶03-DRB1*13∶02-DQB1*06∶04-DPB1*04∶01, seems to have flowed from the Korean peninsula to the Japanese archipelago in the Yayoi period. A stochastic simulation approach indicated that the strong linkage disequilibrium between DQB1*06∶04 and DPB1*04∶01 observed in Japanese cannot be explained without positive selection favoring DPB1*04∶01. The selection coefficient of DPB1*04∶01 was estimated as 0.041 (95% credible interval 0.021–0.077). Our results suggest that DPB1*04∶01 has recently undergone strong positive selection in Japanese population.
doi:10.1371/journal.pone.0046806
PMCID: PMC3463557  PMID: 23056460
6.  Extended LTA, TNF, LST1 and HLA Gene Haplotypes and Their Association with Rubella Vaccine-Induced Immunity 
PLoS ONE  2010;5(7):e11806.
Background
Recent studies have suggested the importance of HLA genes in determining immune responses following rubella vaccine. The telomeric class III region of the HLA complex harbors several genes, including lymphotoxin alpha (LTA), tumor necrosis factor (TNF) and leukocyte specific transcript -1 (LST1) genes, located between the class I B and class II DRB1 loci. Apart from HLA, little is known about the effect of this extended genetic region on HLA haplotypic backgrounds as applied to immune responses.
Methodology/Principal Findings
We examined the association between immune responses and extended class I-class II-class III haplotypes among 714 healthy children after two doses of rubella vaccination. These extended haplotypes were then compared to the HLA-only haplotypes. The most significant association was observed between haplotypes extending across the HLA class I region, ten-SNP haplotypes, and the HLA class II region (i.e. A-C-B-LTA-TNF-LST1-DRB1-DQA1-DQB1-DPA1-DPB1) and rubella-specific antibodies (global p-value of 0.03). Associations were found between both extended A*02-C*03-B*15-AAAACGGGGC-DRB1*04-DQA1*03-DQB1*03-DPA1*01-DPB1*04 (p = 0.002) and HLA-only A*02-C*03-B*15-DRB1*04-DQA1*03-DQB1*03-DPA1*01-DPB1*04 haplotypes (p = 0.009) and higher levels of rubella antibodies. The class II HLA-only haplotype DRB1*13-DQA1*01-DQB1*06-DPA1*01-DPB1*04 (p = 0.04) lacking LTA-TNF-LST1 SNPs was associated with lower rubella antibody responses. Similarly, the class I-class II HLA-only A*01-C*07-B*08-DRB1*03-DQA1*05-DQB1*02-DPA1*01-DPB1*04 haplotype was associated with increased TNF-α secretion levels (p = 0.009). In contrast, the extended AAAACGGGGC-DRB1*01-DQA1*01-DQB1*05-DPA1*01-DPB1*04 (p = 0.01) haplotype was found to trend with decreased rubella-specific IL-6 secretion levels.
Conclusions/Significance
These data suggest the importance of examining both HLA genes and genes in the class III region as part of the extended haplotypes useful in understanding genomic drivers regulating immune responses to rubella vaccine.
doi:10.1371/journal.pone.0011806
PMCID: PMC2910726  PMID: 20668555
7.  DQB1*0602 rather than DRB1*1501 confers susceptibility to multiple sclerosis-like disease induced by proteolipid protein (PLP) 
Background
Multiple sclerosis (MS) is associated with pathogenic autoimmunity primarily focused on major CNS-myelin target antigens including myelin basic protein (MBP), proteolipidprotein (PLP), myelin oligodendrocyte protein (MOG). MS is a complex trait whereby the HLA genes, particularly class-II genes of HLA-DR15 haplotype, dominate the genetic contribution to disease-risk. Due to strong linkage disequilibrium in HLA-II region, it has been hard to establish precisely whether the functionally relevant effect derives from the DRB1*1501, DQA1*0102-DQB1*0602, or DRB5*0101 loci of HLA-DR15 haplotype, their combinations, or their epistatic interactions. Nevertheless, most genetic studies have indicated DRB1*1501 as a primary risk factor in MS. Here, we used 'HLA-humanized' mice to discern the potential relative contribution of DRB1*1501 and DQB1*0602 alleles to susceptibility to "humanized" MS-like disease induced by PLP, one of the most prominent and encephalitogenic target-antigens implicated in human MS.
Methods
The HLA-DRB1*1501- and HLA-DQB1*0602-Tg mice (MHC-II-/-), and control non-HLA-DR15-relevant-Tg mice were immunized with a set of overlapping PLP peptides or with recombinant soluble PLP for induction of "humanized" MS-like disease, as well as for ex-vivo analysis of immunogenic/immunodominant HLA-restricted T-cell epitopes and associated cytokine secretion profile.
Results
PLP autoimmunity in both HLA-DR15-Tg mice was focused on 139-151 and 175-194 epitopes. Strikingly, however, the HLA-DRB1*1501-transgenics were refractory to disease induction by any of the overlapping PLP peptides, while HLA-DQB1*0602 transgenics were susceptible to disease induction by PLP139-151 and PLP175-194 peptides. Although both transgenics responded to both peptides, the PLP139-151- and PLP175-194-reactive T-cells were directed to Th1/Th17 phenotype in DQB1*0602-Tg mice and towards Th2 in DRB1*1501-Tg mice.
Conclusions
While genome studies map a strong MS susceptibility effect to the region of DRB1*1501, our findings offer a rationale for potential involvement of pathogenic DQ6-associated autoimmunity in MS. Moreover, that DQB1*0602, but not DRB1*1501, determines disease-susceptibility to PLP in HLA-transgenics, suggests a potential differential, functional role for DQB1*0602 as a predisposing allele in MS. This, together with previously demonstrated disease-susceptibility to MBP and MOG in DRB1*1501-transgenics, also suggests a differential role for DRB1*1501 and DQB1*0602 depending on target antigen and imply a potential complex 'genotype/target antigen/phenotype' relationship in MS heterogeneity.
doi:10.1186/1742-2094-9-29
PMCID: PMC3344688  PMID: 22316121
EAE/MS; Antigens/Peptides/Epitopes; Neuroimmunology; T Cells; MHC; HLA-Tg mice
8.  Genetic Epidemiology of Glioblastoma Multiforme: Confirmatory and New Findings from Analyses of Human Leukocyte Antigen Alleles and Motifs 
PLoS ONE  2009;4(9):e7157.
Background
Human leukocyte antigen (HLA) class I genes mediate cytotoxic T-lymphocyte responses and natural killer cell function. In a previous study, several HLA-B and HLA-C alleles and haplotypes were positively or negatively associated with the occurrence and prognosis of glioblastoma multiforme (GBM).
Methodology/Principal Findings
As an extension of the Upper Midwest Health Study, we have performed HLA genotyping for 149 GBM patients and 149 healthy control subjects from a non-metropolitan population consisting almost exclusively of European Americans. Conditional logistic regression models did not reproduce the association of HLA-B*07 or the B*07-Cw*07 haplotype with GBM. Nonetheless, HLA-A*32, which has previously been shown to predispose GBM patients to a favorable prognosis, was negatively associated with occurrence of GBM (odds ratio = 0.41, p = 0.04 by univariate analysis). Other alleles (A*29, A*30, A*31 and A*33) within the A19 serology group to which A*32 belongs showed inconsistent trends. Sequencing-based HLA-A genotyping established that A*3201 was the single A*32 allele underlying the observed association. Additional evaluation of HLA-A promoter and exon 1 sequences did not detect any unexpected single nucleotide polymorphisms that could suggest differential allelic expression. Further analyses restricted to female GBM cases and controls revealed a second association with a specific HLA-B sequence motif corresponding to Bw4-80Ile (odds ratio = 2.71, p = 0.02).
Conclusions/Significance
HLA-A allelic product encoded by A*3201 is likely to be functionally important to GBM. The novel, sex-specific association will require further confirmation in other representative study populations.
doi:10.1371/journal.pone.0007157
PMCID: PMC2742900  PMID: 19774073
9.  Identification of antigenic epitopes on human allergens: studies with HLA transgenic mice. 
Environmental Health Perspectives  2003;111(2):245-250.
Environmental factors play an important role in the rise and manifestation of allergic conditions in genetically predisposed subjects. Increased exposure to indoor/outdoor allergens is a significant factor in the development of allergic sensitization and asthma. Recently, strong relationships between the immune response to several highly purified allergens and specific human leukocyte antigen (HLA)-DQ and -DR haplotypes have been reported. The major antigens from clinically important allergens have been cloned and sequenced. However, whether innate structural features of major allergens or peculiar immune recognition of these molecules contribute to the overly robust immune responses is not known. We generated and used transgenic (tg) mice expressing single HLA class II transgene(s) to characterize the allergen epitopes presented by particular HLA class II molecules. Next, we generated in vivo models for asthma in the HLA tg mice by intranasal challenge with allergenic extracts. Furthermore, we used a single epitope to induce an allergic lung inflammation. Our system offers a sophisticated technique for systematically identifying the genetic (individual human class II) and antigenic (individual allergenic epitopes) basis of asthma sensitivity and has important implications for new treatment strategies.
PMCID: PMC1241359  PMID: 12573914
10.  Predicting HLA genotypes using unphased and flanking single-nucleotide polymorphisms in Han Chinese population 
BMC Genomics  2014;15:81.
Background
Genetic variation associated with human leukocyte antigen (HLA) genes has immunological functions and is associated with autoimmune diseases. To date, large-scale studies involving classical HLA genes have been limited by time-consuming and expensive HLA-typing technologies. To reduce these costs, single-nucleotide polymorphisms (SNPs) have been used to predict HLA-allele types. Although HLA allelic distributions differ among populations, most prediction model of HLA genes are based on Caucasian samples, with few reported studies involving non-Caucasians.
Results
Our sample consisted of 437 Han Chinese with Affymetrix 5.0 and Illumina 550 K SNPs, of whom 214 also had data on Affymetrix 6.0 SNPs. All individuals had HLA typings at a 4-digit resolution. Using these data, we have built prediction model of HLA genes that are specific for a Han Chinese population. To optimize our prediction model of HLA genes, we analyzed a number of critical parameters, including flanking-region size, genotyping platform, and imputation. Predictive accuracies generally increased both with sample size and SNP density.
Conclusions
SNP data from the HapMap Project are about five times more dense than commercially available genotype chip data. Using chips to genotype our samples, however, only reduced the accuracy of our HLA predictions by only ~3%, while saving a great deal of time and expense. We demonstrated that classical HLA alleles can be predicted from SNP genotype data with a high level of accuracy (80.37% (HLA-B) ~95.79% (HLA-DQB1)) in a Han Chinese population. This finding offers new opportunities for researchers in obtaining HLA genotypes via prediction using their already existing chip datasets. Since the genetic variation structure (e.g. SNP, HLA, Linkage disequilibrium) is different between Han Chinese and Caucasians, and has strong impact in building prediction models for HLA genes, our findings emphasize the importance of building ethnic-specific models when analyzing human populations.
doi:10.1186/1471-2164-15-81
PMCID: PMC3909910  PMID: 24476119
Major histocompatibility complex (MHC); Human leukocyte antigen (HLA); Single-nucleotide polymorphisms (SNPs)
11.  New Susceptibility and Resistance HLA-DP Alleles to HBV-Related Diseases Identified by a Trans-Ethnic Association Study in Asia 
PLoS ONE  2014;9(2):e86449.
Previous studies have revealed the association between SNPs located on human leukocyte antigen (HLA) class II genes, including HLA-DP and HLA-DQ, and chronic hepatitis B virus (HBV) infection, mainly in Asian populations. HLA-DP alleles or haplotypes associated with chronic HBV infection or disease progression have not been fully identified in Asian populations. We performed trans-ethnic association analyses of HLA-DPA1, HLA-DPB1 alleles and haplotypes with hepatitis B virus infection and disease progression among Asian populations comprising Japanese, Korean, Hong Kong, and Thai subjects. To assess the association between HLA-DP and chronic HBV infection and disease progression, we conducted high-resolution (4-digit) HLA-DPA1 and HLA-DPB1 genotyping in a total of 3,167 samples, including HBV patients, HBV-resolved individuals and healthy controls. Trans-ethnic association analyses among Asian populations identified a new risk allele HLA-DPB1*09∶01 (P = 1.36×10−6; OR = 1.97; 95% CI, 1.50–2.59) and a new protective allele DPB1*02∶01 (P = 5.22×10−6; OR = 0.68; 95% CI, 0.58–0.81) to chronic HBV infection, in addition to the previously reported alleles. Moreover, DPB1*02∶01 was also associated with a decreased risk of disease progression in chronic HBV patients among Asian populations (P = 1.55×10−7; OR = 0.50; 95% CI, 0.39–0.65). Trans-ethnic association analyses identified Asian-specific associations of HLA-DP alleles and haplotypes with HBV infection or disease progression. The present findings will serve as a base for future functional studies of HLA-DP molecules in order to understand the pathogenesis of HBV infection and the development of hepatocellular carcinoma.
doi:10.1371/journal.pone.0086449
PMCID: PMC3919706  PMID: 24520320
12.  HLA and non-HLA genes in Behçet’s disease: a multicentric study in the Spanish population 
Arthritis Research & Therapy  2013;15(5):R145.
Introduction
According to genome wide association (GWA) studies as well as candidate gene approaches, Behçet’s disease (BD) is associated with human leukocyte antigen (HLA)-A and HLA-B gene regions. The HLA-B51 has been consistently associated with the disease, but the role of other HLA class I molecules remains controversial. Recently, variants in non-HLA genes have also been associated with BD. The aims of this study were to further investigate the influence of the HLA region in BD and to explore the relationship with non-HLA genes recently described to be associated in other populations.
Methods
This study included 304 BD patients and 313 ethnically matched controls. HLA-A and HLA-B low resolution typing was carried out by PCR-SSOP Luminex. Eleven tag single nucleotide polymorphisms (SNPs) located outside of the HLA-region, previously described associated with the disease in GWA studies and having a minor allele frequency in Caucasians greater than 0.15 were genotyped using TaqMan assays. Phenotypic and genotypic frequencies were estimated by direct counting and distributions were compared using the χ2 test.
Results
In addition to HLA-B*51, HLA-B*57 was found as a risk factor in BD, whereas, B*35 was found to be protective. Other HLA-A and B specificities were suggestive of association with the disease as risk (A*02 and A*24) or protective factors (A*03 and B*58). Regarding the non-HLA genes, the three SNPs located in IL23R and one of the SNPs in IL10 were found to be significantly associated with susceptibility to BD in our population.
Conclusion
Different HLA specificities are associated with Behçet’s disease in addition to B*51. Other non-HLA genes, such as IL23R and IL-10, play a role in the susceptibility to the disease.
doi:10.1186/ar4328
PMCID: PMC3978908  PMID: 24286189
13.  Relative power of linkage and transmission disequilibrium test strategies to detect non-HLA linked coeliac disease susceptibility genes 
Gut  1999;45(5):668-671.
BACKGROUND—Susceptibility to coeliac disease is genetically determined by possession of specific HLA DQ alleles, acting in concert with one or more non-HLA linked genes. The pattern of familial risk is most parsimonious with a multiplicative model for the interaction between these two classes of genes. Haplotype sharing probabilities across the HLA region in affected sibling pairs suggest that genes within the MHC complex contribute no more than 40% of the sibling familial risk of coeliac disease, making the non-HLA linked gene (or genes) the stronger determinant of coeliac disease susceptibility. Attempts to localise these non-HLA linked genes have been carried out using both linkage and association tests.
AIMS—To review the evidence for the involvement of non-HLA linked genes in coeliac disease, and to compare the relative merits of linkage and transmission disequilibrium tests (TDT) to detect the non-HLA linked gene (or genes) contributing to the development of coeliac disease.
METHODS—Under a range of genetic models the number of affected sibling pairs needed to detect linkage was compared with the number of families required to show a relation between marker and disease, adopting the TDT strategy.
RESULTS AND CONCLUSIONS—Power calculations show that, if there is a single major non-HLA linked susceptibility locus, a non-parametric linkage approach may well prove effective. However, if there are a number of non-HLA susceptibility genes, each with small effect, the sample size necessary for linkage studies will be prohibitive and a systematic search for allelic association should be a more effective strategy.


Keywords: coeliac disease; non-HLA linked genes; linkage; TDT test
PMCID: PMC1727705  PMID: 10517901
14.  A second major histocompatibility complex susceptibility locus for multiple sclerosis 
Annals of Neurology  2007;61(3):228-236.
Objective
Variation in the major histocompatibility complex (MHC) on chromosome 6p21 is known to influence susceptibility to multiple sclerosis with the strongest effect originating from the HLA-DRB1 gene in the class II region. The possibility that other genes in the MHC independently influence susceptibility to multiple sclerosis has been suggested but remains unconfirmed.
Methods
Using a combination of microsatellite, single nucleotide polymorphism, and human leukocyte antigen (HLA) typing, we screened the MHC in trio families looking for evidence of residual association above and beyond that attributable to the established DRB1*1501 risk haplotype. We then refined this analysis by extending the genotyping of classical HLA loci into independent cases and control subjects.
Results
Screening confirmed the presence of residual association and suggested that this was maximal in the region of the HLA-C gene. Extending analysis of the classical loci confirmed that this residual association is partly due to allelic heterogeneity at the HLA-DRB1 locus, but also reflects an independent effect from the HLA-C gene. Specifically, the HLA-C*05 allele, or a variant in tight linkage disequilibrium with it, appears to exert a protective effect (p = 3.3 × 10−5).
Interpretation
Variation in the HLA-C gene influences susceptibility to multiple sclerosis independently of any effect attributable to the nearby HLA-DRB1 gene. Ann Neurol 2007
doi:10.1002/ana.21063
PMCID: PMC2737610  PMID: 17252545
15.  Celiac disease: Prevalence, diagnosis, pathogenesis and treatment 
Celiac disease (CD) is one of the most common diseases, resulting from both environmental (gluten) and genetic factors [human leukocyte antigen (HLA) and non-HLA genes]. The prevalence of CD has been estimated to approximate 0.5%-1% in different parts of the world. However, the population with diabetes, autoimmune disorder or relatives of CD individuals have even higher risk for the development of CD, at least in part, because of shared HLA typing. Gliadin gains access to the basal surface of the epithelium, and interact directly with the immune system, via both trans- and para-cellular routes. From a diagnostic perspective, symptoms may be viewed as either “typical” or “atypical”. In both positive serological screening results suggestive of CD, should lead to small bowel biopsy followed by a favourable clinical and serological response to the gluten-free diet (GFD) to confirm the diagnosis. Positive anti-tissue transglutaminase antibody or anti-endomysial antibody during the clinical course helps to confirm the diagnosis of CD because of their over 99% specificities when small bowel villous atrophy is present on biopsy. Currently, the only treatment available for CD individuals is a strict life-long GFD. A greater understanding of the pathogenesis of CD allows alternative future CD treatments to hydrolyse toxic gliadin peptide, prevent toxic gliadin peptide absorption, blockage of selective deamidation of specific glutamine residues by tissue, restore immune tolerance towards gluten, modulation of immune response to dietary gliadin, and restoration of intestinal architecture.
doi:10.3748/wjg.v18.i42.6036
PMCID: PMC3496881  PMID: 23155333
Celiac disease; Demography; Diagnosis; Pathogenesis; Treatment
16.  Human Leukocyte Antigens and Cellular Immune Responses to Anthrax Vaccine Adsorbed 
Infection and Immunity  2013;81(7):2584-2591.
Interindividual variations in vaccine-induced immune responses are in part due to host genetic polymorphisms in the human leukocyte antigen (HLA) and other gene families. This study examined associations between HLA genotypes, haplotypes, and homozygosity and protective antigen (PA)-specific cellular immune responses in healthy subjects following immunization with Anthrax Vaccine Adsorbed (AVA). While limited associations were observed between individual HLA alleles or haplotypes and variable lymphocyte proliferative (LP) responses to AVA, analyses of homozygosity supported the hypothesis of a “heterozygote advantage.” Individuals who were homozygous for any HLA locus demonstrated significantly lower PA-specific LP than subjects who were heterozygous at all eight loci (median stimulation indices [SI], 1.84 versus 2.95, P = 0.009). Similarly, we found that class I (HLA-A) and class II (HLA-DQA1 and HLA-DQB1) homozygosity was significantly associated with an overall decrease in LP compared with heterozygosity at those three loci. Specifically, individuals who were homozygous at these loci had significantly lower PA-specific LP than subjects heterozygous for HLA-A (median SI, 1.48 versus 2.13, P = 0.005), HLA-DQA1 (median SI, 1.75 versus 2.11, P = 0.007), and HLA-DQB1 (median SI, 1.48 versus 2.13, P = 0.002) loci, respectively. Finally, homozygosity at an increasing number (≥4) of HLA loci was significantly correlated with a reduction in LP response (P < 0.001) in a dose-dependent manner. Additional studies are needed to reproduce these findings and determine whether HLA-heterozygous individuals generate stronger cellular immune response to other virulence factors (Bacillus anthracis LF and EF) than HLA-homozygous subjects.
doi:10.1128/IAI.00269-13
PMCID: PMC3697592  PMID: 23649091
17.  Conditional genotype analysis: detecting secondary disease loci in linkage disequilibrium with a primary disease locus 
BMC Proceedings  2007;1(Suppl 1):S163.
A number of autoimmune and other diseases have well established HLA associations; in many cases there is strong evidence for the direct involvement of the HLA class II peptide-presenting antigens, e.g., HLA DR-DQ for type 1 diabetes (T1D) and HLA-DR for rheumatoid arthritis (RA). The involvement of additional HLA region genes in the disease process is implicated in these diseases. We have developed a model-free approach to detect these additional disease genes using genotype data; the conditional genotype method (CGM) and overall conditional genotype method (OCGM) use all patient and control data and do not require haplotype estimation. Genotypes at marker genes in the HLA region are stratified and their expected values are determined in a way that removes the effects of linkage disequilibrium (LD) with the peptide-presenting HLA genes directly involved in the disease. A statistic has been developed under the null hypothesis of no additional disease genes in the HLA region for the OCGM method and was applied to the Genetic Analysis Workshop 15 simulated data set of Problem 3, which mimics RA (answers were known). In addition to the primary effect of the HLA DR locus, the effects of the other two HLA region simulated genes involved in disease were detected (gene C, 0 cM from DR, increases RA risk only in women; and gene D, 5.12 cM from DR, rare allele increases RA risk five-fold). No false negatives were found. Power calculations were performed.
PMCID: PMC2367484  PMID: 18466509
18.  Analysis of HLA-DRB1,DQA1,DQB1 haplotypes in Sardinian centenarians 
Experimental gerontology  2007;43(2):114-118.
Some genetic determinants of longevity might reside in those polymorphisms for the immune system genes that regulate immune responses. Many longevity association studies focused their attention on HLA (the human MHC) polymorphisms, but discordant results have been obtained. Sardinians are a relatively isolate population and represent a suitable population for association studies. Some HLA-DR and DQ alleles form very stable haplotypes with a strong linkage disequilibrium. In a previous study on Sardinian centenarians we have suggested that HLA-DRB1∗15 allele might be marginally associated to longevity. HLA-DR,DQ haplotypes are in strong linkage disequilibrium and well conserved playing a role in the association to diseases. Hence, we have evaluated, by amplification refractory mutation system/polymerase chain reaction (ARMS-PCR) the HLADQA1 and HLA-DQB1 allele frequencies in 123 centenarians and 92 controls from Sardinia to assess whether the association to HLA-DRB1∗15 allele may be due to the other genes involved in the HLA-DR,DQ haplotypes. The frequencies of HLA-DQA1,DQB1 haplotypes were not significantly modified in centenarians. Nevertheless by evaluating the frequency of DRB1∗15 linked haplotypes, we observed a not significant increase in centenarians of HLA-DQA1∗01,DQB1∗05 and HLA-DQA1∗01,DQB1∗06 haplotypes. These data suggest that these haplotypes might have a role in determining life span expectancy and longevity.
doi:10.1016/j.exger.2007.06.007
PMCID: PMC2645697  PMID: 17714903
Centenarians; HLA-DQA1; HLA-DQB1; Immune response; Longevity; Sardinia
19.  HLA antigen and NK cell activating ligand expression in malignant cells: a story of loss or acquisition 
Seminars in immunopathology  2011;33(4):321-334.
Malignant transformation of cells is often associated with changes in classical and non-classical HLA class I antigen, HLA class II antigen as well as NK cell activating ligand (NKCAL) expression. These changes are believed to play a role in the clinical course of the disease since these molecules are critical to the interactions between tumor cells and components of both innate and adaptive immune system. For some time, it has been assumed that alterations in the expression profile of HLA antigens and NKCAL on malignant cells represented loss of classical HLA class I antigen and induction of HLA class II antigen, non-classical HLA class I antigen and/or NKCAL expression. In contrast to these assumptions, experimental evidence suggests that in some cases dysplastic and malignant cells can acquire classical HLA class I antigen expression and/or lose the ability to express HLA class II antigens. In light of the latter findings as well as of the revival of the cancer immune surveillance theory, a reevaluation of the interpretation of changes in HLA antigen and NKCAL expression in malignant lesions is warranted. In this article, we first briefly describe the conventional types of changes in HLA antigen and NKCAL expression that have been identified in malignant cells to date. Second, we discuss the evidence indicating that, in at least some cell types, classical HLA class I antigen expression can be acquired and/ or the ability to express HLA class II antigens is lost. Third, we review the available evidence for the role of immune selective pressure in the generation of malignant lesions with changes in HLA antigen expression. This information contributes to our understanding of the role of the immune system in the control of tumor development and to the optimization of the design of immunotherapeutic strategies for the treatment of cancer.
doi:10.1007/s00281-011-0270-z
PMCID: PMC3426281  PMID: 21523560
Antigen processing machinery; Cancer; Classical HLA class I antigen; Immune escape; Immune selection; HLA class II antigen; MICA; MICB; NK cell activating ligand; Non-classical HLA class I antigen; ULBP
20.  Activating killer-cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands are significantly increased in autism 
Brain, behavior, and immunity  2012;26(7):1122-1127.
Killer-cell immunoglobulin-like receptor (KIR) proteins are expressed on natural killer (NK) cells and appear important in innate and adaptive immunity. There are about 14 KIR genes on chromosome 19q13.4, composed of those that inhibit and those that activate NK cell killing. Haplotypes have different combinations of these genes meaning that not all genes are present in a subject. There are two main classes of cognate human leukocyte antigen (HLA) ligands (HLA-Bw4 and HLA-C1/C2) that bind to the inhibitory/activating receptors. As a general rule, the inhibitory state is maintained except when virally infected or tumor cells are encountered; however, both increased activation and inhibition states have been associated with susceptibility and protection against numerous disease states including cancer, arthritis, and psoriasis.
Utilizing DNA from 158 Caucasian subjects with autism and 176 KIR control subjects we show for the first time a highly significant increase in four activating KIR genes (2DS5, 3DS1, 2DS1 and 2DS4) as measured by chi square values and odds ratios. In addition, our data suggests a highly significant increase in the activating KIR gene 2DS1 and its cognate HLA-C2 ligand (2DS1+C2; p=0.00003 [Odds Ratio=2.87]). This information ties together two major immune gene complexes, the Human Leukocyte Complex and the Leukocyte Receptor Complex, and may partially explain immune abnormalities observed in many subjects with autism.
doi:10.1016/j.bbi.2012.07.014
PMCID: PMC3469320  PMID: 22884899
killer-cell immunoglobulin-like receptor; KIR genes; KIR haplotypes; human leukocyte antigen; HLA ligands; leukocyte receptor complex; autism; immune dysfunction; natural killer cells
21.  Genotyping HLA-DRB1 and HLA-DQB1 alleles in Japanese patients with normal tension glaucoma 
Molecular Vision  2010;16:1874-1879.
Purpose
Normal tension glaucoma (NTG) is a subtype of glaucoma in which intraocular pressure is within the statistically normal range. NTG may be associated with an immune disorder. The aim of this study was to determine whether specific alleles in the human leukocyte antigen (HLA)-DRB1 and HLA-DQB1 genes correlated with NTG in Japanese patients.
Methods
We genotyped the HLA-DRB1 and HLA-DQB1 alleles in 113 Japanese patients with NTG and in 184 healthy Japanese control subjects using the polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP) Luminex method. We assessed the allelic diversity in patients and controls.
Results
There were no statistically significant differences in the allele frequency of HLADRB1 and HLA-DQB1 between NTG patients and control subjects, and no HLA-DRB1-HLA-DQB1 haplotypes demonstrated any significant association with NTG.
Conclusions
Our findings suggest that HLA-DRB1 and HLA-DQB1 polymorphisms have no significant effect on the development of NTG in Japanese patients.
PMCID: PMC2956698  PMID: 21031025
22.  HLA class II gene associations in African American Type 1 diabetes reveal a protective HLA-DRB1*03 haplotype 
Diabetic Medicine  2013;30(6):710-716.
Aims
Owing to strong linkage disequilibrium between markers, pinpointing disease associations within genetic regions is difficult in European ancestral populations, most notably the very strong association of the HLA-DRB1*03-DQA1*05:01-DQB1*02:01 haplotype with Type 1 diabetes risk, which is assumed to be because of a combination of HLA-DRB1 and HLA-DQB1. In contrast, populations of African ancestry have greater haplotype diversity, offering the possibility of narrowing down regions and strengthening support for a particular gene in a region being causal. We aimed to study the human leukocyte antigen (HLA) region in African American Type 1 diabetes.
Methods
Two hundred and twenty-seven African American patients with Type 1 diabetes and 471 African American control subjects were tested for association at the HLA class II genes, HLA-DRB1, HLA-DQA1, HLA-DQB1 and 5147 single nucleotide polymorphisms across the major histocompatibility complex region using logistic regression models. Population admixture was accounted for with principal components analysis.
Results
Single nucleotide polymorphism marker associations were explained by the HLA associations, with the major peak over the class II loci. The HLA association overall was extremely strong, as expected for Type 1 diabetes, even in African Americans in whom diabetes diagnosis is heterogeneous. In addition, there were unique features: the HLA-DRB1*03 haplotype was split into HLA-DRB1*03:01, which confers greatest susceptibility in these samples (odds ratio 3.17, 95% CI 1.72–5.83) and HLA-DRB1*03:02, an allele rarely observed in Europeans, which confers the greatest protection in these African American samples (odds ratio 0.22, 95% CI 0.09–0.55).
Conclusions
The unique diversity of the African HLA region we have uncovered supports a specific and major role for HLA-DRB1 in HLA-DRB1*03 haplotype-associated Type 1 diabetes risk.
doi:10.1111/dme.12148
PMCID: PMC3709123  PMID: 23398374
23.  Association between HLA Variations and Chronic Hepatitis B Virus Infection in Saudi Arabian Patients 
PLoS ONE  2014;9(1):e80445.
Hepatitis B virus (HBV) infection is a leading cause of liver diseases including cirrhosis and hepatocellular carcinoma. Human leukocyte antigens (HLAs) play an important role in the regulation of immune response against infectious organisms, including HBV. Recently, several genome-wide association (GWAS) studies have shown that genetic variations in HLA genes influence disease progression in HBV infection. The aim of this study was to investigate the role of HLA genetic polymorphisms and their possible role in HBV infection in Saudi Arabian patients. Variations in HLA genes were screened in 1672 subjects who were divided according to their clinical status into six categories as follows; clearance group, inactive carriers, active carriers, cirrhosis, hepatocellular carcinoma (HCC) patients and uninfected healthy controls. Three single nucleotide polymorphisms (SNPs) belonged to HLA-DQ region (rs2856718, rs7453920 and rs9275572) and two SNPs belonged to HLA-DP (rs3077 and rs9277535) were studied. The SNPs were genotyped by PCR-based DNA sequencing (rs2856718) and allele specific TaqMan genotyping assays (rs3077, rs7453920, rs9277535 and rs9275572). The results showed that rs2856718, rs3077, rs9277535 and rs9275572 were associated with HBV infection (p = 0.0003, OR = 1.351, CI = 1.147–1.591; p = 0.041, OR = 1.20, CI = 1.007–1.43; p = 0.045, OR = 1.198, CI = 1.004–1.43 and p = 0.0018, OR = 0.776, CI = 0.662–0.910, respectively). However, allele frequency of rs2856718, rs7453920 and rs9275572 were found more in chronically infected patients when compared to clearance group infection (p = 0.0001, OR = 1.462, CI = 1.204–1.776; p = 0.0178, OR = 1.267, CI = 1.042–1.540 and p = 0.010, OR = 0.776, CI = 0.639–0.942, respectively). No association was found when polymorphisms in HLA genes were compared in active carriers versus cirrhosis/HCC patients. In conclusion, these results suggest that variations in HLA genes could affect susceptibility to and clearance of HBV infection in Saudi Arabian patients.
doi:10.1371/journal.pone.0080445
PMCID: PMC3898905  PMID: 24465366
24.  Complementarity of Binding Motifs is a General Property of HLA-A and HLA-B Molecules and Does Not Seem to Effect HLA Haplotype Composition 
Different human leukocyte antigen (HLA) haplotypes (i.e., the specific combinations of HLA-A, -B, -DR alleles inherited together from one parent) are observed in different frequencies in human populations. Some haplotypes, like HLA-A1-B8, are very frequent, reaching up to 10% in the Caucasian population, while others are very rare. Numerous studies have identified associations between HLA haplotypes and diseases, and differences in haplotype frequencies can in part be explained by these associations: the stronger the association with a severe (autoimmune) disease, the lower the expected HLA haplotype frequency. The peptide repertoires of the HLA molecules composing a haplotype can also influence the frequency of a haplotype. For example, it would seem advantageous to have HLA molecules with non-overlapping binding specificities within a haplotype, as individuals expressing such an haplotype would present a diverse set of peptides from viruses and pathogenic bacteria on the cell surface. To test this hypothesis, we collect the proteome data from a set of common viruses, and estimate the total ligand repertoire of HLA class I haplotypes (HLA-A-B) using in silico predictions. We compare the size of these repertoires to the HLA haplotype frequencies reported in the National Marrow Donor Program (NMDP). We find that in most HLA-A and HLA-B pairs have fairly distinct binding motifs, and that the observed haplotypes do not contain HLA-A and -B molecules with more distinct binding motifs than random HLA-A and HLA-B pairs. In addition, the population frequency of a haplotype is not correlated to the distinctness of its HLA-A and HLA-B peptide binding motifs. These results suggest that there is a not a strong selection pressure on the haplotype level favoring haplotypes having HLA molecules with distinct binding motifs, which would result the largest possible presented peptide repertoires in the context of infectious diseases.
doi:10.3389/fimmu.2013.00374
PMCID: PMC3827838  PMID: 24294213
haplotypes; HLA antigens; selection; genetic; peptide binding; bioinformatics; computational biology
25.  The HLA Region and Autoimmune Disease: Associations and Mechanisms of Action 
Current Genomics  2007;8(7):453-465.
The HLA region encodes several molecules that play key roles in the immune system. Strong association between the HLA region and autoimmune disease (AID) has been established for over fifty years. Association of components of the HLA class II encoded HLA-DRB1-DQA1-DQB1 haplotype has been detected with several AIDs, including rheumatoid arthritis, type 1 diabetes and Graves’ disease. Molecules encoded by this region play a key role in exogenous antigen presentation to CD4+ Th cells, indicating the importance of this pathway in AID initiation and progression. Although other components of the HLA class I and III regions have also been investigated for association with AID, apart from the association of HLA-B*27 with ankylosing spondylitis, it has been difficult to determine additional susceptibility loci independent of the strong linkage disequilibrium (LD) with the HLA class II genes. Recent advances in the statistical analysis of LD and the recruitment of large AID datasets have allowed investigation of the HLA class I and III regions to be re-visited. Association of the HLA class I region, independent of known HLA class II effects, has now been detected for several AIDs, including strong association of HLA-B with type 1 diabetes and HLA-C with multiple sclerosis and Graves’ disease. These results provide further evidence of a possible role for bacterial or viral infection and CD8+ T cells in AID onset. The advances being made in determining the primary associations within the HLA region and AIDs will not only increase our understanding of the mechanisms behind disease pathogenesis but may also aid in the development of novel therapeutic targets in the future.
doi:10.2174/138920207783591690
PMCID: PMC2647156  PMID: 19412418
Genes; autoimmunity & HLA

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