Vitamin D receptor gene polymorphisms and HLA DRB1*04 cosegregation in Saudi type 2 diabetes patients

The vitamin D receptor (VDR) gene has been involved in the modulation of susceptibility to inflammatory and autoimmune conditions, and could play a role in the pathogenesis of type 2 diabetes mellitus (T2DM). Susceptibility to T2DM was recently also suggested to associate with HLA alleles. We evaluated possible correlations between VDR polymorphisms, HLA alleles, and risk for development of T2DM by analyzing 627 individuals (368 T2DM patients and 259 healthy control subjects) part of a well-characterized cohort followed in Riyadh, Kingdom of Saudi Arabia. Genomic DNA was genotyped for the VDR gene single nucleotide polymorphisms of Fok-1, Taq-1, ApaI, and Bsm-I. Analyses were run by allelic discrimination real-time PCR. HLA genotyping was performed as well by PCR using sequence-specific primers, whereas cytokine production was evaluated by FACS. Results showed T2DM to be significantly associated with the VDR Taq1 (rs731236-AG) and Bsm-I (rs1544410-CT) genotypes, and the VDR rs1544410-T allele. Cosegregations resulting in significant increases of T2DM odds ratio were detected between Taq1 and Bsm-I VDR polymorphisms and HLA DRB1*04. Notably, the VDR polymorphisms observed to be more frequent in T2DM patients correlated with increased VDR expression and IL-12 production, as well as with metabolic parameters of susceptibility to T2DM, including serum cholesterol and high-density lipoprotein levels. VDR polymorphisms are present in T2DM, and correlate with HLA DRB1*04 and with immunologic and metabolic parameters; results from this study add T2DM to the list of diseases that are likely modulated by an HLA/VDR interaction.     

Modern European cranial variables and blood polymorphisms show comparable spatial patterns

Spatial patterns in cranial traits for modern European populations are compared with patterns described by Sokal et al. (1989) for blood polymorphisms. Spatial patterns in these variables are described from both one-dimensional and directional autocorrelation correlograms. Manhattan distances computed among one-dimensional correlograms are used (1) to cluster variables with similar patterns and (2) to test the hypothesis that these clusters are to some extent accounted for by the type of variable. The one-dimensional correlograms for cranial traits do not show a significant contrast with either red cell antigens or the set of blood polymorphisms that excludes HLA. The only contrast that accounts for any of the cluster structure among one-dimensional correlograms is that between HLA and non-HLA variables. A cluster analysis of the directional correlograms demonstrates that cranial traits reflect patterns comparable to those for blood polymorphisms. This finding implies that patterns in cranial variables can be accounted for by the same, or similar, population processes as those inferred from patterns in blood polymorphisms. The implications of this finding for the likely origin of the northwest-southeast cline seen in some modern blood polymorphisms and modern cranial variables, but not in Neolithic cranial variables, are discussed.     

Viral adaptation to host immune responses occurs in chronic hepatitis B virus (HBV) infection, and adaptation is greatest in HBV e antigen-negative disease

Hepatitis B virus (HBV)-specific T-cell responses are important in the natural history of HBV infection. The number of known HBV-specific T-cell epitopes is limited, and it is not clear whether viral evolution occurs in chronic HBV infection. We aimed to identify novel HBV T-cell epitopes by examining the relationship between HBV sequence variation and the human leukocyte antigen (HLA) type in a large prospective clinic-based cohort of Asian patients with chronic HBV infection recruited in Australia and China (n = 119). High-resolution 4-digit HLA class I and II typing and full-length HBV sequencing were undertaken for treatment-naïve individuals (52% with genotype B, 48% with genotype C, 63% HBV e antigen [HBeAg] positive). Statistically significant associations between HLA types and HBV sequence variation were identified (n = 49) at 41 sites in the HBV genome. Using prediction programs, we determined scores for binding between peptides containing these polymorphisms and associated HLA types. Among the regions that could be tested, HLA binding was predicted for 14/18 (78%). We identified several HLA-associated polymorphisms involving likely known anchor residues that resulted in altered predicted binding scores. Some HLA-associated polymorphisms fell within known T-cell epitopes with matching HLA restriction. Enhanced viral adaptation (defined as the presence of the relevant HLA and the escaped amino acid) was independently associated with HBeAg-negative disease (P = 0.003). Thus, HBV appears to be under immune pressure in chronic HBV infection, particularly in HBeAg-negative disease.     

Association of MHC and rheumatoid arthritis: HLA polymorphisms in phenotypic variants of rheumatoid arthritis

Genes in the human leukocyte antigen (HLA) region remain the most powerful disease risk genes in rheumatoid arthritis (RA). Several allelic variants of HLA-DRB1 genes have been associated with RA, supporting a role for T-cell receptor-HLA-antigen interactions in the pathologic process. Disease-associated HLA-DRB1 alleles are similar but not identical and certain allelic variants are preferentially enriched in patient populations with defined clinical characteristics. Also, a gene dosing effect of HLA-DRB1 alleles has been suggested by the accumulation of patients with two RA-associated alleles, especially in patient subsets with a severe disease course. Therefore, polymorphisms in HLA genes are being explored as tools to dissect the clinical heterogeneity of the rheumatoid syndrome. Besides HLA polymorphisms, other risk genes will be helpful in defining genotypic profiles correlating with disease phenotypes. One such phenotype is the type of synovial lesion generated by the patient. HLA genes in conjunction with other genetic determinants may predispose patients to a certain pathway of synovial inflammation. Also, patients may or may not develop extraarticular manifestations, which are critical in determining morbidity and mortality. HLA genes, complemented by other RA risk genes, are likely involved in shaping the T-cell repertoire, including the emergence of an unusual T-cell population characterized by the potential of vascular injury, such as seen in extraarticular RA.     

Association of MHC and rheumatoid arthritis. HLA polymorphisms in phenotypic variants of rheumatoid arthritis

Genes in the human leukocyte antigen (HLA) region remain the most powerful disease risk genes in rheumatoid arthritis (RA). Several allelic variants of HLA-DRB1 genes have been associated with RA, supporting a role for T-cell receptor-HLA-antigen interactions in the pathologic process. Disease-associated HLA-DRB1 alleles are similar but not identical and certain allelic variants are preferentially enriched in patient populations with defined clinical characteristics. Also, a gene dosing effect of HLA-DRB1 alleles has been suggested by the accumulation of patients with two RA-associated alleles, especially in patient subsets with a severe disease course. Therefore, polymorphisms in HLA genes are being explored as tools to dissect the clinical heterogeneity of the rheumatoid syndrome. Besides HLA polymorphisms, other risk genes will be helpful in defining genotypic profiles correlating with disease phenotypes. One such phenotype is the type of synovial lesion generated by the patient. HLA genes in conjunction with other genetic determinants may predispose patients to a certain pathway of synovial inflammation. Also, patients may or may not develop extraarticular manifestations, which are critical in determining morbidity and mortality. HLA genes, complemented by other RA risk genes, are likely involved in shaping the T-cell repertoire, including the emergence of an unusual T-cell population characterized by the potential of vascular injury, such as seen in extraarticular RA.     

Quantity of HLA-C surface expression and licensing of KIR2DL+ natural killer cells

Natural killer (NK) cells require interaction of inhibitory surface receptors with human leukocyte antigen (HLA) ligands during development to acquire functional competence in a process termed "licensing." The quantity of HLA required for this process is unknown. Two polymorphisms affecting HLA-C surface expression (rs9264942 and rs67384697) have recently been identified, and shown to influence progression of HIV infection. We typed a cohort of healthy donors for the two HLA-C-related polymorphisms, KIR2DL1 and KIR2DL3, and their respective HLA-C ligands and analyzed how HLA ligands influenced licensing status of killer cell immunoglobulin-like receptor (KIR)+ NK cells in terms of degranulation and cytokine production in response to HLA-deficient target cells. The presence of respective HLA class I ligands increased the function of KIR2DL1+ and KIR2DL3+ NK cells in a dose-dependent manner. In contrast, neither of the HLA-C-related polymorphisms nor the quantity of cell surface HLA-C had any significant effect on NK cell function. Interestingly, HLA-Cw7-an HLA-C allele with low surface expression-licensed KIR2DL3+ NK cells more strongly than any other KIR2DL3 ligand. The quantity of cell surface HLA-C does not appear to influence licensing of NK cells, and the HLA-C-related polymorphisms presumably influence HIV progression through factors unrelated to NK cell education.     

Passive sexual transmission of human immunodeficiency virus type 1 variants and adaptation in new hosts

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences.     

Widespread impact of HLA restriction on immune control and escape pathways of HIV-1

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine.     

Evidence of viral adaptation to HLA class I-restricted immune pressure in chronic hepatitis C virus infection

Cellular immune responses are an important correlate of hepatitis C virus (HCV) infection outcome. These responses are governed by the host's human leukocyte antigen (HLA) type, and HLA-restricted viral escape mutants are a critical aspect of this host-virus interaction. We examined the driving forces of HCV evolution by characterizing the in vivo selective pressure(s) exerted on single amino acid residues within nonstructural protein 3 (NS3) by the HLA types present in two host populations. Associations between polymorphisms within NS3 and HLA class I alleles were assessed in 118 individuals from Western Australia and Switzerland with chronic hepatitis C infection, of whom 82 (69%) were coinfected with human immunodeficiency virus. The levels and locations of amino acid polymorphisms exhibited within NS3 were remarkably similar between the two cohorts and revealed regions under functional constraint and selective pressures. We identified specific HCV mutations within and flanking published epitopes with the correct HLA restriction and predicted escaped amino acid. Additional HLA-restricted mutations were identified that mark putative epitopes targeted by cell-mediated immune responses. This analysis of host-virus interaction reveals evidence of HCV adaptation to HLA class I-restricted immune pressure and identifies in vivo targets of cellular immune responses at the population level.     

Analysis of the contribution of HLA genes to genetic predisposition in inflammatory bowel disease.

Crohn disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) of unknown etiology. First-degree relatives of IBD patients have a 10-fold increase in risk of developing the same disease, and distinct associations between specific HLA types and both CD and UC have been reported. We have evaluated the contribution of genes at the HLA locus to susceptibility in IBD by linkage analysis of highly informative microsatellite polymorphisms in 43 families with multiple affected cases. No evidence for linkage of HLA to IBD was obtained under any of the four models tested. Analysis of HLA haplotype sharing in affected relatives indicated that the relative risk to a sibling conferred by the HLA locus was 1.11 in UC and 0.75 in CD, with upper (95%) confidence limits of 2.41 and 1.37, respectively. This suggests that other genetic or environmental factors are responsible for most of the familial aggregation in IBD.     

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.     

山东临朐人群HLA等位基因多态性 与幽门螺杆菌感染关系的研究

为了研究山东临朐地区健康人白细胞抗原（HLA）Ⅰ类等位基因多态性与幽门螺杆菌（Hp）感染的关系,运用序列特异性引物聚合酶链反应（PCR SSP）的方法,检测Hp阳性人群（90例）和Hp阴性人群（49例）的HLA Ⅰ类和Ⅱ类等位基因。结果在HLA Ⅰ类（A、B、CW）的共68个等位基因多态中,发现在感染及非感染人群中存在4个显著性差异的位点;在HLA Ⅱ类（DRB1、DQB1和DRB3、DRB4、DRB5）的共22个等位基因多态中,没有发现显著性差异的位点。A*02等位基因频率,Hp阳性低于阴性人群（OR,0.56;95%CI,0.33～0.94;P,0.029）;B*48等位基因频率,Hp阳性低于阴性人群（OR,0.15;95%CI,0.03～0.72;P,0.007）;CW*08〖STBZ〗等位基因频率,Hp阳性低于Hp阴性人群（OR,0.32;95% CI,0.15～0.69;P,0.003）;CW*15等位基因频率,Hp阳性高于Hp阴性人群（OR,5.11;95%CI,0.63～40.90;P,0.024）。结果表明HLA Ⅰ类等位基因的多态性可能与山东临朐地区Hp的易感性有关;HLA Ⅱ类等位基因的多态性可能与其无关。HLA Ⅰ类等位基因中,CW*15可能是Hp感染的易感基因;A*02、B*48和CW*08可能是保护性基因。Abstract: In order to analyze the relationship of HLA polymorphisms and the infection of H.pylori in the population of Linqu County in Shandong Province,polymerase chain reaction with sequence specific primers(PCR SSP) was used to determine the alleles of HLA typeⅠand Ⅱ in 90 Hp positive persons and 49 Hp negative controls.The results showed that among the 68 alleles of HLA typeⅠ,4 alleles were found significantly different between Hp positive and Hp negative population,while no significant difference was found among the 22 alleles of HLA typeⅡ.Hp positive persons had a lower allele frequency of A*02（OR=0.56,95% CI=0.33～0.94;P=0.029）, B*48（OR=0.15,95% CI=0.03～0.72;P=0.007）,CW*08（OR=0.32,95% CI=0.15～0.69;P=0.003）and a higher allele frequency of CW*15（OR=5.11,95% CI=0.63～40.90;P=0.024）compared with Hp negative controls.Our results indicated that the polymorphisms of HLA typeⅠis involved in the genetic susceptibility of Hp infection in Linqu County,while the polymorphisms of HLA typeⅡ may have no relationship with the genetic susceptibility of Hp infection.It was shown that among the alleles of HLA typeⅠ,CW*15might be a susceptible gene of Hp infection while A*02,B*48 and CW*08might be protective genes.     

Polymorphism in the RD (D6S45) gene

Summary The RD (D6S45) gene in the class III region of the HLA major histocompatibility complex encodes a protein normally containing 24 consecutive basic-acidic dipeptide repeats. We determined the frequency of variations in the number of repeats by use of the polymerase chain reaction. Of 107 subjects 7 (3.3%) carried genes encoding 22 or 23 repeats. There was no difference in the frequency of such polymorphisms between normal individuals and those with systemic lupus erythematosus, a disease associated with other polymorphisms in the class III region of HLA. The frequency of polymorphisms in proteins with oligopeptide repeats may provide useful information concerning functional constraints on repeat number.     

HLA-DP and HLA-DO genes in presumptive HLA-identical siblings: structural and functional identification of allelic variation

We analyzed HLA class II genomic polymorphisms in three families in which bone marrow transplantation was performed between individuals presumed to be HLA identical, but in which unexplained mixed lymphocyte culture reactivity was observed. These families were characterized by classical HLA serology, MLC, and DP typing. In each family, a pair of "HLA-identical" siblings demonstrated a small proliferative response in bidirectional MLC. Southern blotting analysis performed with cDNA probes for DQ alpha, DP alpha, and DP beta identified DP genomic differences in each case. Hybridization of Bgl II-digested genomic DNA with a DP alpha cDNA probe revealed three prominent polymorphic fragments (7.7, 5.8, and 3.7 kb), which discriminated between presumptive identical siblings and indicated crossover events within HLA. Similarly, hybridization of SstI-digested genomic DNA with a DP beta cDNA probe, although resulting in a more complex pattern, identified DP genomic disparity between the presumed HLA identical siblings. Hybridization of SstI-digested DNA from two families with evidence of DP recombination was performed by using an oligonucleotide probe specific for the newly described HLA class II gene DO beta. Two major polymorphic fragments, at 6.2 and 3.3 kb, segregated in these families and localized the crossovers flanking the DO beta gene between the DQ and DP loci. The contribution of the antigenic differences marked by these HLA DP and DO DNA polymorphisms to allorecognition in MLR and in graft-vs-host disease are discussed.     

Associations between restriction fragment length polymorphisms detected with a probe for human 21-hydroxylase (21-OH) and two clinical forms of 21-OH deficiency

Summary DNAs from unrelated healthy individuals and unrelated individuals affected with 21-hydroxylase deficiency (congenital and late-onset adrenal hyperplasia) were digested with seven restriction enzymes and hybridized with a cDNA probe specific for human 21-hydroxylase genes. Associations were found between restriction fragments and the two forms of the disease: (i) The late onset form is associated with a double dose of a 14 kb fragment generated by Eco RI and with a triple dose of a 3.2 kb fragment generated by TaqI in patients with HLA B14 haplotypes; (ii) The classical congenital form is negatively associated with the 14 kb fragment and with a 3.7 kb fragment generated by TaqI in patients with HLA Bw47 haplotypes. A 3.2 kb TaqI fragment is negatively associated with the HLA B8 haplotypes. The other five enzymes tested give no polymorphisms or polymorphisms without correlation with the two forms of the disease.     

Genetic epidemiology: Giant cell arteritis and polymyalgia rheumatica

Giant cell arteritis (GCA) (temporal arteritis) and polymyalgia rheumatica (PMR) are common, frequently related conditions in people generally over 50 years of age. Most studies have shown an association of GCA with HLA-DRB1^*04 alleles. As regards isolated PMR, however, the HLA class II genetic susceptibility varies from one population to another. Besides associations with HLA, tumor necrosis factor appears to influence susceptibility to both conditions. Genetic polymorphisms have also been considered to be important candidates as factors of susceptibility to GCA and PMR. In this regard, gene polymorphisms for ICAM-1 (intercellular adhesion molecule 1), RANTES (regulated upon activation, normal T cell expressed, and presumably secreted), and interleukin (IL)-1 receptor antagonist seem to play a role in the pathogenesis of GCA and PMR in some populations. However, additional studies are required to clarify the genetic influence on susceptibility to these conditions.     

Identification of a telomeric boundary of the HLA region with potential for predisposition to human narcolepsy

We report on a study performed to determine a boundary of the region with the potential to contribute to the predisposition to human narcolepsy (the susceptibility region) in the human leukocyte antigen (HLA) region. We investigated a Japanese narcolepsy family, in which a de novo chromosomal recombination occurred between the HLA-DRB1 and HLA-B genes in the proband. The recombinant chromosome carrying HLA-DRB1*1501 was transmitted to the affected child and grandchild, suggesting that a strong genetic factor(s) predisposing to the disorder was (were) present on the chromosome, and that the recombination breakpoint could be regarded as a boundary to the susceptibility region. To search for the breakpoint, we carried out allele typing at various polymorphic sites, e.g., microsatellite repeat polymorphisms, restriction fragment length polymorphisms, and single-nucleotide polymorphisms in the HLA region, and examined haplotypes with the polymorphic sites in the family members. Haplotype analyses revealed that the recombination breakpoint was present approximately 50 kb to the telomeric side of the palmitoyl-protein thioesterase-2 (PPT2) gene in the HLA class III region. From the gene map of the HLA region, the cyclic AMP response element-binding protein-related protein gene (CREB-RP) appeared to be located at the telomeric end in the 50-kb region. Therefore, the data presented here suggest that the susceptibility region for the disorder in the family is present on the centromeric side of the CREB-RP gene in the recombinant Chromosome 6 carrying HLA-DRB1*1501.     

Association of HLA-DRB alleles and pulmonary tuberculosis in North Chinese patients

Human leukocyte antigen (HLA) plays a central role in the regulation of the immune response. HLA class II molecules are essential for T cell-mediated adaptive immunity and present peptide antigens to CD4(+) T cells. Because of its important role in the immune response and its high degree of polymorphism, the HLA system is associated with many diseases. We examined the polymorphisms of HLA-DRB alleles and the sequences of the HLA-DRB promoter region in 97 unrelated patients with pulmonary tuberculosis and in 62 unrelated normal controls of the Han nationality from North China, using PCR with sequence-specific primers and PCR direct sequencing. We found that the frequency of HLA-DRB1*15 was significantly higher in the pulmonary tuberculosis group than in the healthy control group. The P value was 0.001, and the odds ratio was 3.793. The pulmonary tuberculosis group had the same HLA-DRB1 promoter region sequences as the control group. We concluded that the HLA-DRB1*15 allele is associated with pulmonary tuberculosis in the Han nationality from North China. The HLA-DRB1 promoter region sequences had no association with the development of pulmonary tuberculosis.     

Genetic epidemiology. Giant cell arteritis and polymyalgia rheumatica

Giant cell arteritis (GCA) (temporal arteritis) and polymyalgia rheumatica (PMR) are common, frequently related conditions in people generally over 50 years of age. Most studies have shown an association of GCA with HLA-DRB1 *04 alleles. As regards isolated PMR, however, the HLA class II genetic susceptibility varies from one population to another. Besides associations with HLA, tumor necrosis factor appears to influence susceptibility to both conditions. Genetic polymorphisms have also been considered to be important candidates as factors of susceptibility to GCA and PMR. In this regard, gene polymorphisms for ICAM-1 (intercellular adhesion molecule 1), RANTES (regulated upon activation, normal T cell expressed, and presumably secreted), and interleukin (IL)-1 receptor antagonist seem to play a role in the pathogenesis of GCA and PMR in some populations. However, additional studies are required to clarify the genetic influence on susceptibility to these conditions.