Genome-Wide Analysis of Genetic Risk Factors for Rheumatic Heart Disease in Aboriginal Australians Provides Support for Pathogenic Molecular Mimicry.

Background: Rheumatic heart disease (RHD) after group A streptococcus (GAS) infections is heritable and prevalent in Indigenous populations. Molecular mimicry between human and GAS proteins triggers proinflammatory cardiac valve-reactive T cells. Methods: Genome-wide genetic analysis was undertaken in 1263 Aboriginal Australians (398 RHD cases; 865 controls). Single-nucleotide polymorphisms were genotyped using Illumina HumanCoreExome BeadChips. Direct typing and imputation was used to fine-map the human leukocyte antigen (HLA) region. Epitope binding affinities were mapped for human cross-reactive GAS proteins, including M5 and M6. Results: The strongest genetic association was intronic to HLA-DQA1 (rs9272622; P = 1.86 × 10-7). Conditional analyses showed rs9272622 and/or DQA1*AA16 account for the HLA signal. HLA-DQA1*0101_DQB1*0503 (odds ratio [OR], 1.44; 95% confidence interval [CI], 1.09-1.90; P = 9.56 × 10-3) and HLA-DQA1*0103_DQB1*0601 (OR, 1.27; 95% CI, 1.07-1.52; P = 7.15 × 10-3) were risk haplotypes; HLA_DQA1*0301-DQB1*0402 (OR 0.30, 95%CI 0.14-0.65, P = 2.36 × 10-3) was protective. Human myosin cross-reactive N-terminal and B repeat epitopes of GAS M5/M6 bind with higher affinity to DQA1/DQB1 alpha/beta dimers for the 2-risk haplotypes than the protective haplotype. Conclusions: Variation at HLA_DQA1-DQB1 is the major genetic risk factor for RHD in Aboriginal Australians studied here. Cross-reactive epitopes bind with higher affinity to alpha/beta dimers formed by risk haplotypes, supporting molecular mimicry as the key mechanism of RHD pathogenesis.

A population-wide applicable HLA-DQ2 and DQ8 genotyping using DNA from dried blood spots and duplex allele-specific qPCR amplification.

Genotyping of HLA-DQ2 and DQ8 haplotypes is important for diagnosis or for screening of early risk detection of celiac disease or type 1 diabetes. Usually, venous blood DNA extraction and expensive and time consuming amplification are used, that hinder population-wide studies. We assayed a friendly HLA-DQ2 and DQ8 genotyping procedure using a combination of DNA from dried blood spot (DBS) and duplex allele-specific qPCR amplification using SYBR Green. DNA was extracted using home-made buffers and compared to an extraction commercial kit. Duplex reactions by qPCR were designed using each Tm allele amplicon for reference samples (positive HLA-DQ2 or DQ8) with allele-specific primers. DBS samples from 558 children (7.99 ± 2.47 y) were collected. The DNA final yield obtained by the home-made extractive procedure was higher than from the commercial kit (1.11 ± 0.56 vs 0.23 ± 0.14 µg), while the quality was similar for both DNA samples. There was concordance in the amplification profiles for DNA samples obtained with both methods. All of four alleles from DQ2 and DQ8 haplotypes were accurately identified in duplex reactions. By using DBS samples and DNA extraction home-made procedure, the costs were reduced by 60%. The whole procedure is cost-effective for HLA-DQ2 and DQ8 genotyping.

Gliadin antibodies in older population and neurological and psychiatric disorders.

OBJECTIVES: A variety of neurological and psychiatric disorders have recently been linked to coeliac disease and gluten sensitivity. We here explored whether persistently positive gliadin antibodies (AGA) and coeliac-type HLA increase the risk of gluten sensitivity-related neurological and psychiatric manifestations. The study was carried out in an older population who had consumed gluten for decades but who had no previous coeliac disease diagnosis. MATERIALS AND METHODS: The original study population comprised 4272 randomly selected older individuals, of whom 2089 had AGA and transglutaminase 2 antibodies (antiTG2) measured twice within a 3-year interval. Forty-nine persistently AGA-positive but antiTG2-negative subjects with coeliac-type HLA and 52 randomly selected persistently AGA- and antiTG2-negative age- and sex-matched controls were clinically examined for neurological disorders. The Psychological General Well-Being (PGWB) questionnaire, the SF-36 health survey questionnaire and the Depression Scale (DEPS) were employed to evaluate psychological well-being. The medical files of all the study subjects were analysed for previous illnesses. RESULTS: Persistently AGA-positive but antiTG2-negative older subjects carrying coeliac disease-type HLA did not evince significantly more neurological symptoms or diseases than AGA-negative control subjects (P = 0.682, P = 0.233). There were no statistically significant differences between AGA-positive and AGA-negative groups in psychological well-being and quality of life when measured by PGWB (P = 0.426), SF-36 questionnaires (P = 0.120) and DEPS (P = 0.683). CONCLUSIONS: At population level, persistent AGA positivity did not indicate gluten sensitivity-related neurological and psychiatric disorders.

Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes.

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.

[Analysis of occurrence of DRB and DQ alleles in sarcoidosis and tuberculosis from Northern Poland]. / Analiza czetosci wystepowania alleli DRB i DQ u chorych na sarkoidoze i gruzlice pluc z terenu pólnocnej Polski.

INTRODUCTION: Sarcoidosis is a multisystem disorder of unknown etiolgy. Pathologic similarities between SA and tuberculosis (TB) suggest M. tuberculosis antigen(s) as causative agents. It seems likely that in the genetically different predisposed hosts, the same antigen(s) may cause the development of sarcoid or tuberculous immune response. AIM: The aim of this study was to compare the frequency of occurrence of HLA class II alleles in SA, TB and in the healthy individuals. MATERIAL AND METHODS: To test a difference in haplotypes associated with both diseases, we compared the distribution of DQA1 and DQB1 alleles in 45 SA patients, 62 TB patients and in 143 healthy volunteers, using a PCR-SSP "low (DRB1, DQB1) and high (DQA1) resolution" method. RESULTS: Our results revealed that DRB1*03, DRB1*11, DQB1*02 i DQA1*0501 in Stage I of SA with Löfgrens syndrom (Ls) and DRB1*15, DQA1*0102, DQA1*0103 in Stage II of SA were more common, whereas DRB1*16, DRB1*04, DRB1*08, DQB1*02, DQB1*03, DQB1*05, DQA1*0102, DQA1*0301 in Ls and DQB1*02, DQB1*03, DQB1*05, DQA1*0102, DQA1*0301 in Stage II were less common than in the controls but after Bonferroni correction occurrence of DRB1*04, DQB1*02, DQB1*03, DQB1*05 and DQA1*0102, DQA1*0301, DQA1*0501 was significantly differ. In TB group, DRB1*16, DRB1*14, DQB1*05 i DQA1*0303 were more frequent and DRB1*11, DQB1*02, DQA1*0201, DQA1*0505 less frequently present as compared to the controls, but after correction DRB1*16, DQB1*02, DQB1*05, DQA1*0303, DQA1*0505 were significantly different. In SA, DRB1*11, DQB1*02 i DQA1*0201, DQA1*0501, DQA1*0505 in Ls and DRB1*15, DRB1*11, DQA1*0102 in Stage II were more common and DRB1*16, DRB1*04, DRB1*14, DQB1*03, DQB1*05, DQB1*06, DQA1*0301, DQA1*0302, DQA1*0303 in Ls and Stage II were less frequent than in the TB group. DQB1*02, DQA1*0501 (Ls) and DRB1*15 (Stage II) were more frequently present in SA than in TB, even after Bonferroni correction. CONCLUSIONS: In summary, we identified associations of HLA class II alleles in SA and TB with expression pattern specific and different for each group. In most cases, in SA patients frequency of HLA class II alleles occurrence is opposite to the frequency in TB patients.

HLA-DRB1*1501, -DQB1*0301, -DQB1*0302, -DQB1*0602, and -DQB1*0603 alleles are associated with more severe disease outcome on MRI in patients with multiple sclerosis.

The most important confirmed genetic factor of susceptibility to multiple sclerosis (MS) has been identified in the HLA class II region. The hypothesis that several genes, including HLA class II, may influence the prognosis of patients with MS has been proposed. In a recent study, using low intermediate resolution typing, we found that some HLA alleles may predict disease severity as assessed by magnetic resonance imaging (MRI) measures. The aim of this study was to examine the relationship between high-resolution typing of HLA alleles and disease severity as measured by brain MRI quantitative markers of demyelinating and destructive pathology in patients with MS. In 41 MS patients (27 relapsing-remitting, 7 secondary progressive, and 7 primary progressive), we performed high-resolution typing of alleles HLA-DRB1*04, -DQB1*03, -DRB1*15, -DQB1*06, and of haplotypes -DRB1*04-DQB1*03 and -DRB1*15-DQB1*06. These alleles and haplotypes were associated with higher susceptibility to MS in a recently published case-control study conducted in the Friuli-Venezia-Giulia region, Italy. Of 41 included patients, 13 were men and 28 were women. Mean age was 43.3 (SD 11.4) years, mean disease duration 10.3 (SD 7.8) years, and mean EDSS 2.3. DNA extraction and genomic typing were obtained with the sequence-specific primers method using primer pairs that amplified the HLA alleles. All patients underwent a 1.5-T MRI examination of the brain. Disease severity was assessed by clinical measures [Expanded Disability Status Scale (EDSS)] and MRI measures. T2- and T1-lesion volumes (LVs) and brain atrophy measures [fractions of brain parenchyma (BPF), gray matter (GMF), and white matter (WMF)] were calculated. We used general linear model analysis (GML), controlled for age, disease duration, and treatment status, to compare the MRI measures according to allele and haplotype status. The following significant results were found: HLA-DRB1*1501 positive patients had significantly lower GMF (0.493 vs 0.526, p < 0.001), lower BPF (0.784 vs 0.815, p = 0.018), and higher T1-LV (2.8 vs 0.7ml, p = 0.036); -DQB1*0301 positive patients had significantly higher T2-LV (34.1 vs 0.7 ml, p = 0.041), and showed a trend for lower BPF (0.790 vs 0.846, p = 0.064); -DQB1*0302 positive patients had significantly lower T1-LV (2.4 vs 0.9 ml, p = 0.016); and -DQB1*0602 positive patients had significantly lower GMF (0.492 vs 0.521, p = 0.007) and lower BPF (0.781 vs 0.811, p = 0.023). No differences were found in the indices of MRI disease severity according to HLA haplotype associations. Both in correlation and in regression analyses, we observed significant associations between HLA-DRB1*1501 and lower GMF and BPF and higher T1-LV, between -DQB1*0301 and higher T2-LV and disease duration, between -DQB1*0302 and lower GMF and higher T1- and T2-LV, between -DQB1*0602 and lower GMF and BPF, and between -DQB1*0603 and higher T1-LV and EDSS. High-resolution HLA genotyping analysis revealed a robust relationship between alleles HLA-DRB1*1501, -DQB1*0301, -DQB1*0302, -DQB1*0602, and -DQB1*0603, and more severe damage on inflammatory and neurodegenerative MRI measures.

Lack of association of HLA class II alleles with peanut allergy.

BACKGROUND: Peanut allergy is a common and severe phenotype of food allergy with a strong genetic component; HLA class II polymorphisms are attractive candidate genes for this disorder. OBJECTIVE: To determine possible genotypic associations of HLA class II with peanut allergy and attempt replication of previously reported associations. METHODS: Sibling pairs discordant for peanut allergy were genotyped (low resolution) by polymerase chain reaction-based methods to 7 DQ and 18 DR allele groups. A chi2 analysis was undertaken against sibling controls with statistical adjustment for multiple analyses. RESULTS: Seventy-three children with confirmed peanut allergy (mean age, 6.5 years; male, 72%; asthma, 58%; atopic dermatitis, 62%; allergic rhinitis, 67%; other food allergies, 41%) and 75 of their siblings who eat peanut (mean age, 8 years; male, 52%; asthma, 12%; atopic dermatitis, 22%; allergic rhinitis, 37%; other food allergy, 7%) were genotyped. Distribution of DQ7 (29% of children with peanut allergy vs 47% sibling controls) was statistically significantly different (P = .04) before statistical correction for multiple comparisons was made by multiplying them by the number of alleles tested (and not statistically significant after correction; P = .30). Distribution of DR11 was nearly statistically significant without statistical adjustment (26% with peanut allergy vs 41% of sibling controls; P = .07; corrected P = 1.3). Alleles that were previously reported to have a weak association with peanut allergy (DRB1 *03, *08; DQB1 *0302, *04) were not verified in this cohort (unadjusted P > .44). CONCLUSIONS: We could not establish an association between the HLA class II alleles evaluated in this cohort of sibling pairs discordant for peanut allergy.

HLA-DQB1 sequencing-based typing using newly identified conserved nucleotide sequences in introns 1 and 2.

Sequencing-based typing (SBT) human leukocyte antigen (HLA) class I and II genes should examine entire exon sequences where polymorphisms lie. Primers for the amplification of complete exons therefore anneal in introns and their design relies on accurate intron sequences being available. We decided to develop a SBT method for HLA-DQB1 using amplification primers which anneal in introns 1 and 2, yet the amount of intron sequence data previously available in databases was sparse. Therefore, we undertook a systematic sequencing of introns 1 and 2 using DNA from cell lines homozygous for DQB1. This study confirmed an earlier report that the non-coding regions of this gene are the most polymorphic seen in the human genome. Intron sequences within an allele group were largely identical, the exceptions being DQB1*0301 differing from other DQB1*03 allele groups and DQB1*0601 differing from all other DQB1*06 alleles. A retroviral Alu element, related to the AluYa5a2 subfamily, was identified uniquely inserted in intron 2 of DQB1*02 alleles. For the typing approach, six amplification primers were designed based on conserved allele group sequences covering all of the HLA DQB antigens, and two sequencing primers were also designed which anneal in intron 2. This method has proved to be very robust and has been used as part of a referral DNA sequencing service for a number of years.

HLA class II involvement in HIV-associated Toxoplasmic encephalitis development.

UNLABELLED: A total of 220 individuals were included in this study, 112 HIV-seronegative healthy individuals and 108 HIV-1-infected patients involving: 18 AIDS patients with Toxoplasmic encephalitis (AIDS-TE), 49 AIDS patients without TE, and 41 asymptomatic patients, were genotyping for DR and DQ loci by molecular biology techniques. Fisher's Exact test was used for statistical analysis. HLA-DQB*0402 and DRB1*08 alleles were associated with a high risk to develop opportunistic infections with neurological involvement, mainly Toxoplasma encephalitis in relationship with subjects healthy (OR = 20.43; Pc = 7.0 x 10(-6) and OR = 11; Pc = 2.6 x 10(-4), respectively); in relationship with AIDS no TE (OR = 6.98; Pc = 0.028 and OR = 4.85; P = 0.012, Pc = 0.14) and with patients in asymptomatic stage (OR = 61.50, Pc = 8.4 x 10(-6) and OR = 19.38; Pc = 3.9 x 10(-4)), respectively. CONCLUSIONS: It was concluded that the presence of HLA-DQB*0402 and DRB1*08 alleles in HIV-1-positive patients could be considered risk factors for developing neurological opportunistic infections, mainly Toxoplasmic encephalitis.

In silico identification of supertypes for class II MHCs.

The development of epitope-based vaccines, which have wide population coverage, is greatly complicated by MHC polymorphism. The grouping of alleles into supertypes, on the basis of common structural and functional features, addresses this problem directly. In the present study we applied a combined bioinformatics approach, based on analysis of both protein sequence and structure, to identify similarities in the peptide binding sites of 2225 human class II MHC molecules, and thus define supertypes and supertype fingerprints. Two chemometric techniques were used: hierarchical clustering using three-dimensional Comparative Similarity Indices Analysis fields and nonhierarchical k-means clustering using sequence-based z-descriptors. An average consensus of 84% was achieved, i.e., 1872 of 2225 class II molecules were classified in the same supertype by both techniques. Twelve class II supertypes were defined: five DRs, three DQs, and four DPs. The HLA class II supertypes and their fingerprints given in parenthesis are DR1 (Trp(9beta)), DR3 (Glu(9beta), Gln(70beta), and Gln/Arg(74beta)), DR4 (Glu(9beta), Gln/Arg(70beta), and Glu/Ala(74beta)), DR5 (Glu(9beta), Asp(70beta)), and DR9 (Lys/Gln(9beta)); DQ1 (Ala/Gly(86beta)), DQ2 (Glu(86beta), Lys(71beta)), and DQ3 (Glu(86beta), Thr/Asp(71beta)); DPw1 (Asp(84beta) and Lys(69beta)), DPw2 (Gly/Val(84beta) and Glu(69beta)), DPw4 (Gly/Val(84beta) and Lys(69beta)), and DPw6 (Asp(84beta) and Glu(69beta)). Apart from the good agreement between known binding motifs and our classification, several new supertypes, and corresponding thematic binding motifs, were also defined.

Association of HLA-DQA1 and DQB1 genes with vitiligo in Chinese Hans.

BACKGROUND: Vitiligo is an acquired depigmentary disorder of the skin and hair which results from selective destruction of melanocytes. Serological typing and genotyping of human leukocyte antigen (HLA) have shown discrepancies in HLA associations with vitiligo in different ethnic populations. METHODS: Polymerase chain reaction sequence-specific primer (PCR-SSP) method was used to analyze the distribution of HLA-DQA(1) and -DQB(1) alleles among 187 patients with vitiligo and 273 healthy controls through Epi Info version 6 package (Centers for Disease Control and Prevention, Atlanta, GA, USA). RESULTS: The frequencies of HLA-DQA1*0302 (OR = 1.98, P(c) < 0.01), -DQB1*0303 (OR = 3.14, P(c) < 0.001), and -DQB1*0503 (OR = 3.36, P(c) < 0.05) alleles were significantly increased in patients with vitiligo compared with controls, and HLA-DQA(1)*0501 (OR = 0.40, P(c) < 0.01) allele frequency was highly decreased. HLA-DQA1*0302 (OR = 5.19, P(c) < 0.001), -DQA1*0601 (OR = 2.95, P(c) < 0.05), -DQB1*0303 (OR = 4.50, P(c) < 0.001), and -DQB1*0503 (OR = 6.69, P(c) < 0.001) alleles were positively associated, whereas HLA-DQA1*0501 (OR = 0.05, P(c) < 0.001) allele was negatively associated with childhood vitiligo patients, and HLA-DQB1*0303 (OR = 2.76, P(c) < 0.001) allele was positively associated with adult vitiligo patients compared with controls. The frequency of HLA-DQB1*0303 (OR = 3.72, P(c) < 0.001) allele was significantly increased in localized vitiligo patients vs. controls, whereas HLA-DQA1*0302 (OR = 2.47, P(c) < 0.01), -DQB1*0303 (OR = 2.67, P(c) < 0.01), and -DQB1*0503 (OR = 4.46, P(c) < 0.01) allele frequencies were significantly increased and -DQA1*0501 (OR = 0.27, P(c) < 0.01) allele frequency was highly decreased in generalized vitiligo patients. CONCLUSIONS: HLA-DQA1*0302, -DQA1*0601, -DQB1*0303, and -DQB1*0503 alleles could be susceptible alleles of vitiligo, while HLA-DQA1*0501 allele could be a protective allele in Chinese Hans. There may be different genetic backgrounds between vitiligo patients of childhood and adult, localized and generalized.

HLA-DQA1 and -DQB1 alleles in Latino and African American children with diabetes mellitus.

Few studies have described the genetics of childhood diabetes mellitus (DM) in US minorities. High-risk DQA1 and DQB1 alleles (DQA1*0301, DQB1*0201, and DQB1*0302 in African Americans and Latinos, and DQA1 *0501 in African Americans) were identified from previous studies and tested in 45 African American and 26 Latino patients from the population-based Chicago Childhood Diabetes Registry, and in 50 healthy race-matched controls. Sixteen of the African American patients and three Latinos had youth-onset type 2 DM and were analyzed separately. In African Americans with type 1 DM, both DQA1*0102 and DQB1*0602 were protective (p < 0.0001), and the susceptibility alleles DQA1*0301 and DQB1*0201 were more frequent than in controls (p < 0.01). In Latinos, DQA1*0301 and DQB1*0302 were marginally increased in patients with DM1 compared to controls; no individual DQA1 or DQB1 allele was protective. Patients with DM1 were significantly more likely to carry one or two high-risk DQA1 alleles in both populations; they were also more likely than controls to carry at least one high-risk DQB1 allele. The odds ratio for the ability to form at least two high-risk DQA1-DQB1 heterodimers (cis and/or trans) was 7.9 (95% CI: 1.7-40.0) for African Americans and 5.7 (1.3-25.6) for Latinos with DM1. African American patients with DM2 were not statistically different from controls, and were less likely to carry four high-risk susceptibility alleles than patients with DM1 (p = 0.002). Many of the HLA-DQ associations previously documented in non-Hispanic White populations also are found in African Americans and Latinos with DM1, although some differences exist.

HLA typing in Taiwanese patients with oral submucous fibrosis.

BACKGROUND: A significant association of certain human leukocyte antigens (HLA) and haplotypic pairs with oral submucous fibrosis (OSF) has been reported. However, controversial result of no HLA association with OSF has also been reported. In this study, the phenotype and haplotype frequencies of HLA-A, -B, -C, -DRB1, and -DQB1 in 135 Taiwanese OSF patients were calculated and compared with those in 540 healthy control Taiwanese. METHODS: The analysis of HLA-A, -B, and -C antigens, and of HLA-DRB1 and -DQB1 alleles in OSF patients and healthy control subjects, was performed by serologic typing and DNA typing using polymerase chain reaction with sequence-specific primers (PCR-SSP), respectively. RESULTS: We found that the phenotype frequency of HLA-B76 (3.0%) in OSF patients was significantly greater than that (0%) in healthy control subjects (corrected P (Pc) = 0.000). In addition, the haplotype frequencies of HLA-B48/Cw7 (3.0%), -B51/Cw7 (6.7%), and -B62/Cw7 (8.2%) in OSF patients were significantly greater than the corresponding haplotype frequencies (0, 0.7, and 1.9%, respectively) in healthy control subjects (Pc = 0.000). The relative risk (RR) values of haplotypes B51/Cw7 (9.57) and B62/Cw7 (4.7) were greater than the RR values of phenotypes B51 (1.81), B62 (2.31), and Cw7 (1.91) in OSF patients. In addition, the etiologic fraction (EF) value of haplotype B51/Cw7 (0.63) was higher than the EF values of phenotypes B51 (0.2) and Cw7 (0.59). CONCLUSIONS: We conclude that some Taiwanese areca quid (AQ) chewers with particular HLA phenotypes and haplotypes are prone to have OSF. In addition, some particular HLA haplotypes may play more important roles than the individual HLA phenotypes for the genetic susceptibility to OSF. However, the significantly increased HLA phenotype B76 and three of the common HLA haplotypes detected are present in only about 20% of incident cases of OSF.

The relationship between nonresponse to hepatitis B vaccine and HLA genotype/haplotype.

OBJECTIVE: To study the relationship between the nonresponse to hepatitis B vaccine and HLA genotype/heplotype in Chinese population and provide the evidence for explaining the genetic mechanism of this nonresponse. METHODS: Our research focused on the relationship between nonresponse to Hepatitis B vaccine and HLA-DRB1, DRB3, DRB4, DRB5 and DQB1 genotype/haplotype in Chinese population, collected from a community in Guangxi Zhuang Autonomous Region. The group specific amplification was employed to characterize 107 individuals' genotype and haplotype of HLA clusters. Different models statistics such as relative risk test, correlation test and linkage disequilibrium analysis were used to analyze the data. RESULTS: The results showed that there is a linkage disequilibrium between nonresponse to Hepatitis B vaccine and HLA haplotype DR4, 1122 (DRB1 * 0401- 22, 1122)-DR53 (DRB4 * 0101101, 0102/3)-DQB4 (DQB1 * 04). CONCLUSION: In Chinese population, nonresponse to hepatitis B vaccine is highly associated with special HLA haplotye.

Susceptibility locus for non-obstructive azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604.

Non-obstructive azoospermia is a male infertility characterized by no or little sperm in semen as a result of a congenital dysfunction in spermatogenesis. Previous studies have reported a higher prevalence of particular human leukocyte antigen (HLA) antigens in non-obstructive azoospermia. As the expression of the RING3 gene located in the HLA class II region was predominant in the testis, mainly around spermatids and pachytene spermatocytes, it is tempting to speculate that RING3 is one of the strong candidate genes responsible for the pathogenesis of the disease. In this study, the genetic polymorphism in the RING3 gene was investigated by the direct sequencing technique. As a result, a total of 14 single nucleotide polymorphisms were identified. Among them, six were localized in the coding region but none of them was accompanied by an amino-acid substitution. No significant difference in the allelic distribution at these 14 polymorphic sites was observed between the patients and healthy controls, suggesting that the susceptible gene for non-obstructive azoospermia is not the RING3 gene. Then, in order to map the susceptibility locus for non-obstructive azoospermia precisely within the HLA region, 11 polymorphic microsatellite markers distributed from the SACM2L gene just outside the HLA class II region (187 kb telomeric of the DPB1 gene) to the OTF3 gene in the HLA class I region were subjected to association analysis in the patients. Statistical analysis of distribution in the allelic frequency at each microsatellite locus demonstrated that the pathogenic gene for non-obstructive azoospermia is located within the HLA-DR/DQ subregion. In fact, DRB1*1302 and DQB1*0604 were found to be strongly associated with non-obstructive azoospermia by polymerase chain reaction-based DNA typing. Further, haplotype analysis suggested that the DQB1*0604 allele may play a decisive role in the pathogenesis of non-obstructive azoospermia.