Serology, restriction fragment length polymorphism, and sequence analysis of a unique HLA class II antigen, DR5x6.

We analyzed a new class II HLA haplotype, which we have designated DR5x6, by serology, restriction fragment length polymorphism (RFLP), and sequence analysis. As the name DR5x6 implies, the antigen is serologically closely related to both DR5 and DRw6. RFLP analysis of this haplotype suggests a close similarity with DRw11 haplotypes. The DNA sequences encoded by the second exon of its DRB1, DRB3, and DQB1 genes were also determined. Comparison of these sequences with those of alleles at these loci in other haplotypes suggests that this haplotype could have evolved from a DRw11 ancestor haplotype (DRw11-DRw52b (Dw25)-DQw7) by means of: (a) a gene conversion at the DRB1 locus involving DRw8 (Dw8.3) as the sequence donor, plus a point mutation or a gene conversion involving DR4-Dw4; and (b) a recombination event by which this haplotype would have acquired the DRw5a (Dw24) allele at the DRB3 locus.

DR6 in Koreans. DR11 frequently acts as a recipient gene to create DR13 alleles.

DR6 is a complex allele family composed of at least 16 different alleles. Although 25% of Koreans express DR6 alleles, this allele family has not been well studied in the population. DNA samples obtained from 252 unrelated individuals were screened by PCR using Taq DNA polymerase and a DRB1 group-specific PCR primer set that amplifies the polymorphic second exon of DR3, DR11, and DR6 DRB1 alleles. To identify the DR6 allelic frequencies in this population, PCR-positive samples were further analyzed by dot-blot hybridization using digoxigenin-labeled SSOPs. In this process, a new DRB1 allele was identified by its unique hybridization pattern and was further characterized by direct sequencing after PCR. The new DRB1 sequence is similar to DRB1*1101, differing at codon 47 (TAC[Tyr]/TTC[Phe]) and at codon 58 (GCC[Ala]/GAG[Glu]). Based on sequence comparisons as well as its DRB3 and DQ associations, the new allele may have arisen by a gene conversion event from DRB1*1101. The resultant DR molecule bears DR6 serologic determinants as determined by serologic typing and, based on sequence, is probably a DR13 and not a DR14 allele. These data suggest that the DR11 allele has frequently acted as a recipient gene in the gene conversion events that created the subfamily of DR13 alleles, DRB1*1303, *1304, *1305, and the new allele described here.

Chronic active autoimmune hepatitis in children. Strong association with a particular HLA-DR6 (DRB1*1301) haplotype.

The association of HLA antigens and type I or "lupoid" CAH-C was investigated in a population of 52 Argentinian Caucasoid patients. When compared with a population of normal individuals of the same ethnic group (n = 197), a significant increase of HLA-DR6 was observed (68.6% in patients vs 17.3% in controls; RR = 12.3, chi 2 = 52.4, pc = 0.00001). DNA typing showed that the HLA-DRB1*1301 allele was present in 32 out of 33 HLA-DR6 patients (66.6% of all the C-CAH patients vs 10.5% in controls; RR = 16.2, chi 2 = 111.3, pc = 0.00001). Analysis of HLA-DQB1 alleles also showed a significant increase of DQB1*0603 (RR = 15.4, chi 2 = 106.5, pc = 0.00001), an allele found in strong linkage disequilibrium with DRB1*1301. The association of CAH-C with this particular HLA-DR6 haplotype has not been previously described for the adult onset CAH. This different HLA predisposition, together with the fact that extrahepatic autoimmune diseases occur frequently only in the adult form of the disease, suggest that the immunopathogenic mechanisms involved in the development of these diseases may be different.

The complexity of DRw6 and DR5 haplotypes in American blacks demonstrated by serology, cellular typing, and restriction fragment length polymorphism analysis.

This study describes the diversity of DRw6 and DR5 haplotypes in the American black population using serology, cellular typing, and restriction fragment length polymorphism (RFLP) analysis. DRw6 (DRw13 and DRw14) and DR5 (DRw11 and DRw12) haplotypes are observed at a high frequency in this population (DRw6: 32%, DR5: 30%). Many of these haplotypes express undefined HLA-D specificities and unusual DQ and DRw52 associations which previously have not been well characterized or reported (e.g., DRw13, DQw5, DRw52c, D-; DRw13, DQw2, DRw52a, D-; DRw11, DQw5, DRw52c, D-). Serologic analysis of class II alleles in American blacks suggests the presence of DRw13, DRw11 and DQw6 allelic variants and demonstrates the difficulty in defining DRw6 and DR5 in this population. The class II genes from four American black families expressing many of the novel DRw13, DRw14, DRw11, and DRw12 haplotypes defined by serology and mixed leukocyte culture were further characterized by RFLP analysis. The data presented here along with other published data identify at least eight DRw13 haplotypes (DRw13A-DRw13H) in the human population. Five of these haplotypes exhibit an undefined HLA-D specificity. Three DRw14 haplotypes (DRw14A-DRw14C) and eight DR5 haplotypes (DRw11A-DRw11E and DRw12A-DRw12C) were also identified. The novel DRw6 and DR5 haplotypes observed in American blacks may arise from differences in DRB1, DQA1, and DQB1 genes as well as from differences in the combinations of alleles of these genes encoded by a haplotype. The serologic and RFLP analyses suggest that some DRw13 and DRw11 haplotypes represent transitional steps between DRw13 and DRw11 in the evolutionary pathway which generated the DRw52 family.

Irregular polymerase chain reaction-sequence-specific oligonucleotide hybridization patterns reveal seven new HLA-DRB1 alleles related to DR2, DR3, DR6, DR8, and DR11. Implications for sequence-specific priming.

In the past 3 years we have typed over 7000 individuals for HLA-DRB using a nonradioactive PCR-SSO method. The use of locally developed computer programs simplified data input and the interpretation of the DRB PCR-SSO readings. In this way we detected a number of samples with unexpected hybridization patterns. DRB1 exon 2 segments of these samples were amplified, cloned, and sequenced and appeared to identify seven new DRB alleles: DRB1*0304, a DRB1*0301 variant, was observed in three unrelated Caucasoid individuals; DRB1*1606, which is very similar to *1603; DRB1*1113 is a *1101 variant with some *1401 sequences; DRB1*1310 is *1301-like; DRB1*1311 is similar to *1305 and *1307; DRB1*1416 is a *1401 sequence with a HV3 derived from *1301; DRB1*0808 was found in an Ethiopian individual. Next, we studied the effectiveness of PCR-SSP typing of the newly defined DRB1 alleles. Only two variants were distinguished as odd by PCR-SSP and two were typed as regular specificities. Three alleles were not amplified by the primer sets used. As compared to PCR-SSO, the PCR-SSP typing method using currently available typing kits clearly has limitations as far as the recognition of new and variant alleles is concerned. The products of some of these new alleles may be distinguished using conventional serology.

Towards covering immunological genes with highly informative markers: a trans-species approach.

To establish a highly informative screening system for immunologically relevant genes ("immunoprinting") we co-amplified via polymerase chain reaction (PCR) polymorphic exons plus adjacent intronic simple repetitive dinucleotide stretches in the T-cell receptor (Tcr) Vb6 and Major Histocompatibility Complex (MHC)-DRB loci in man and several ungulate species. In both gene families the basic structure of the simple repeat was found to be preserved for more than 70 x 10(6) years in all investigated species. The simple repeats exhibit extensive length variability. Distinct exon sequences are correlated with a defined repeat length and substructure. In addition, PCR and the oligonucleotides for typing were applicable to a broad range of species from different mammalian orders. Multiplex PCR of different members of the Tcr Vb6 family and MHC-DRB resulted in a complex pattern similar to an oligolocus fingerprint. Hence immunoprinting can be employed for searching for associations of immunologically relevant genes with diseases even across species barriers.

Vitiligo is associated with HLA-DR4 in black patients. A preliminary report.

We have determined the HLA-DR and HLA-DQ phenotypes of 24 black patients with vitiligo and compared these with phenotypes of 143 local black controls. HLA-DR4 was significantly increased in patients, 38% vs 11% for controls. HLA-DQw3 was also increased in patients, 58% vs 32% for controls and may be explained in part by linkage disequilibrium with HLA-DR4. When patients were subgrouped according to family history of autoimmune disease and compared with controls, the increase in HLA-DR4 and HLA-DQw3 segregated with a positive family history. HLA-DRw6 in patients with a negative family history of autoimmune disease (64%) was significantly greater than the 10% in patients with a positive family history. When patients were subgrouped according to age at onset of disease, HLA-DR4 was increased in those with early onset of disease (younger than 20 years) while HLA-DRw6 was greater in patients who were older at onset of disease. These findings support the hypothesis of an immunogenetic influence on the expression of vitiligo in black patients with vitiligo.

[Study of HLA-II antigens in chronic hepatitis C and B and in acute hepatitis B]. / Estudio de los antígenos HLA-II en las hepatitis crónicas C y B y en las hepatitis agudas B.

The aim of this study was to analyze the variability of the HLA-II system in a series of patients with chronic hepatitis B, chronic hepatitis C and acute hepatitis B to know whether there is any relationship between the polymorphism of the HLA system, the different types of hepatitis and the evolution of the infection. HLA-II antigens were determined by a PCR technique in serum samples of 24 controls, 22 cases of chronic hepatitis C, 38 cases of chronic hepatitis B and 11 with acute hepatitis B. The prevalence of the HLA-DR4 antigen was lower in the cases of chronic hepatitis B (10.5%) and C (13.6%) than in the controls (33.3%), particularly the DRB1*0401 allele (p = NS). The prevalence of HLA-DR6 was similar in chronic hepatitis B (42.1%) and acute hepatitis B (45.5%). Predominance of the DRB1*1301 and DRB1*1302 alleles were, however, observed in acute hepatitis B (36.4%) versus chronic hepatitis B (13%). These data suggest that immunologic factors such as HLA antigens may influence in the susceptibility to infection by HBV and HCV. The use of PCR techniques which discriminate between the different alleles of the HLA antigens may provide better knowledge of the immune response.

[Relationship between human leukocyte antigen-DRB1 allele gene polymorphism and intrahepatic cholestasis of pregnancy].

OBJECTIVE: To study the relationship between human leukocyte antigen-DRB1 (HLA-DRB1) allele genes polymorphism and intrahepatic cholestasis of pregnancy (ICP). METHODS: Forty-two patients with ICP were tested for HLA-DRB1 allele genes polymorphism with the polymerase chain reaction technique and sequence specific oligonucleotide (PCR-SSO) probes hybridization, 56 normal pregnant women as control group were also tested. In addition, the phenotype frequencies of HLA-DRB1 alleles were compared with it's clinical character in patients with ICP. RESULTS: The higher frequencies were observed for alleles DR9, DR12 and DR4 in both groups. DR6 alleles were detected in 14 cases out of 42 patients. Patients with ICP had a significantly higher frequency of the allele DR6 when compared to control group (16.7% vs 3.6%), with a relative risk (RR) as 6.5 (P < 0.01). No significant differences were observed between the frequencies of other detected HLA-DRB1 alleles in both groups. There was no association between HLA-DR6 allele and the level of liver function and cholylglycine in ICP. CONCLUSION: The study showed that HLA-DR6 gene might be one of the susceptibility genes to ICP.

Human leukocyte antigens and systemic lupus erythematosus: a protective role for the HLA-DR6 alleles DRB1*13:02 and *14:03.

Many studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic lupus erythematosus (SLE) have been performed. However, few protective associations with HLA-DRB1 alleles have been reported. Here, we sought protective, as well as predispositional, alleles of HLA-DRB1 in Japanese SLE patients. An association study was conducted for HLA-DRB1 in Japanese SLE patients. Relative predispositional effects were analyzed by sequential elimination of carriers of each allele with the strongest association. We also explored the association of DRB1 alleles with SLE phenotypes including the presence of autoantibody and clinical manifestations. Significantly different carrier frequencies of certain DRB1 alleles were found to be associated with SLE as follows: increased DRB1*15:01 (P = 5.48×10⁻¹°, corrected P (Pc) = 1.59×10⁻8, odds ratio [OR] 2.17, 95% confidence interval [CI] 1.69-2.79), decreased DRB1*13:02 (P = 7.17×10⁻5, Pc = 0.0020, OR 0.46, 95% CI 0.34-0.63) and decreased DRB1*14:03 (P = 0.0010, Pc = 0.0272, OR 0.34, 95% CI 0.18-0.63). Additionally, the "*15:01/*13:02 or *14:03" genotype tended to be negatively associated with SLE (P = 0.4209, OR 0.66), despite there being significant positive associations with *15:01 when present together with alleles other than *13:02 or *14:03 (P = 1.79×10⁻¹¹, OR 2.39, 95% CI 1.84-3.10). This protective effect of *13:02 and *14:03 was also confirmed in SLE patients with different clinical phenotypes. To the best of our knowledge, this is the first report of a protective association between the carrier frequencies of HLA-DRB1*13:02 and *14:03 and SLE in the Japanese population.

HLA-DR typing in polyarticular juvenile idiopathic arthritis: a study from a tertiary care hospital in northern India.

INTRODUCTION: Many studies of human leukocyte antigen (HLA) association with juvenile idiopathic arthritis (JIA) have reported conflicting results, which were probably related to ethnic differences. Moreover, in India, studies on HLA-DR typing on JIA, particularly polyarticular JIA, is lacking. OBJECTIVE: The aim of our study was to reveal the frequency of HLA DR types in a cohort of polyarticular JIA in northern India. METHODS: Fifty-two polyarticular JIA patients were included as per the recent International League of Associations for Rheumatology classification, 2001. HLA-DR typing was performed in 21 patients (18 rheumatoid factor [RF]+ and three RF-) by a DNA-based polymerase chain reaction method for the determination of HLA alleles using sequence specific primers (SSP). The results were compared with that of 23 healthy controls of the same age and sex. RESULTS: HLA-DR4 was present in five cases (23%) in the diseased group while only in one case (4.3%) in the control group with a relative risk of 5.47, but when compared with only RF+ polyarticular JIA, HLA-DR4 was found to be significantly high (27.7% vs. 4.43%; P < 0.05) with a relative risk of 6.3. Further, DR4, DR1, DR2, DR9, DR10 were also non-significantly high in these patients with relative risk of 3.2 for DR9 and 1.8 for DR10. In contrast, HLA-DR6 was seen only in 5.5% of polyarticular JIA cases, whereas it was present in 39% of controls (P < 0.05), a showing negative association. CONCLUSION: HLA-DR4 codes for susceptibility to RF+ polyarticular JIA with a six-fold risk, whereas HLA-DR6 offers protection.

HLA-DR6 association confers increased resistance to T. rubrum onychomycosis in Mexican Mestizos.

BACKGROUND: Onychomycosis is multifactorial in origin. Studies have suggested an autosomal dominant pattern of inheritance and human leukocyte antigen DR4 (HLA-DR4) has been shown to protect against onychomycosis in an Ashkenazi Jewish population. AIM: This study investigates HLA class II association in a Mexican Mestizo population with Trichophyton rubrum onychomycosis. METHODS: This was a prospective case-control study. Mexican Mestizos with a clinical diagnosis of onychomycosis and culture positive for T. rubrum were recruited, together with age- and sex-matched controls. First-degree relatives were also investigated for onychomycosis. Case-control samples were HLA typed by polymerase chain reaction sequence-specific primer based analysis. RESULTS: Twenty-one cases and 42 controls were recruited with a mean age of 40 years (range: 18-58 years). HLA-DR6 was found in seven (33%) cases and 19 (45%) controls [P < 0.023, odds ratio (OR) = 0.088, 95% confidence interval (CI): 0.01-0.71]. Six (29%) cases and three (7%) controls had an affected child (P < 0.043, OR = 9.15, 95% CI: 1.07-78.31), and 13 (62%) cases and 12 (29%) controls had an affected first-degree relative (P < 0.02, OR = 4.0, 95% CI: 1.1-14.3). CONCLUSIONS: These results suggest that HLA-DR6 confers protection against the development of onychomycosis in a Mexican Mestizo population. Having an affected first-degree relative significantly increases the risk of onychomycosis, suggesting genetic susceptibility.

Prognostic factors for survival in scleroderma associated pulmonary arterial hypertension.

OBJECTIVE: Identification of prognostic factors for survival in systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH) is necessary for appropriate monitoring, interventions, and timely referral for lung transplantation. Our objectives were (1) to identify factors associated with survival in SSc-PAH and (2) to evaluate the methodologic quality of prognostic studies against current standards. METHODS: A systematic review was performed to identify studies evaluating factors associated with survival in SSc-PAH. The methodologic quality of each study was evaluated using a methodologic quality index. RESULTS: HLA-DRw6 (RR 54.52, p = 0.01), HLA-DRw52 (RR not reported, p = 0.02), initial systolic pulmonary artery pressure (sPAP) > 60 mmHg (HR 3.60, 95% CI 1.42, 9.15), elevated mean right atrial pressure (mRAP) (HR 20.7, p = 0.0001), and shorter time between SSc onset and observed PAH (5.24 vs 9.93 yrs, p < 0.01) were associated with decreased survival. Age > 50 years (HR 2.34, 95% CI 0.54, 10.2), male sex (HR 2.02, 95% CI 0.65, 6.20), limited subtype (HR 2.37, 95% CI 0.68, 8.20), pulmonary fibrosis [Kaplan-Meier (KM) curves, p = 0.3], change in pulmonary vascular resistance (KM curves, p = 0.8), anti-centromere (HR 1.67, 95% CI 0.66, 4.26) and anti-ScL-70 (HR 0.28, 95% CI 0.03, 1.99) antibodies were not definitively associated with survival. Attributes of participants, prognostic factors, and outcome measures were well reported. Study attrition, confounding, and analysis were not well reported. CONCLUSION: HLA-DRw52 and -DRw6, initial sPAP > 60 mmHg, mRAP, and shorter time between SSc onset and observed PAH were associated with decreased survival; however, methodologic quality of study reporting was variable. Prognostic factor research is needed using current methodologic standards.

Primary sclerosing cholangitis is associated with extended HLA-DR3 and HLA-DR6 haplotypes.

Primary sclerosing cholangitis (PSC) is associated with the human leukocyte antigen (HLA)-DRB1*0301-DQA1*0501-DQB1*0201 (DR3) and HLA-DRB1*1301-DQA1*0103-DQB1*0603 (DR6) haplotypes. Recently, the extended HLA class I region has been found to harbour genes that modulate or confer susceptibility independently of the HLA class II genes in several immune-mediated diseases. The aim of the present study was to evaluate the influence of genes in the extended HLA class I region on susceptibility to PSC. Seven microsatellite markers (MIB, D6S265, D6S2222, D6S464, D6S2223, D6S2225 and D6S2239) were analysed together with HLA class II alleles in 219 Norwegian patients with PSC and 282 random controls. To control for associations because of linkage disequilibrium (LD), 142 HLA-DR3 homozygous and 187 DR6-positive controls were included. The unstratified analysis showed significant associations with the alleles MIB*349 [odds ratio (OR) = 3.0, corrected P value (P(c)) = 3 x 10(-12)], D6S265*122 (OR = 1.7, P(c)= 0.004), D6S464*209 (OR = 1.8, P(c)= 0.03) and D6S2225*147 (OR = 2.7, P(c)= 4 x 10(-6)), which were mainly secondary to the DR3 association. When stratifying for DR6, an association with the D6S265*122 allele was still observed (OR = 3.7, P(c)= 0.0004). In the presence of the D6S265*122 allele, the risk to develop PSC conferred by DR6 was increased four times compared with the risk conferred by DR6 alone. In addition, a novel negative association of PSC with DR11 was observed (OR = 0.21, P(c)= 2 x 10(-4)). In conclusion, our study shows that a gene in LD with D6S265 contributes to susceptibility to develop PSC in individuals carrying DR6. Moreover, we found that the PSC-associated DR3 haplotype extends more telomeric than that previously reported. We also report a possible protective effect of DR11 on development of PSC.

Particular genetic variants of ligands for natural killer cell receptors may contribute to the HLA associated risk of primary sclerosing cholangitis.

BACKGROUND/AIMS: Combinations of killer immunoglobulin-like receptors (KIRs) and HLA class I ligands that reduce natural killer (NK) cell inhibition have been shown to increase risk for autoimmune diseases. We aimed to clarify to what extent such combinations influence susceptibility to primary sclerosing cholangitis (PSC). METHODS: Three hundred and sixty-five Scandinavian PSC patients and 368 healthy controls were genotyped for the presence or absence of genes encoding all KIRs using a PCR-SSP approach. KIR binding site variation of HLA-A, -B and -C was also determined. RESULTS: The KIR gene frequencies were similar among patients and controls. However, the frequency of HLA-Bw4 and -C2, which are ligands for the inhibitory KIRs 3DL1 and 2DL1, respectively, was significantly reduced in PSC patients as compared with controls (38.2% vs. 54.7%, P(corrected)[P(c)]=0.0006 and 42.7% vs. 56.9%, P(c)=0.009, respectively). Two HLA risk haplotypes in PSC (carrying DRB1*0301 or DRB1*1501, respectively) were devoid of both of these alleles, and carried the 5.1 variant of the major histocompatibility complex class I chain-related A (MICA) gene previously reported to influence PSC susceptibility. CONCLUSIONS: Particular variants of ligands for NK cell receptors encoded at three neighbouring genes in the HLA complex may contribute to PSC associations observed in this genetic region.

Hepatitis C virus-associated oral lichen planus: is the geographical heterogeneity related to HLA-DR6?

BACKGROUND: The association between hepatitis C virus (HCV) and oral lichen planus (OLP) is more common in the Mediterranean area and Japan, possibly because of immunogenetic factors. METHODS: Intermediate-resolution HLA-DRB typing by hybridization with oligonucleotide probes was performed in 31 Italian OLP patients with HCV infection, in 45 Italian OLP and in 48 British OLP patients without HCV infection. As healthy controls we included data from 145 unrelated Italian and 101 unrelated British bone marrow donors. RESULTS: Italian HCV+ve OLP patients possessed the HLA-DR6 allele more frequently than Italian and British OLP patients without HCV infection (51.6% vs. 17.7% vs. 16.7%; P corrected = 0.028 and 0.017, respectively). There was no difference in the frequency of the HLA-DR6 allele between Italian and British control subjects. CONCLUSIONS: The present data suggest that HLA-DR6 may be responsible for the peculiar geographic heterogeneity of the association between HCV and OLP.

Six new DR52-associated DRB1 alleles, three of DR8, two of DR11, and one of DR6, reflect a variety of mechanisms which generate polymorphism in the MHC.

We have sequenced DNA from six new DR52-associated DRB1 alleles initially detected by PCR/SSOP analysis. Three DR8 associated alleles differed from previously known alleles by single nucleotide substitutions. DRB1*0807 and DRB1*0811 both vary from DRB1*08021 at codon 57 resulting in two different amino acids at this residue. DRB1*0807 was identified in samples of Brazilian origin while *0811 was identified among samples from the Tlingit Native American population of Southeast Alaska. DRB1*0814, identified in a family of Chinese origin, differed from DRB1*08032 at codon 12 at both the nucleotide and the amino acid level. In addition, two alleles of DR11, DRB1*1113 and *1119, were each detected in Caucasian individuals. DRB1*1113 differs from other DR11 alleles at codons 37, 67, 70 and 74, while DRB1*1119 differs from *1101 by a single nucleotide substitution at codon 67. Finally, DRB1*1418 was detected in a sample from an Asian or Pacific Islander and shares sequences with several other DR52-associated DRB1 alleles. These six DRB1 alleles appear to have been generated by either gene conversion events, DRB1*1113 and *1418, or by point mutations, DRB1*0814, *0807, *0811 and *1119, although the single nucleotide substitutions found in the latter three alleles are also present in at least one other DRB1 allele and, therefore, could have been the product of gene conversions.