Group-specific amplification of cDNA from DRB1 genes. Complete coding sequences of partially defined alleles and identification of the new alleles DRB1*040602, DRB1*111102, DRB1*080103, and DRB1*0113

We present here the complete coding sequences, previously unavailable, of the DRB1 alleles DRB1*030102, *0306, *040701, *0408, *1327, *1356, *1411, *1446, *1503, *1504, *0806, *0813, and *0818. For cDNA isolation, new group-specific primers located at the 5′UT and 3′UT regions were used to carry out allele-specific amplification and a convenient method for determining full-length sequences for DRB1 alleles. Complete coding sequencing of samples previously typed as DRB1*0406, DRB1*080101, and DRB1*1111 revealed new alleles with noncoding nucleotide changes at exons 1 and 3. In addition, we found a novel allele, DRB1*0113, whose second exon carries a sequence motif characteristic of DRB1*07 alleles. The predicted class II haplotypic associations of all alleles are reported and discussed.     

Identification of the DRB1*1313 allele in a sibling pair

Sequence-specific oligonucleotide probe hybridization (PCR/SSOP), which is now routinely used in HLA laboratories to type for alleles of the class II loci, can also detect the presence of previously unknown alleles. In the course of clinical typing for DRB1, we identified two samples with probe hybridization patterns appropriate for DRB1*1313, a previously submitted sequence that had been withdrawn. Direct sequencing showed that the two individuals carried an allele with an exon 2 sequence identical to that previously submitted for DRB1*1313.     

Identification by sequencing based typing and complete coding region analysis of three new HLA class II alleles: DRB3*0210, DRB3*0211 and DQB1*0310

The study of HLA class II polymorphism by direct exon 2 DNA sequencing analysis has been established to be a reliable and accurate high-resolution typing procedure. This approach shows some advantages in relation to previous methods, polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and sequence-specific primers (PCR-SSP), basically due to the capability of analysis for the complete sequenced genomic region, including non-polymorphic motifs. DRB3 and DQB1 sequencing based typing (SBT) in unrelated bone marrow donor searching allowed us to detect three new alleles. The complete coding region sequences were characterised from cDNA. Two new DRB3 alleles, DRB3*0210 and DRB3*0211, were described in two Caucasian bone marrow donors. Both sequences showed single point mutations regarding DRB3*0202, producing amino acid replacements at positions 51 (Asp to Thr) and 67 (Leu to Ile), respectively. These two point mutations can be found in other DRB alleles, and suggest that gene conversion would be involved in the origin of both alleles. A new DQB1 sequence was found in a Spanish patient that showed two nucleotide differences, positions 134 and 141, with regard to its close similar DQB1*03011 allele. Only substitution at position 134 provoked amino acid replacement at residue 45, Glu to Gly. This single amino acid change would be involved in the lack of serologic recognition of this new molecule by DQ7-specific reagents.     

Presence of the DRB4*0103102N null allele in different DRB1*04-positive individuals

The DRB4 gene encoding the DR53 antigen is present in DRB1*04-, DRB1*07- and DRB1*09-positive individuals. Eight allelic variants of DRB4 have been recognized, 5 resulting in an expressed DR53 antigen and 3 belonging to the null alleles. So far the DRB4*0103102N null allele had been found exclusively in individuals carrying the haplotype DR7,-DQ9. High-resolution typing of HLA class II by polymerase chain reaction using sequence-specific primers (PCR-SSP) and/or sequence-based typing of kidney patients and their families revealed the presence of the DRB4*0103102N null allele segregating with DRB1*04 and DQB1*03 in 4 different families. Three different haplotypes on which the null allele was located, were recognized by family studies: DRB1*0401, DQB1*0301; DRB1*0402, DQB1*0302 and DRB1*0404, DQB1*0302. Determination of the DR53 specificity of antisera reacting with DR53-positive individuals has always been difficult due to the simultaneous presence of DR4, 7 or 9. Identification of DR4-positive DR53-negative individuals as described here, provided the serological reactions with DR53-antisera and revealed the antibody specificities in the antisera used.     

HLA-DRB1~* 1501与我国北方汉族儿童结核病的易感性研究

为了研究中国北方儿童结核病与HLAⅠ类基因的关联,我们采用PCR-SSO方法检测了97例北方汉族结核患儿和91例正常对照的HLA-DRBl,DQAl,DQBl等位基因.发现中国北方汉族儿童结核病与HLA-DRBl·1501有显著关联.进一步比较DR分子结构发现β链第86位氨基酸对结核病的易感性可能有重要意义.     

Detection of four novel alleles: DRB1*1130, DRB1*13072, DRB1*1315 and DRB1*1331

Four new DR52-associated DRB1 alleles are described. One allele, DRB1*1130, is a hybrid between a DRB3*02 allele and a DRB1*11011 allele. The other alleles, DRB1*13072, DRB1*1315, and DRB1*1331, are simple reshufflings of known polymorphic motifs.     

Two DR2-associated novel alleles arose from the silent mutation of codon 72: DRB1*16012, DRB5*01012

Two DRB1*02-associated alleles, DRB1*16012 and DRB5*01012, are described. Both alleles carry the same silent substitution at codon 72.     

T细胞识别HLA-DRB1*0901分子显示TCR BV基因取用的高度局限性

目的分析ＤＲ９纯合细胞刺激下ＤＲ９阴性正常人ＴＣＲＢＶ基因片段的取用格局,确定和分离出ＤＲ９关联性疾病中的自身反应性Ｔ细胞克隆。方法常规方法获取ＰＢＭＣ经ＰＣＲ－ＳＳＯ和ＰＣＲ－ＲＦＬＰＤＮＡ分型,确定ＤＲ、ＤＱ、ＤＰ等位基因后,进行单向混合淋巴细胞培养,抽提总ＲＮＡ并合成第一链ｃＤＮＡ,定量ＰＣＲ检测２２种ＴＣＲＢＶ基因片段的取用格局。结果Ｔ细胞对ＤＲ９分子的识别和扩增具有寡克隆性,２例都有ＢＶ７和ＢＶ１６片段的优势取用,而片段ＢＶ１９和ＢＶ１０则在２例中分别表达。结论实验结果表明,共有ＢＶ７和ＢＶ１６片段的优势取用是ＤＲ９抗原等分子经直接途径激活了带有相应ＴＣＲＢＶ细胞的结果;而片段ＢＶ１９和ＢＶ１０在２例中分别表达可能是不同ＨＬＡ背景的ＡＰＣ间接递呈ＤＲ９抗原的结果。     

Extensive HLA class II studies in 58 non-DRB1*15 (DR2) narcoleptic patients with cataplexy

Narcolepsy is a sleep disorder that has been shown to be tightly associated with HLA DR15 (DR2). In this study, 58 non-DR15 patients with narco-lepsy-cataplexy were typed at the HLA DRB1, DQA1 and DQB1 loci. Subjects included both sporadic cases and narcoleptic probands from multiplex families. Additional markers studied in the class II region were the promoters of the DQA1 and DQB1 genes, two CA repeat polymorphisms (DQCAR and DQCARII) located between the DQA1 and DQB1 genes, three CA repeat markers (G51152, T16CAR and G411624R) located between DQB1 and DQB3 and polymorphisms at the DQB2 locus. Twenty-one (36%) of these 58 non-DR15 narcoleptic patients were DQA1*0T02 and DQBI*0602, a DQ1 Subtype normally associated with DRB1*15 in DR2-positive narcoleptic subjects. Additional microsatellite and DQA1 promoter diversity was found in some of these non-DR15 but DQB1*0602-positive haplotypes but the known allele specific codons of DQA1*0102 and DQB1*0602 were maintained in all 21 cases. The 37 non-DQA1*0102/DQB1*0602 subjects did not share any particular HLA DR or DQ alleles. We conclude that HLA DQA1*0102 and DQB1*0602 are the most likely primary candidate susceptibility genes for narcolepsy in the HLA class II region.     

抗磷脂抗体阳性SLE患者HLA DRB1、DQA1、DQB1基因单倍型研究

本文采用ＰＣＲ ＲＦＬＰ技术对抗磷脂抗体阳性（ＡＰＡ＋）ＳＬＥ患者ＨＬＡ ＤＲＢ１、ＤＱＡ１ 和ＤＱＢ１ 基因进行分型研究, 同时以上海地区汉族随机人群作对照, 发现这类病人的ＤＲＢ１＊ ０８０３ ＤＱＡ１＊ ０１０３ ＤＱＢ１ ＊０６０１ 单倍型频率显著增高（ Ｐ＜ ０－０１） 相对危险率为４－４５, 说明该疾病与此单倍型存在着很强的关联。     

HLA DRB1*DQB1* haplotype in HTLV-I-associated familial infective dermatitis may predict development of HTLV-I-associated myelopathy/tropical spastic paraparesis

A possible causal association between infective dermatitis and HTLV-I infection was reported in 1990 and confirmed in 1992. We now report familial infective dermatitis (ID) occurring in a 26-year-old mother and her 9-year-old son. The mother was first diagnosed with ID in 1969 at the age of 2 years in the Dermatology Unit at the University Hospital of the West Indies (U.H.W.I.) in Jamaica. The elder of her 2 sons was diagnosed with ID at the age of 3 years, also at U.H.W.I. Both mother and son are HTLV-I-seropositive. A second, younger son, currently age 2 years, is also HTLV-I-seropositive, but without clinical evidence of ID. Major histocompatibility complex (MHC), class II, human leucocyte antigen (HLA) genotyping documented a shared class II haplotype, DRB1*DQB1* (1101-0301), in the mother and her 2 sons. This same haplotype has been described among Japanese patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and has been associated with a possible pathologically heightened immune response to HTLV-I infection. The presence of this haplotype in these familial ID cases with clinical signs of HAM/TSP may have contributed to their risk for development of HAM/TSP. The unaffected, HTLV-I-seropositive, younger son requires close clinical follow-up. © 1995 Wiley-Liss, Inc.     

Sequencing of a novel HLA-DRB1*07 allele with a new polymorphism at amino acid position 21 (DRB1*0715).

DRB1*0715 differs from DRB1*070101 in a point nucleotide mutation at codon 21 (ACG→AAG), which produces an amino acid residue change from T to K.     

The HLA DQB 1*0502 allele is neutrally associated with insulin-dependent diabetes mellitus in the Sardinian population

In the Sardinian population a very high incidence of insulin-dependent diabetes mellitus (IDDM) and the lack of HLA-DR2 protective effect due to the high frequency of the A2, Cw7, B17, 3F31, DR2, DQw1 extended haplotype has been reported. This haplotype, carrying a Serine at position 57 of the DQB1*0502 allele, has been previously reported to be underrepresented in patients when compared to controls. In order to provide an explanation for this finding, we defined by RFLP analysis the HLA haplotype of 45 Sardinian IDDM patients and 49 controls. All DR-2DQw1 subjects were molecularly characterized at the HLA DQA and DQB loci. All DR2-positive patients and the vast majority of the DR2-positive controls had the DQB1*0502 allele at the DR2-linked DQB1 locus, with no statistically significant difference between the two groups. All DQA1 genes were the ones expected, with only two exceptions. Nine out of 10 of the DR2-positive patients were compound heterozygotes for DQB1*0201/DQB1*0502 alleles; only this allele combination was significantly increased (p less than 0.0003). Our data suggests that a) the DQB1*0502 allele is neutral for IDDM development and b) the susceptibility to IDDM in our DR2-positive patients is related to the compound heterozygous state between the neutral DQA1*0102/DQB1*0502 and the susceptibility DQA1*0501/DQB1*0201 alleles.     

Unusual association of the DRB4 null allele, DRB4*0103102N, with HLA DRB1*0402 in a sample of Austrian patients

Tissue typing for HLA class II antigens is routinely performed by serological, and/or DNA-based methods. Accuracy, absence of cross-reactivity and controllable level of resolution are striking advantages of molecular methods. However, a disadvantage of molecular typing, compared to serological methods, is the identification of unexpressed alleles. Whereas serology allows a more or less direct insight into antigen presence, molecular biology is an indirect method and results must also be interpreted by considering the biological pathways of protein expression. We believe that identification of nonexpressed MHC alleles is of importance for transplantation, since nonexpressed MHC allele positive individuals could give rise to antibody formation against the respective expressed MHC product in donor tissue. Usually, the nonexpressed DRB4*0103102N is encountered in association with DRB1*0701-DQB1*03032, which facilitates correct DNA typing. Here we describe the unusual association of this unexpressed DRB4*0103102N, with DRB1*0402-DQB1*0302 in a sample of Austrian patients.     

HLA-DRB1, DQA1 and DQB1 DNA polymorphisms in healthy Algerian and genetic relationships with other populations

Abstract: This study presents the results of HLA-DRB1, -DQA1, and -DQB1 sequence-specific oligonucleotide probe (SSOP) typings for a population sample of 47 individuals originating from Western Algeria. Allele and haplotype frequencies, as well as linkage disequilibria are computed by the standard methods used for the XIth International Histocompatibility Workshop data. A total of 24 alleles are detected at the DRB1 locus, where a very high heterozygosity level (0.914) is found. The highest DRB1 frequencies are 0.160, DRB1*1101, and 0.138, for DRB1*0301 and DRB1*0701. The DQA1 and DQB1 loci are less polymorphic. Among the 8 DQA1 alleles detected, DQA1*0501 is highly predominant with a frequency of 0.383. Thirteen DQB1 alleles are observed among which DQB1*0301 and DQB1*0201 are the most frequent (0.351 and 0.245, respectively). Three haplotypes predominate clearly: DRB1*1101-DQA1*0501-DQB1*0301 (0.138), DRB1*0701-DQA1*0201-DQB1*0201 (0.128) and DRB1*0301-DQA1*0501-DQB1*0201 (0.117). The two latter are among the most frequent haplotypes found in European and North American Caucasoid populations, but the DQA1*0501-DQB1*0201 association is not significant in Algerians. The genetic distances computed for each locus among a set of populations from different continents are significantly correlated to geography. They indicate that the Algerians are very close to South European populations, particularly to Sardinians, Italians, Romanians and French, with some intermediate characteristics between Europeans and sub-Saharan Africans. These results may serve as reference for future studies of HLA and disease in the Algerian population.     

Sensitivity to bee and wasp venoms: association with specific IgE responses to the bee and wasp venom and HLA DRB1 and DPB1

Background Stings from bees and wasps can cause systemic reactions which can be fatal in some individuals. In these venom-sensitive patients, specific IgE to the venom is produced and is considered to participate in the adverse reactions. This immune response requires antigen presentation by human leucocyte antigens (HLA) class II molecules, which includes DR and DP, which are present on antigen presenting cells. Objective To test for associations between HLA class II DRB1 and DPB1 alleles and life- threatening sensitivity to both bee and wasp venoms. To establish further whether any associations are independent of the atopy phenotype. Methods A total of 33 bee- and 44 wasp-venom-sensitive patients was studied. DRB1 genotypes were determined by single stranded oligonucleotide (SSO) probing of PCR products, and DPB1 genotypes by amplified fragment length polymorphism (AFLP) analysis. Total and specific IgE were measured using the Pharmacia Immunocap, FEIA. Patients with specific IgE to the venom antigens only were termed monosensitive and those with additional specific IgE to HDM and/or GP were termed polysensitive. Results Allele frequencies were compared to an unrelated control population. The 33 bee- sensitive patients had a greater prevalence of DRB 1*07 alleles than the control subjects, 26% vs 14%, with an odds ratio (OR) of 2.1 (95%CI, 1.2–3.7, P= 0.015, corrected for multiple comparisons, p_c > = ns). This association was confined to the 15 monosensitive bee patients, who had a 43% DRB 1*07 allele frequency when compared with 11% in the 18 polysensitive bee patients, OR 6.1 (95%CI, 1.73–22, P= 0.004, p_c= 0.05), and when compared with a control group of non-venom subjects, 43% vs 16%, OR 3.9 (95% CI, 1.72–9.0, P= 0.002, p_c= 0.02). The 44 wasp-sensitive patients had an increase in the DRB1*11 allele when compared with the control subjects, 13% vs 6%, with an OR 2.2 (95%CI, 1.0–4.6, P= 0.04, p_c= NS), and a decreased prevalence of DRB 1*04 alleles, 10% vs 19%, with an OR 0.33 (95%CI, 0.24–0.99, P= 0.04, p_c= NS), but these were not significant when multiple comparisons were taken into account. The DPB1 alleles were not significantly different between the venom sensitive patients and the controls. Conclusion Patients monosensitive to bee venom had a significantly greater prevalence of DRB 1 *07 alleles than the non-venom, control population suggesting that IgE responses in these patients may, in part be controlled by immune response HLA class II genes. These results are also suggestive of wasp-sensitive patients having a higher prevalence of DRB1*11 and a lower prevalence of DRB 1*04 than the control population.     

HLA DRB1*130101-DQB1*060101 haplotype is associated with acute chest syndrome in sickle cell anemia patients

We investigated the association of human leukocyte antigens (HLA) class II alleles and haplotypes with the pathogenesis of acute chest syndrome (ACS) in 186 sickle cell anemia (SCA) patients, of whom 58 had documented ACS (new pulmonary infiltrate, fever, and other associated clinical events) and 128 with a negative history of ACS, serving as controls. HLA DRB1* and -DQB1* genotyping was performed by polymerase chain reaction–sequence-specific priming. Of the DRB1* and DQB1* alleles analyzed, only DRB1*130101 ( Pc  < 0.001) was positively associated with ACS. DRB1*130101-DQB1*060101 haplotype was more prevalent among ACS patients ( P  = 0.018), thus conferring disease susceptibility. Specific HLA alleles and haplotypes may influence ACS risk in SCA patients, and specific HLA genotypes may be useful markers for identifying high-risk SCA ACS patients.     

High-resolution DNA typing in immunoglobulin A deficiency confirms a positive association with DRB 1*0301, DQBl*02 haplotypes

Selective immunoglobulin A (IgA) deficiency, the most common form of primary immunodeficiency, is related to the HLA genes. Previous studies demonstrated associations with particular HLA-DR-DQ haplotypes and a neutral amino acid at position 57 of the DQß chain was implicated in the susceptibility to selective IgA deficiency. In this study we reanalyzed the reported findings by high-resolution DNA typing of the loci DRB1, DQB1 and DQA1. We compared the typing results of 74 IgA-deficient individuals, detected by screening of blood donors, with those taken from 111 healthy controls. Results confirmed a strong positive association with DRB 1*0301, DQB1*02 and a negative association with DRB1*1501, DQB 1*0602. Considering the molecular interactions between HLA class II alleles and the peptides bound we conclude that the amino acid at position 57 of the DQß chain may contribute to the susceptibility to selective IgA deficiency, but not determine it. An extended statistical analysis strengthened the hypothesis that selective IgA deficiency might be communicated by the distinct haplotype DRB1*0301, DQB1*02.