High-resolution HLA typing for the DRB3/4/5 genes by sequence-based typing

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence-based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene-specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR-SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.     

HLA-A*11 and novel associations in Malays and Chinese with systemic lupus erythematosus

We investigated the association of the HLA genes in Malaysian patients with systemic lupus erythematosus (SLE) and their associations with the clinical manifestations in 160 SLE patients (99 Chinese and 61 Malays) and 107 healthy control individuals (58 Chinese and 49 Malays) were studied. Sequence specific primer amplification (PCR-SSP) phototyping techniques were used to analyse 25 HLA-A allele groups, 31 HLA-DR allele groups and 9 HLA-DQ allele groups. Appreciable increases in allele frequencies of HLA-A*11, DRB1*0701, DRB1*1601-1606, DRB5*01-02 and DQB1*05, and decrease in HLA-DRB1*1101-1121, 1411, DRB1*1201-3, DRB1*1301-22, DRB3*0101, 0201, 0202, 0203, 0301 and DQB1*0301, 1304 in SLE patients compared with healthy control individuals. However, after Bonferroni correction (p(c)<0.05) only HLA-A*1101, 1102, DRB5*01-02, DQB1*05, DRB1*1201-3, DRB3*0101, 0201, 0202, 0203, 0301 and DQB1*0301, 0304 remained significant. Allele frequencies of DRB1*0701 and DRB4*0101101, 0102, 0103, DQB1*05, DRB1*1301-22, DRB3*0101, 0201, 0202, 0203, 0301 and DQB1*0301, 0304 were significantly increased in Malay SLE patients compared with healthy control individuals. In contrast, Chinese SLE patients had increased allele frequencies of DRB1*1601-1606, DQB1*05, DRB1*1201-3, DRB3*0101, 0201, 0202, 0203, 0301, DRB3*0101, 0201, 0202, 0203, 0301 and DQB1*0301, 0304 compared with healthy control individuals. HLA-A*6801-02 and DRB1*1601-1606 frequencies appeared elevated in a subset of patients with serositis and DRB1* 0401-1122 frequency was elevated in those displaying neurologic disorder. However, unequivocal evidence of these associations would require investigation of substantially larger cohorts. On the whole, our findings suggest that HLA allele associations with SLE are race specific in Malays and Chinese.     

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 DR-DQ-encoded genetic determinants of childhood-onset type 1 diabetes in Finland: an analysis of 622 nuclear families

The diabetes predisposing effect of HLA genes is defined by a complex interaction of various haplotypes. We analyzed the disease association of HLA DRB1-DQA1-DQB1 genotypes in a large nuclear family cohort (n = 622) collected in Finland. Using the affected family based artificial control approach we aimed at characterizing all detectable disease-specific HLA haplotype and genotype effects. The DRB1*0401-DQB1*0302 haplotype was the most prevalent disease susceptibility haplotype in the Finnish population followed by (DR3)-DQA1*05-DQB1*02 and DRB1*0404-DQB1*0302. DRB1*0405-DQB1*0302 conferred the highest disease risk, although this haplotype was very rare. The DRB1*04-DQB1*0304 was also associated with increased disease risk, an effect detected for the first time in the Finnish population. The following haplotypes showed significant protection from the disease and are listed in decreasing order of the strength of their effect: (DR7)-DQA1*0201-DQB1*0303, (DR14)-DQB1*0503, (DR15)-DQB1*0602, DRB1*0403-DQB1*0302, (DR13)-DQB1*0603, (DR11/12/13)-DQA1*05-DQB1*0301, (DR1)-DQB1*0501. In addition to the DRB1*0401/0404-DQB1*0302/(DR3)-DQA1*05-DQB1*02 genotype and DRB1*04-DQB1*0302 homozygous genotypes, heterozygous combinations DRB1*0401-DQB1*0302/(DR13)-DQB1*0604, approximately /(DR8)-DQB1*04, approximately /(DR9)-DQA1*03-DQB1*0303, approximately /(DR1)-DQB1*0501 and approximately /(DR7)-DQA1*0201-DQB1*02 were also disease-associated. As a new finding in this population, the (DR3)-DQA1*05-DQB1*02 homozygous and (DR3)-DQA1*05-DQB1*02/(DR9)-DQA1*03-DQB1*0303 heterozygous genotypes conferred disease susceptibility. Similarly, the DRB1*0401-DQB1*0302/(DR13)-DQB1*0603 genotype was disease predisposing, implying that DQB*0603-mediated protection from diabetes is not always dominant. Comparison of our findings with published data from other populations indicates a significant disease-specific heterogeneity of the (DR8)-DQB1*04, (DR7)-DQA1*0201-DQB1*02 and (DR3)-DQA1*05-DQB1*02 haplotypes.     

中国汉族人群供-受者HLA-A、B、Cw、DRB1和DQB1高分辨等位基因频率分布及错配比例的研究

目的 从基因高分辨水平,分析中国汉族人群供-受者人类白细胞抗原(human leukocyte antigens,HLA)-A、B、Cw、DRB1、DQB1各位点等位基因频率和分布的多态性;及供-受者等位基因匹配情况.方法 采用基因测序分型(sequence based typing,SBT)、序列特异性寡核苷酸探针法(sequence specific oligonueleotide probe,SSOP)和序列特异性引物法(sequence specific primer,SSP),对2540名中国汉族人的(其中1168名受者,1372名供者)DNA标本进行HLA高分辨基因分型,并作统计学处理.结果 2540份样本中共检测到44种HLA-A等位基因,频率高于0.05的A*1101、A*2402、A*0201、A*0207、A*3303、A*0206、A*3001共占80.4%;81种HLA-B等位基因,频率高于0.05的B*4001、B*4601、B*5801、B*1302、B*5101共占43.0%;44种HLA-Cw等位基因,频率高于0.05的Cw*0702、Cw*0102、Cw*0304、Cw*0801、Cw*0602、Cw*0303、Cw*0302、Cw*0401共占80.3%;61种HLA-DRB1等位基因,频率高于0.05的DRB1*0901、DRB1*1501、DRB1*1202、DRB1*0803、DRB1*0701、DRB1*0405、DRB1*0301、DRB1*1101共占70.1%;22种HLA-DQB1等位基因,频率高于0.05的DQB1*0301、DQB1*0303、DQB1*0601、DQB1*0602、DQB1*0202、DQB1*0302、DQB1*0401、DQB1*0502、DQB1*0201共占87.4%.这5个位点均处于杂合子缺失状态,其中A、B、DRB1位点符合HardyWeinberg平衡(Hardy-Weinberg equi1ibrium,HWE)(P＞0.05);Cw、DQB1位点偏离HWE(P＜0.05);排除个别基因型观察值与期望值偏差较大外,这5个位点均符合HWE.在供-受者数据的比较中,HLA全相合(10/10)的比例仅22.4%;单个等位基因错配(9/10)的比例为24.6%;两个等位基因错配(8/10)的比例为26.3%.结论 中国汉族人群高分辨水平HLA-A、B、Cw、DRB1,DQB1等位基因频率及分布特点,对非亲缘造血干细胞移植供者检索有重要参考价值;并为中华骨髓库数据入库和利用提供遗传学依据.

Abstract：

Objective To analyze the allele frequencies and polymorphism of human leukocyte antigens (HLA) -A, B, Cw, DRB1 and DQB1 between donors-recipients on high-resolution typing; and to analyze the matching and mismatching proportion between donors and recipients. Methods HLA highresolution types were determined by sequence based typing (SBT), sequence specific oligonucleotide probe (SSOP) and sequence specific primer (SSP) on 2540 unrelated Chinese Han individuals including 1168 recipients and 1372 donors, then statistical analyses were carried out. Results Forty-four HLA-A alleles were detected, and among them the frequencies of A * 1101, A * 2402, A * 0201, A * 0207, A * 3303, A *0206 and A * 3001 exceeded 0.05, and accounted for 80.4%. Eighty-one HLA-B alleles were detected, and frequencies of B * 4001, B * 4601, B * 5801, B * 1302 and B * 5101 exceeded 0. 05, and accounted for 43. 0% of total. There were 44 HLA- Cw alleles, among them the frequencies of Cw * 0702, Cw * 0102,Cw * 0304, Cw * 0801, Cw * 0602, Cw * 0303, Cw * 0302 and Cw * 0401 exceeded 0.05, and were 80.3 %of total. There were 61 HLA-DRB1 alleles, the frequencies of DRB1 * 0901, DRB1 * 1501, DRB1 * 1202,DRB1 * 0803, DRB1 * 0701, DRB1 * 0405, DRB1 * 0301 and DRB1 * 1101 exceeded 0. 05, and were 70. 1% of total. Finally, 22 HLA-DQB1 alleles were detected, the frequencies of DQB1 * 0301, DQB1 *0303, DQB1 * 0601, DQB1 * 0602, DQB1 * 0202, DQB1 * 0302, DQB1 * 0401, DQB1 * 0502 and DQB1 *0201 exceeded 0. 05, and they were 87.4% of total. All the five loci were of heterozygote deficiency. The HLA-A, B and DRB1 loci conformed to Hardy-Weinberg equilibrium (HWE) (P＞0. 05); but HLA-Cw and HLA-DQB1 loci did not (P＜0.05). Except several particular genotypes, all the five loci conformed to HWE. After comparing data between donors and recipients, only 22.4% of recipients found HLA matched donors (10/10); 24. 6% of recipients found single HLA allele mismatched donors (9/10); 26. 3% of recipients had two HLA alleles mismatched donors (8/10). Conclusion The characteristics of allele frequencies and polymorphism of HLA-A, B, Cw, DRB1 and DQB1 on high-resolution typing in Chinese Han population is valuable for donor searching in unrelated hematopoietic stem cell transplantation, and it provides genetic basis for donor registry and usage of donor resource for Chinese Marrow Donor Program.     

应用SBT直接测序分型法研究中国南方汉族人群HLA五个基因座单倍型

目的 研究中国南方汉族人群HLA-A、B、Cw、DRBl、DQBl等位基因多态性及单倍型的分布特征.方法 应用聚合酶链反应-直接测序分型(polymerase chain reaction sequence-based typing,PCR-SBT)法对186名中国南方汉族健康人群HLA-A、B、Cw、DRBl、DQBl进行基因分型.结果 检出的HLA-A、B、Cw、DRBl、DQBl等位基因分别有28、49、24、29、20种.经统计分析A*0207-B*4601(10.81%),A*3303-B*5801(6.14%),B*4601-DRBl*0901(6.22%),B*4001*DRBl*0901(3.78%),DRBl*0901-DQBl*0303(12.16%)和DRBl*1202-DQBl*0301(8.38%)单倍型呈强连锁不平衡单倍型(RLF≥0.5,X<'2>>3.84,P<0.05);A*0207-B*4601-Cw*0102(10.75%),A*3303-B*5801-Cw*0302(5.14%),A*0207-B*4601-DR*0901(5.07%),A*3303-B*5801-DRBl*0301(2.96%),A*0207-B*4601-Cw*0102-DRBl*0901-DQBl*0303(4.87%)和A*1101-B*1301-Cw*0304-DRBl*1501-DQBl*0601(2.43%)单倍型分别是中国南方汉族人群常见单倍型.结论 中国南方汉族人群HLA 5个基因座单倍型分布具有高度的遗传多态性且有其自身分布特点.本研究获得的较完整的HLA 5个基因座单倍型分布数据,将为人类学、HLA疾病相关性和器官移植等研究提供遗传学参考数据.     

Sequencing of a new HLA-DR4 allele with an unusual residue at position 88 that does not affect T cell allo recognition.

It is rather common to encounter new HLA alleles identified by unorthodox patterns observed during low resolution typing performed with sequence specific oligonucleotide probes (SSOP). One of the best examples is locus DRB1, where allelic subtypes are characterized by a combination of a limited number of residues located in three hypervariable regions of exon 2. HLA-DR oligotyping analysis of a female caucasoïd bone marrow donor of the Lyon registry led to the identification of an individual that typed as DRB1^* 11, DRB3^* 02, DRB4^* 01, DQB1^* 0301-0302. This donor was however typed by serology as DR11 DR4, DR52 DR53, DQ7 DQ8. PCR-SSP typing for DR4 subtype revealed an amplification pattern typical for DRB1^* 0404. After PCR amplification of genomic DNA and sequencing the entire exon 2, a new DRB1 allele was identified : DRB1^* 04var that is identical to DRB1^* 0404, except for one nucleotide at codon 88 resulting in a Ser ==> Arg exchange. This mutation had prevented amplification with the DR generic primers. The donor inherited this new DRB1^* 04var from her mother who presented the same molecular characteristics. The complete SSOP typing of the implicated haplotype is : A^*68011, Cw^*0704, B^*4402, DRB1^* 04var, DRB4^* 0103, DQA^* 0301, DQB1^* 0302, DPB1^* 0601.

Cellular typing by three HTCs-DRB1^*0404/DW14 from the 9th Workshop showed that this DRB1^* 04var typed exactly like a DW14 cell (the S.R.R. being 40%, 34%, 36%, respectively). This suggests that residue 88 does not affect T cell recognition possibly due to its location at the far end of the alpha helix flanking the peptide binding groove.     

DQA103 subtypes have different associations with DRB1 and DQB1 alleles

Polymorphisms outside the hypervariable regions of HLA class II alleles that do not affect the peptide-binding site are probably not under selective pressure and could therefore be useful as markers of the evolutionary pathways of the HLA class II haplotypes. We have analyzed such a polymorphism in the variants of DQA1^*03, which differ at residue 160 encoded in exon 3. Our study included homozygous BCLs of the 10th IHWS and samples of a multiracial panel of 723 unrelated subjects which were also typed for allelic variations in exon 2 by hybridization with SSOP. BCLs having DQA1^*03 and 131 selected DQA1^*03-positive samples were typed for the dimorphism in exon 3 that distinguishes DQA1^*0301 and DQA1^*0302. DQA1^*0301 was found to be exclusively associated with DQB1^*0302, while samples carrying DQB1^*0201, 0301, 0303, and 0401 always had DQA1^*0302. A few haplotypes carrying DQB1^*0302 had DQA1^*0302. The fact that DQA1^*0301 is completely included in DQB1^*0302, and not vice versa, suggests that DQA1^*0301 may have arisen from a mutation in a haplotype containing DQA1^*0302-DQB1^*0302. DQB1^*0302 was found to be associated with all DR4 subtypes, suggesting possibly that the current variants of DRB1-DR4 may be of more recent origin. DRB1^*0405 was the only subtype of DR4 which was not associated with DQA1^*0301 and had multiple associations with the DQB1 alleles, therefore, perhaps representing the oldest allele of this group.     

系统性红斑狼疮临床表现与HLA Ⅱ类单倍型关联的研究

目的 探讨系统性红斑狼疮（ＳＬＥ）易感基因致病的模式。方法 利用多聚酶链反应／特异寡核控针杂交（ＰＣＲ／ＳＳＯＰＨ）方法检测１１３例确诊ＳＬＥ病人的ＨＬＡⅡ基因型并进行单倍型分析。结果 ＳＬＦ病人的单倍型具有特定的结构特征,即以２个或３个重型ＳＬＥ相关基因共同组成１个单倍型;反之,２个或３个轻型ＳＬＥ相关基因组成另１个单倍型;重型基因和轻型基因之间很少有强连锁不平衡。ＤＱＡ１＊０３０１－ＤＱＢ１＊     

Complex interaction between HLA DR and DQ in conferring risk for childhood type 1 diabetes.

Type 1 (insulin-dependent) diabetes mellitus is associated with HLA DR and DQ factors, but the primary risk alleles are difficult to identify because recombination events are rare in the DQ-DR region. The risk of HLA genotypes for type 1 diabetes was therefore studied in more than 420 incident new onset, population-based type 1 diabetes children and 340 age, sex and geographically matched controls from Sweden. A stepwise approach was used to analyse risk by relative and absolute risks, stratification analysis and the predispositional allele test. The strongest relative and absolute risks were observed for DQB1*02-DQA1*0501/DQB1*0302-DQA1*0301 heterozygotes (AR 1/46, P < 0.001) or the simultaneous presence of both DRB1*03 and DQB1*0302 (AR 1/52, P < 0.001). Stratification analysis showed that DQB1*0302 was more frequent among DRB1*04 patients than DRB1*04 controls (P < 0.001), while DRB1*03 was more frequent among both DQA1*0501 (P < 0.001) and DQB1*02 (P < 0.001) patients than respective controls. The predispositional allele test indicated that DRB1*03 (P < 0.001) would be the predominant risk factor on the DRB1*03-DQA1*0501-DQB1*02 haplotype. In contrast, although DQB1*0302 (P < 0.001) would be the predominant risk factor on the DRB1*04-DQA1*0301-DQB1*0302 haplotype, the predispositional allele test also showed that DRB1*0401, but no other DRB1*04 subtype, had an additive risk to that of DQB1*0302 (P < 0.002). It is concluded that the association between type 1 diabetes and HLA is due to a complex interaction between DR and DQ since (1) DRB1*03 was more strongly associated with the disease than DQA1*0501-DQB1*02 and (2) DRB1*0401 had an additive effect to DQB1*0302. The data from this population-based investigation suggest an independent role of DR in the risk of developing type 1 diabetes, perhaps by providing diseases-promoting transcomplementation molecules.     

中国湖北汉族HLA—Ⅱ类等痊基因频率的群体调查

调查中国湖北汉族人群ＨＬＡ－Ⅱ类基因频率。方法,用聚合酶链反应／序列特异性引物和聚合酶链反应／限制性片段长度多态性技术,对中国湖北汉族１６８名正常个体进行了ＨＬＡ－ＤＲＢ１（ｎ＝１６８）、ＨＬＡ－ＤＱＢ１（ｎ＝１６０）、ＨＬＡ－ＤＰＢ１（ｎ＝９３）基因的多态性检测。结果共检出３９种ＤＲＢ１、１５种ＤＱＢ１和１７种ＤＰＢ１等位基因型别,等位基因频率较高的分别是：ＤＲＢ１＊０９０１（ｇｅｎｅｆｒｅｑｕ     

Specific amplification of the HLA-DRB4 gene from c-DNA. Complete coding sequence of the HLA alleles DRB4*0103101 and DRB4*01033

We present the complete coding sequence of the HLA alleles DRB4*0103101 and DRB4*01033 derived from the lymphoblastoid cell line G081, established from an individual of Spanish Gypsy ethnic origin. This cell was typed by PCR-SSP and reverse SSO as DRB4*0103101 but further characterization of the DRB4 gene by sequence-based typing (SBT) demonstrated heterozygosity at codon 78 (TAC, TAT). With the aim of confirming this polymorphism, RNA isolated from G081 was subjected to RT-PCR using primers designed to recognize specifically the 5' and 3' UT regions of HLA-DRB4 and the product was cloned and sequenced. Nucleotide sequences derived from seven clones confirmed the heterozygosity of G081, as they corresponded to two open reading frames of 801 nucleotides that matched either DRB4*0103101 or the recently described DRB4*01033, for which a partial sequence, spanning exons 2 and 3, has been reported. The phenotype of G081 (A*01; B*0702, *1302/1303; Cw*0602, *07; DRB1*0403, *0701; DRB4*0103101, *01033; DQB1*0202, *0302; DQA1*0201, *0301) is consistent with a proposed association of DRB4*01033 with DRB1*0403 and DQB1*0302.     

HLA profile of three ethnic groups living in the North-Western region of Russia

HLA class II alleles were determined by PCR-SSO and PCR-SSP typing of DNA samples from 55 Nentsy, 81 Saami and 73 Pomor individuals from the North-European part of Russia. The results were compared with similar data from Russians. A high frequency of the DRB1*04-DQA1*0301-DQB1*0302 haplotype and a low frequency of the DRB1*11-DQA1*0501-DQB1*0301 haplotype, observed in all three ethnic groups, may indicate a common aboriginal component in their ancestry. Saami and Pomors displayed a similar pattern of allele and haplotype distribution, with the exception of the DRB1*04-DQA1*0304-DQB1*0301 haplotype, which was significantly higher among Saami compared Nentsy, Pomors and Russians. Nentsy individuals had a particularly high frequency of the DRB1*09-DQA1*0301-DQB1*0303 and the DRB1*12-DQA1*0501-DQB1*0301 haplotypes. Genetic distances and correspondence analysis show that Pomors have a close relationship with Norwegians and Finns, whereas Nentsy and Saami are more closely related to Oriental populations.     

HLA class II antigens in South African Blacks with type I diabetes

Type 1 diabetes mellitus is poorly characterised in many African communities, including South Africa, where little is known of the disease epidemiology. This study aimed to identify the HLA class II alleles associated with type 1 diabetes in a group of Zulu subjects in Durban, KwaZulu-Natal by PCR-SSP. The HLA alleles associated with type 1 diabetes included HLA-DQB*0302 (P<0.0001), DRB1*O9 (P<0.0001), DRB1*04 (P=0.002), DRB1*0301 (P=0.003), DQB*02 (P=0.004) and DQA*03 (P=0.035). Estimated haplotypes positively associated with type 1 diabetes included HLA-DRB1 *0301-DQA*0501, DRB1*04-DQA*03, DRB1*04-DQB*0302, DRB1*0301-DQB*0201, DQA*0501-DQB*0201 and DQA*03-DQB*0302. These findings are similar to those reported from Zimbabwe and other populations with type 1 diabetes.     

Sequence-based typing of HLA class II DQB1

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.     

Comprehensive typing of DR52 (DRB3)-associated DRB1 and DRB3 alleles by PCR-RFLP

Abstract: The DR52-associated DRB1 and DRB3 alleles were resolved by PCR-RFLP. Second exon was amplified using four primer pairs (groups 1–4) for DRB1 and a pair for DRB3 alleles. Except for three endonucleases, all others had either none or only one site for a specific amplified product. Group 1 primers amplify 10 DRB1 alleles (DRB1*0302, 1101, 1302, 1303, 1305, 1307, 1402, 1403, 1407 and 1409). All but one pair, DRB1*1402 from 1409, could be resolved using seven endonucleases (ApaI, SacII, FokI, AvaII, BsaAI, BsrBI and SfaNI). Group 2 consisted of four alleles (DRB1*1201, 1202, 1404 and 1411) that can be resolved along with co-amplified DRB1*0804 and 0806 using five endonucleases (AvaII, SacII, FokI, HaeII and RsaI). Group 3 primers amplify 15 DRB1 alleles (DRB1*0301, 0303, 1102, 1103, 1104, 1107, 1301, 1304, 1306, 1308, 1401, 1405, 1406, 1408 and 14-New), which can be resolved using nine enzymes (KpnI, AvaII, FokI, SacII, HaeII, BsrBI, SfaNI, DdeI and RsaI). BsrBI, a new endonuclease, can resolve DRB1*1301 from 1306 and the previously unresolved allele DRB1*1103 from 1104. DRB1*1410, co-amplified with DR4 group-specific primers, is resolved with PstI which cleaves all DR4 alleles but not DRB1*1410. All four DRB3 alleles (DRB3*0101, 0201, 0202 and 0301) and their heterozygotes are resolved using two endonucleases, RsaI and HphI. Thirty-four DR52-associated alleles and their heterozygotes can be unambiguously resolved, except for DRB1*1402 from 1409. Thus, PCR-RFLP remains an effective method for high-resolution HLA-DR typing. Furthermore, PCR-RFLP can complement the evolving PCR-SSP method for allele-specific typing using a minimal number of restriction endonucleases.     

HLA-DR and -DQ associations with insulin-dependent diabetes mellitus in a population of Turkey

Genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) has been shown to be associated with MHC in many studies. To extend this data with a population with relatively low IDDM incidence, MHC DRB, DQA, and DQB have been investigated by polymerase chain reaction and sequence specific oligonucleotide probe hybridization (PCR/SSO) in 178 IDDM patients from Turkey and compared to 248 healthy controls. Significant differences are detected between IDDM and control groups in the frequencies of DRB1*0402 DQA1*03 DQB1*0302 (28.1% vs. 5.2%, p < 0.0001, OR: 7.1) and DRB1*0301 DQA1*0501 DQB1*02 (57% vs. 18.1%, p < 0.0001, OR: 6.1). Among the negative associations, the most strong ones are with DRB1*1401 DQA1*0101 DQB1*0503 (0.6% vs. 8.9%, p < 0.0001, OR: 0.1), DRB1*1502 DQA1*0103 DQB1*0601 (1.1% vs. 7.7%, p = 0.0023, OR: 0.1), DRB1*1301 DQA1*0103 DQB1*0603 (0.6% vs. 6.9%, p = 0.0039, OR: 0.2) and DRB1*1101 DQA1*0501 DQB1*0301 (3.9% vs. 12.1%, p < 0.0001, OR: 0.2). When the DRB, DQA or DQB genotypes of the susceptible alleles are compared, the most strong susceptibility marker of the disease is found to be DRB1*0301/*04 (31.4% vs. 2.8%, p < 0.0001, OR: 15.8) and among these, heterozygote genotype DRB1*0301/*0401 (4.5% vs. 0, p = 0.0008, OR: 24.8).These results confirm the positive associations with IDDM previously observed in other Caucasian populations and reveal many negative and strong associations which maybe underlining several characteristics that distinguish Turkish diabetics form other Caucasians.     

HLA-DR and -DQ alleles in Italian patients with melanoma

Abstract: Controversial data have been reported about HLA alleles and susceptibility to melanoma. Our investigation was undertaken to analyze the relationship between HLA alleles distribution in patients with melanoma and susceptibility to the tumor, in order to study the possible correlation between HLA class II DQA1, DQB1 and DRB1 genes involved in immune recognition, and melanoma, usually considered a highly immunogenic tumor. We therefore typed by means of PCR-SSP (sequence-specific primers) 53 Italian patients and 53 healthy random controls coming from the same geographic area. We observed a decrease of all haplotypes bearing DQB1*0301, DQB1*0302 and DQB1*0303 alleles but not of haplotype DRB1*11;DQA1*0501;DQB1*0301. Our results seem to support the hypothesis of a protective role of some DQ3-bearing haplotypic combinations in melanoma.     

Complex interaction between HLA DR and DQ in conferring risk for childhood type 1 diabetes

Type 1 (insulin-dependent) diabetes mellitus is associated with HLA DR and DQ factors, but the primary risk alleles are difficult to identify because recombination events are rare in the DQ–DR region. The risk of HLA genotypes for type 1 diabetes was therefore studied in more than 420 incident new onset, population-based type 1 diabetes children and 340 age, sex and geographically matched controls from Sweden. A stepwise approach was used to analyse risk by relative and absolute risks, stratification analysis and the predispositional allele test. The strongest relative and absolute risks were observed for DQB1*02-DQA1*0501/DQB1*0302-DQA1*0301 heterozygotes (AR 1/46, P < 0.001) or the simultaneous presence of both DRB1*03 and DQB1*0302 (AR 1/52, P < 0.001). Stratification analysis showed that DQB1*0302 was more frequent among DRB1*04 patients than DRB1*04 controls (P < 0.001), while DRB1*03 was more frequent among both DQA1*0501 (P < 0.001) and DQB1*02 (P < 0.001) patients than respective controls. The predispositional allele test indicated that DRB1*03 (P < 0.001) would be the predominant risk factor on the DRB1*03-DQA1*0501-DQB1*02 haplotype. In contrast, although DQB1*0302 (P < 0.001) would be the predominant risk factor on the DRB1*04-DQA1*0301-DQB1*0302 haplotype, the predispositional allele test also showed that DRB1*0401, but no other DRB1*04 subtype, had an additive risk to that of DQB1*0302 (P < 0.002). It is concluded that the association between type 1 diabetes and HLA is due to a complex interaction between DR and DQ since (1) DRB1*03 was more strongly associated with the disease than DQA1*0501-DQB1*02 and (2) DRB1*0401 had an additive effect to DQB1*0302. The data from this population-based investigation suggest an independent role of DR in the risk of developing type 1 diabetes, perhaps by providing diseases-promoting transcomplementation molecules.     

Discovery of HLA-DRB1*0331 in a Taiwanese marrow donor and the importance of sequence-based typing in a rare or previously unrecognized allele

We describe here a novel HLA-DRB1* allele, DRB1*0331, observed from a Taiwanese bone marrow donor using DNA sequence-based typing (SBT) method. The 'new' allele differs from DRB1*0306 and DRB1*0325 by one nucleotide at positions 196 and 227, respectively. Nucleotide mutations caused amino acid substitutions from N to Y at codon 37 and from F to Y at codon 47, as compared with amino acid sequence encoded by the DRB1*030101 allele. The donor was first typed as DRB1*0403/0406/0439/0441/0446/0451/0452 (NMDP code DRB1*04XX) and DRB1*0304/0323/0325 (NMDP code DRB1*03APDA) by sequence-specific oligonucleotide (SSO) typing kit. Subsequent typing of the donor by high-resolution sequence-specific primer (SSP) protocol indicated DRB1*0403 and DRB1*0306. The anomalous result of DRB1*03 was resolved by SBT and recognized as DRB1*0331. We concluded that SSP or SSO alone may mistype a precedent unrecognized allele and that two different typing techniques or SBT may have to be employed to safe guard true HLA typing when rare alleles are encountered at the first time.