DRB1 Typing Research Articles

Major Histocompatibility Complex Class‐II Alleles in Primary Biliary Cirrhosis

The major histocompatibility complex (MHC) class-II alleles at the DRB1, DQB1 and DPB1 loci were investigated in 40 patients with primary biliary cirrhosis (PBC) and 43 local healthy controls. Restriction fragment length polymorphism (RFLP) was used for DRB1 typing. DQB1 and DPB1 regions were amplified using the polymerase chain reaction (PCR) and then probed with 32P-labelled allele-specific oligonucleotide probes. There was an increased frequency of DR8 (10% compared to 4% in controls), and a decreased frequency of DR2 (18% compared with 28% in controls) in patients with PBC, but the differences were not significant. There were no significant differences for the other DR alleles, the seven DQ alleles, or the eight DP alleles. In conclusion, no significant MHC class-II associations with primary biliary cirrhosis have been demonstrated.

MOLECULAR COMPATIBILITY AND RENAL GRAFT SURVIVAL—THE HLA DRB1 GENOTYPING1

HLA DRB1 allelic types were determined by using sequence-specific oligonucleotides for the analysis of 91 renal donor-recipient pairs that were followed for 30 months. The Kaplan-Meier method was used to evaluate graft survivals (GS) of the matched and mismatched groups. The degree of compatibility was measured by allelic type matches and mismatches of the pairs. Furthermore, HLA DR genotypes were categorized into five groups, namely DR1, DR2, DR4, the group that also has the DRw52 allele, and the group of DR7, DR9, and DR10. All types within a group were considered a match. Serotypes of the same study group were also included for comparison. Associations of superior GS with compatible DRB1 allelic types, serotypes, and grouped DRB1 types were observed. A significantly higher GS rate was found in donor-recipient pairs when HLA DRB1 types were analyzed by group mismatching (P = 0.03) rather than type mismatching (P > 0.34). Because of the numerous allelic types that can be assigned on the basis of nucleotide sequence variations, a larger number of donor-recipient pairs is required to derive statistically significant results.

Correlation of mixed lymphocyte culture reactivity with DRB and DQB genomic typing in marrow transplantation: [formula omitted], S Vasisth, LE Adams LR Whitacre, K Balakrishnan. Children's Hospital Medical Center and Hoxworth Blood Center, University of Cincinnati, Cincinnati, Ohio

Correlation of mixed lymphocyte culture reactivity with DRB and DQB genomic typing in marrow transplantation: [formula omitted], S Vasisth, LE Adams LR Whitacre, K Balakrishnan. Children's Hospital Medical Center and Hoxworth Blood Center, University of Cincinnati, Cincinnati, Ohio

Simultaneous DRB1, DRB3 and DRB5 typing by chemiluminescent reverse dot blot: [formula omitted], P DeTogni, D Chaplin and T Mohanakumar, HLA Laboratory, Barnes Hospital and Washington University School of Medicine, Departments of Surgery and Pathology and Howard Hughes Medical Institute, St. Louis, MO

Simultaneous DRB1, DRB3 and DRB5 typing by chemiluminescent reverse dot blot: [formula omitted], P DeTogni, D Chaplin and T Mohanakumar, HLA Laboratory, Barnes Hospital and Washington University School of Medicine, Departments of Surgery and Pathology and Howard Hughes Medical Institute, St. Louis, MO

Linkage between HLA-DRB1 and -DRB3 types in the Japanese population analyzed by oligonucleotide genotyping

We analyzed linkage between HLA-DRB1 and -DRB3 types in 219 Japanese donors by oligonucleotide genotyping. In the Japanese population, DRB1 ∗1201 was linked with DRB3 ∗0101 in all donors analyzed; in contrast, most Caucasian DRB1 ∗1201 is known to be linked with DRB3 ∗02(01/02) ( ∗0201 or ∗0202). However, most DRB1 ∗1202 was linked with DRB3 ∗0301. Thus, the two DRw12-related DRB1 types are linked with DRB3 types distinct from each other. All the three DRw14-related DRB1 types, DRB1 ∗1401, DRB1 ∗1402, and DRB1 ∗1405, were linked with DRB3 ∗02(01/02) in the Japanese population, contrasting with the known linkage between DRB1 ∗1402 and DRB3 ∗0101 in other ethnic populations. The serologically “blank” DR type, DRB1 ∗1403, was linked with DRB3 ∗0101. Other DRB1 types, DRB1 types, DRB1 ∗0301, DRB1 ∗11(01/04) ( ∗1101 or ∗1104), and DRB1 ∗13(01/ 02) ( ∗1301 or ∗1302) in the Japanese population were linked mostly with the same DRB3 types, like those known in other ethnic populations.

HLA class II “typing”: Direct sequencing of DRB, DQB, and DQA genes

Routine clinical HLA class II typing is based largely on serological and cellular methods. These methods have many drawbacks that have led to the evaluation of molecular approaches to typing, including restriction fragment length polymorphism studies and oligotyping. We present here an alternative molecular approach, sequence-based typing (SBT), that allows direct determination of the sequences of all HLA class II polymorphic genes, thus providing the most detailed information currently possible in this regard. The data presented here using SBT are based on direct sequencing of polymerase chain reaction (PRC)-amplified DRB, DQB, and DQA cDNAs using a limited number of oligonucleotides. The oligonucleotides are designed to allow simultaneous determination of allelic sequences in any hetrozygote as well as characterization of DRB isotypic complexity. Two types of amplification oligonucleotides (nonconserved and/or conserved) are used for DRB typing, which involves a maximum of four simultaneous cDNA/PCR/sequencing reactions. The first of these reactions only uses conserved oligonucleotides and is designed to detect all the different DRB transcripts present in any given heterozygote; the other three reactions use nonconserved oligonucleotides and are designed to ensure the unambiguous interpretation of the most complex DRB heterozygote combinations. Characterization of DQA1 and DQB1 sequences can be performed by using conserved oligonucleotides and only involves one reaction per locus. We have applied SBT to 43 homozygous cell lines and to 38 different heterozygote combinations that had previously been serologically typed. In all cases we were able to determine the allelic composition at DRB1, DRB3/4/5 and/or DQB1, and DQA1 loci of these cell lines and subjects; our results, analyzed by blind protocol, were consistent with the serological phenotypes. SBT can be extended to class I and class III genes and is automatable. We believe that this strategy deserves further evaluation as a possible HLA typing method. Human Immunology 33, 69–81 (1992)

PCR‐SSO TYPING FOR HLA‐DRB ALLELES

Polymerase chain reaction (PCR) amplification of the second exon of DRB genes from genomic DNA is described. Sequence-specific oligonucleotide (SSO) probes have been designed to allow for the typing of DRB alleles. The methods are described in detail and the results of a trial on both homozygous typing cells and restriction fragment length polymorphism (RFLP)-typed local caucasoid controls are given. The ease of use of this form of DRB typing is emphasized and potential complications are discussed.

HLA-DQA1 and HLA-DQB1 genes may jointly determine susceptibility to develop multiple sclerosis

Serologic DR typing and genomic DRB1, DQA1, DQB1, DPA1, and DPB1 typing using sequence-specific oligonucleotides were performed in 69 multiple sclerosis (MS) patients and 181 healthy controls on in vitro amplified DNA. The frequencies of DR2 as well as the DR2-associated DQA1 ∗0102 and DQB1 ∗0602 alleles were increased whereas DR7 was decreased among MS patients. The distribution of DR4 subtypes as well as DP alleles were similar in patients and healthy controls. All but one of 23 DR4-positive MS patients carried the DQB1 ∗0302 allele, whereas five of five DR7-positive MS patients carried the DQB1 ∗0303 allele. Of the MS patients, 99% compared to 79% of the controls carried DQA1 alleles encoding glutamine at residue 34, while 97% of the MS patients compared to 72% of the controls carried DQB1 alleles encoding DQB chains sharing long polymorphic streches. A combination of such DQA1 and DQB1 alleles was carried by 96% of the MS patients and 60% of the controls, suggesting an association between MS and a combination of particular DQA1 alleles and DQB1 alleles. The corresponding DQαβ heterodimers may have in common an ability to bind a particular peptide

HLA A Class II (DR, DQ) in Japanese Patients with Type 1 Diabetes Mellitus

DNA restriction fragment length polymorphism (RFLP) typing of HLA-DR and DQ alleles of 60 Japanese type 1 (insulin dependent) diabetic patients and 115 controls was performed. RFLP typing of DRB1 showed increased frequency of DR9 and decreased frequencies of DR2 and DRW6 among patients compared to controls. In the RFLP typing of BamHI-digested DNA to DQ beta probe (BamHI-DQB1), the incidence of the 10.26 kb fragment, which represents either DQW4, DQW8 or DQW9, was markedly elevated in the patients, whereas the incidence of DQW6 was reduced. The predicted DR-DQ haplotype study revealed that DR4-DQW4 or DQW8, DRW8-DQW4 or DQW8 and DR9-DQW9 may contribute to susceptibility to type 1 diabetes. When serological typing of the 13 DRW8 patients was performed, all the 11 DRW8 patients carrying DQW4 or DQW8 (BamHI-10.26 kb) were positive for DQW3. These results indicated that the HLA-DQ locus may play an important role in the development of type 1 diabetes in the Japanese as well as other ethnic groups and that the DRW8-DQW8 haplotype may predispose to the disease in Japan.

The application of oligonucleotide probes to HLA Class II typing of the DRB sub‐region

A plan for DRB typing at the sequence level is detailed. Only one polymerase chain amplification reaction is needed and the application of a limited number of short oligonucleotide probes allows an almost complete definition of DRB alleles. The scheme was tested on 40 homozygous cell-lines selected to cover a wide range of specificities, and 40 RFLP-typed controls. The results are presented and discussed. The simplicity and accuracy of this scheme are emphasized.