DP Typing Research Articles

Localization of the recombination points in a family with two DR/DP recombinations.

In a family with a maternal DR/GLO recombination, cellular DP typing showed it to be located between DR and DP. RFLP studies done during the 9th international histocompatibility workshop gave anomalous segregation patterns of DPA and DPB bands that could be interpreted as being due to a second, paternal DR/DP recombination. This assumption was confirmed later by PCR-SSO typing. A more precise mapping has been done by new markers showing the maternal recombination to be within the TAP2 locus and the paternal recombination to be between DQB1 and DQB3. This supports earlier suggestions of a hot spot of recombination in the TAP region. The recombinations involve parental haplotypes that presently show DR/DP linkage disequilibrium in the French population and it is proposed that DR/DP recombinations occur randomly while B/DR recombinations preferentially occur on haplotypes without strong linkage disequilibrium. Existing DR/DP linkage disequilibria in a given population will thus be broken down with time. The mixed lymphocyte culture response towards an isolated DP difference was tested in this and another DR/DP recombinant family. It showed that an alloresponse towards DP may be highly variable and this suggests that it might be important to define the rules for the strength of this reaction and the possible implications for allotransplantation.

DP polymorphism in HLA-A1,-B8,-DR3 extended haplotypes associated with membranoproliferative glomerulonephritis and systemic lupus erythematosus.

We and others have shown an association between autoimmune disorders and the major histocompatibility complex extended haplotype HLA-A1,-B8,-SCO1,-DR3. The primary gene or genes within this haplotype conferring such susceptibility, however, have not been defined. In this study, we tested the hypothesis that linkage disequilibrium in this haplotype extends through the DP locus, and that DP type may be linked to membranoproliferative glomerulonephritis (MPGN) and systemic lupus erythematosus (SLE). DP and DQ typing was performed by restriction fragment length polymorphism analysis for 43 chromosomes (19 healthy controls, 9 SLE, 15 MPGN) bearing the -A1,-B8,-SCO1,-DR3 extended haplotype. Although all were DQw2, a variety of DP types were identified (DPw1, 0.26; DPw2, 0.09; DPw3, 0.14; DPw4, 0.44). Although DPw1 was represented on extended haplotypes with greater frequency than on 113 non-A1,-B8,-SCO1,-DR3 haplotypes (0.26 vs. 0.03; P < 0.001), there were no significant differences between healthy individuals with this haplotype and those with autoimmune disease. We conclude that the strong linkage disequilibrium of this haplotype breaks down between the DQ and DP loci. Loci important to disease susceptibility, therefore, are more likely to occur telomeric to DP.

HLA-DPA1 and HLA-DPB1 in rheumatoid arthritis and its subsets.

The aim of this study was to examine the relationship between HLA-DP and susceptibility to articular and extra-articular features (Felty's syndrome and vasculitis) of rheumatoid arthritis (RA). The possible association of DP types with severity of articular disease was also analysed. No statistically significant associations were observed between HLA-DP alleles and articular or extra-articular features of RA, or to the severity of the arthritis when p was corrected for the number of alleles tested.

One-step molecular HLA-DR prescreening employing a set of 14 sequence specific oligonucleotides in a non-radioactive tetramethylammonium chloride hybridization protocol.

A novel non-radioactive protocol for molecular generic HLA-DR typing is introduced, employing sequence specific oligonucleotides (SSOs) enzymatically 3'-labelled with biotin-14-dATP via terminal deoxynucleotidyltransferase in a tetramethylammonium chloride hybridization procedure. The detection reaction is carried out, using streptavidin conjugated horseradish peroxidase which is bound to the SSOs, in combination with a light emitting detection system. Fourteen SSOs and one control SSO are employed for generic HLA-DR typing in a one-step protocol. In order to demonstrate the suitability of this procedure, 5 homozygous typing cell lines and samples of 11 pretyped individuals which include most serologically defined HLA-DR specificities (DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9, 10, 52, and DR53) are analysed with the panel of 14 SSOs. The typing results show that this protocol, which avoids the use of radioisotopes, combines high specificity and easy handling. It also allows typing of poorly amplified samples because even after longer exposition times no false positive signals were observed and is particularly suitable for routine molecular HLA-DR typing on the generic level. In addition it can easily be adapted to DP and DQ typing or DR subtyping.

Influence of HLA-DR2, HLA-DPw4, and T cell receptor alpha chain genes on the susceptibility to multiple sclerosis.

In view of our recent report that T cell receptor (TCR) alpha chain restriction fragment length polymorphism (RFLP) is associated with multiple sclerosis (MS), we have assessed the possibility that HLA-DR2, HLA-DPw4, and TCR alpha chain RFLPs may interact to increase the risk of developing MS. Detection of TCR alpha chain polymorphisms, and HLA-DR and -DP typing were carried out by RFLP analysis on MS patients and healthy controls from the Melbourne metropolitan area. Interaction effects among these loci in producing susceptibility to MS were assessed by hierarchial log-linear analysis. Although HLA-DR2 was significantly associated with MS (chi 2 = 9.30, P = 0.002), no interactive effect between MS and HLA-DPw4 was observed. Significant interactions were observed with MS and both C alpha and V alpha, with the strongest effect seen with C alpha (chi 2 = 21.30, P less than 0.001). The combination of DR2/C alpha imparted a relative risk of 47. However, when the data were analysed for four and three way interactions, no significant effects were seen with MS, DR2, DPw4, V alpha, and C alpha, implying that the combined presence of these polymorphic markers is not essential for increasing susceptibility to MS.

Severe acute graft-versus-host disease by HLA-DPB1 disparity in recombinant family of bone marrow transplantation between serologically HLA-identical siblings: An application of the polymerase chain reaction—restriction fragment length polymorphism method

It has remained to be established that matching of the HLA-DP antigen plays a key role in bone marrow transplantation (BMT), mainly due to the difficulty of the primed lymphocyte test (PLT) method for DP typing. We previously reported an efficient technique for HLA class II genotyping by digestion of polymerase chain reaction (PCR)-amplified genes with restriction fragment length polymorphisms (RFLP) endonucleases (PCR-RFLP method). DNAs from 46 recipients and corresponding donors in serologically HLA-identical sibling-BMT cases were DP typed by this PCR-RFLP method. Of the 46 cases, five (10.9%) were genetically DP mismatched (recombinant frequency between the DR-DQ and DP subregions was at least 2.7% per meiosis), providing an important opportunity to look at the effect of the disparity only seen in the DP antigen on BMT. Three of the four DP-mismatched BMT cases that could be evaluated developed severe acute graft-versus-host disease, suggesting that DP disparity played an important role in BMT.

Genetic differences shown by HLA typing among Japanese patients with euthyroid Graves' ophthalmopathy, Graves' disease and Hashimoto's thyroiditis: genetic characteristics of euthyroid Graves' ophthalmopathy

Euthyroid Graves' ophthalmopathy (EO) is an ophthalmic disorder without persistent hyperthyroidism. To elucidate genetic differences among EO, Graves' disease (Gr) and Hashimoto's thyroiditis (H), we analysed HLA-A, B, C, DR, DQ, D and DP types in 23 Japanese EO patients, 88 Gr patients, 46 H patients and 186 control subjects utilizing assays of lymphocyte cytotoxicity and restriction fragment length polymorphism (RFLP). When compared with the control subjects, EO patients showed significant associations with HLA B40 (w61), DR9, DQw3, and Dw15 (P less than 0.01) and with HLA B12 and Cw1 (P less than 0.05). When allowance was made for the number of antigens tested, only DQw3 was significant. Significant differences were found between EO and Gr (DPw2), and between EO and H (Cw1) even after correction of P values. Comparisons between EO and related subgroups of Gr confirmed the heterogeneity of EO again. It is concluded from these results that EO is associated with different HLA types from Gr and H.

LIMITED HETEROGENEITY OF THE HLA CLASS II CONTRIBUTION TO SUSCEPTIBILITY TO RHEUMATOID ARTHRITIS IS SUGGESTED BY POSITIVE ASSOCIATIONS WITH HLA—DR4, DR1 AND DRwlO

Most patients with rheumatoid arthritis (RA) exhibit an HLA class II epitope found on most DR4 and DR1 molecules. We have investigated the possibility of other associations being present in the minority of patients lacking these antigens. One hundred and eighty patients with classical or definite RA and 100 controls were assigned HLA-DR, DQ and DP types by a DNA-based system using sequence-specific oligonucleotides to probe amplified class II alleles amplified by the polymerase chain reaction. The expected associations with DR4 (relative risk 10, P less than 0.0001) and DR1 (relative risk 2.3, P less than 0.001) were observed and only 13% of individuals lacked both of these alleles compared with 54% of age- and race-matched controls. A significant association with DRw10 (P = 0.02) was also observed in the DR4/DR1-negative RA group (3/23 patients compared with 0/54 DR4/DR1-negative controls). No novel associations with other DR, DQ, or DP alleles were evident in contrast to some previous studies. The third allelic hypervariable region of the DR beta chain of DRw10 contains a similar amino acid sequence to that found in many DR4 and DR1 molecules. These results extend this correlation and suggest that this susceptibility determinant may account for the HLA-linked susceptibility in 89% of our cases of RA.

Reassessment of HLA association with celiac disease in special reference to the DP association

Patients with the late-onset form of celiac disease have been studied for HLA association by conventional serology (DR and DQ typing) and by oligonucleotide probing with gene amplification (DP typing). Patients and controls were sampled in the Bologna area of northern Italy. Almost all patients were positive for DQw2 (94%), being DR3 positive (72%) and/or DR7 positive (65%). The proportion of DR3/7 heterozygotes in the patients was significantly increased over that expected from the Hardy-Weinberg equilibrium. No positive association with DR5 and no significant increase of DR5/7 heterozygotes were observed. Among the DP alleles reported to exhibit an association with celiac disease in other populations, only DPB3 showed a moderate increase of a borderline significance, not attributable to a linkage disequilibrium with DQw2.

A new PCR-RFLP DP typing method tested in homozygous and heterozygous cells

A new PCR-RFLP DP typing method tested in homozygous and heterozygous cells

Routine HLA DP typing which technique to choose?

Routine HLA DP typing which technique to choose?

Incidence and specificity of HLA-DP antibodies in pregnancy sera

A total of 630 human pregnancy sera were investigated for HLA-DP antibodies (ab) by monoclonal antibody-specific immobilization of leukocyte antigens (MAILA) using monoclonal antibody (mAB) B7/21.2 and selected B-lymphoblastoid cell lines from the Tenth International Histocompatibility Workshop reference panel. DP-specific abs were detected in 86 of 330 sera (26.1%) of a retrospective series selected for positive reactions in lymphocytotoxicity screening, and in 29 of 300 unselected sera (9.7%) of a prospective series. Approximately 80% of DP-reactive sera were also positive in lymphocytotoxicity test for class I and/or class II ab. On the other hand, sera containing lymphocytotoxic ab for both class I and class II ab revealed the highest incidence of DP ab (35%). Out of 115 DP-reactive sera, 28 clearly presented one or more DP specificities. Absorption/elution studies revealed complex patterns of reactivity in some sera which were similar to those of known mABs and possibly reflect supertypic DP specificities and/or serological cross-reactions. Serological DP typing of individuals by the MAILA technique appears promising.

A new approach for the analysis of HLA class II polymorphism: ‘HLA oligotyping’

Histocompatibility typing allows the matching of patients and donors in organ transplantation, and the accuracy of HLA matching influences to a great extent the clinical outcome. Recent breakthroughs in the molecular biology of HLA class II genes have revealed that the degree of HLA diversity and polymorphism is in fact much greater than was expected on the basis of the traditional serological HLA typing assays. In parallel, it has become possible to analyse this extensive polymorphism directly at the level of the HLA class II genes and of their DNA sequences. We have described a DNA typing procedure referred to as ‘HLA oligotyping’ which is based on the hybridisation of allele and loci specific oligonucleotide probes. This procedure has now become operational on a large scale and this review describes the principles and major applications of the technique. It consists in the hybridisation of DNA with informative sequence-specific oligonucleotide probes, following an amplification of DNA in vitro by the polymerase chain reaction (PCR). The use of this highly sensitive technique for HLA-DR, -DQ and -DP typing is discussed, focusing on the clinical applications in the field of organ transplantation, particularly for bone marrow transplantation with unrelated donors. It now allows the unambiguous identification of all HLA subtypes, including those that cannot be recognised otherwise, and it represents powerful complement to current methods of HLA typing. Finally this methodology is widely used in HLA-disease association studies, aiming at the characterisation of HLA class II epitopes involved in the susceptibility or resistance to autoimmune diseases.

一側性大脳障害例の頭振り後眼振について

The head-shaking test (HST) and the caloric test were used in the examination of 31 patients with unilateral internal capsule vascular lesions demonstrated by CT scan.Monophasic, biphasic and triphasic types of HSAN were observed in 13 patients (42%). In subtype A (3 patients, 10%) the first phase of nystagmus was to the side contralateral to that of the lesion, and in subtype B (10 patients, 32%) it was to the same side. The DP type of HSAN, which becomes slightly weaker and continues for at least 30 seconds, was observed in 14 patients (45%), 12 subtype A (39%) and 2 patients subtype B (6%). The first phase inhibited type of HSAN was seen in 1 patient subtype B (3%). No HSAN was observed in 3 patients (10%).Slow-phase velocity (SPV) could be determined in 30 patients; in 16 the caloric test caused DP nystagmus to the side of the lesion and in 14 to the contralateral side. There was no relationship between the direction or the SPV of the first nystagmus in the monophasic, biphasic or triphasic type of HSAN and the direction or the value of DP (%) in the caloric nystagmus. There was also no relationship between the direction of the DP type of HSAN and the direction or the value of DP (%) in the caloric nystagmus.Our results suggest that HSAN in patients with unilateral internal capsule lesions is due to a tonic imbalance, caused by the lesion, of the cortico-fugal tonic fibers ipsilaterally to the vestibulo-ocular system with eyes open in darkness.

A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.

The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing.

RFLP-defined HLA-DP polymorphism in four ethnic groups

Patterns of BglII, MspI, and TaqI DPα and DPß hybridizing restriction fragment length polymorphisms are compared with primed lymphocyte typing—determined specificities in 28 10th International Histocompatibility Workshop core cell lines. Correlation of specific RFLP patterns with most recognized DP types is confirmed, although the RFLPs do not distinguish DPw3 from DPw6. Analysis of DP-region nucleotide sequence data shows that the observed RFLPs are those expected to hybridize to the DPα and ß probes and are thus not due to cross-hybridization to other genes. The distribution of RFLPs in Chinese, Micronesian, South Indian, and white Australian populations is described. The most frequent DP specificity in Chinese is DPw5. In the other populations DPw4 is the most common specificity, although DPw6 is also relatively common in Micronesians. Four new DPα RFLP patterns and three new DPß patterns are described. There are also numerous unusual combinations of DPα and DPß alleles particularly in the South Indian population.

Discrimination of antibodies against antigens of different MHC loci in human sera by monoclonal antibody-specific immobilization of leukocyte antigens

To detect human antibodies against antigens of different major histocompatibility complex loci, particularly of class II specificity, a newly developed enzyme immunoassay for platelet antibodies was adapted for the use of lymphocytes as target cells. Periphral blood lymphocytes, phytohemagglutinin-stimulated T cells, or Epstein-Barr virus-transformed B cells were simultaneously incubated with a monomorphic class- or locus-specific monoclonal antibody and the human antibody to be investigated. After solubilization, cell lysates were transferred to an enzyme-linked immunosorbent assay tray coated with a goat anti-mouse Ig antibody. Following immobilization of the monoclonal antibody antigen complexes, human major histocompatibility complex antibodies were detected by addition of enzyme-labeled goat anti-human Ig. By means of this technique human antibodies against different major histocompatibility complex molecules present in the same sample could be clearly distinguished. Application of the monoclonal antibody-specific immobilization of lymphocyte antigens assay is presented by several examples. Of these, identification of DP-specific antibodies as well as serological DP typing are of particular interest.

Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP-alpha and DP-beta loci.

Abstract Allelic sequence variation of the HLA DP-alpha and DP-beta genes has been analyzed in a panel of 34 DP-typed cell lines. The polymorphic second exon of these genes was specifically amplified in vitro by the polymerase chain reaction method, using the thermostable DNA polymerase of Thermus, aquaticus. The analysis of M13 clones containing the amplified DP-beta sequences revealed a total of 14 allelic variants. In general, specific allelic DP-beta sequences were associated with each of the defined DPw1-w6 types, with beta allele subtypes revealed for the DPw2 and DPw4 specificities. An additional six DP-beta alleles which did not correlate with any of the T cell-defined specificities (DP "blanks") were also identified. Only the two previously characterized alleles of DP-alpha were detected. These observations suggest that the T cell-defined DP specificities are determined by polymorphic residues on the beta-chain. The sequence polymorphisms in DP-beta are clustered in a few specific regions, and can be detected using sequence-specific oligonucleotide probes and polymerase chain reaction amplified DNA in a rapid dot-blot format. This approach provides a simple and informative method of DP typing. The DP-beta sequences derived from four DP-typed celiac disease patients were compared with the distribution of DP-beta alleles in control individuals.

The susceptibility to insulin-dependent diabetes mellitus is associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3,4 heterozygotes.

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P = 6 x 10(-6)): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P = 0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P = 0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Two alleles detected for HLA-DZ alpha on the basis of a polymorphic restriction site in the third exon.

The restriction enzyme ApaI has been used to define Restriction Fragment Length Polymorphism (RFLP) within the DZ alpha gene. Digestion of genomic DNA of 96 individuals with ApaI reveals the existence of two different patterns. Both homozygous and heterozygous individuals were detected. Hardy-Weinberg analysis indicates that DZ alpha is part of a di-allelic system with allele frequencies of 50%. DZ alpha polymorphism in the population studied does not correlate with serologically determined class I and HLA-DR, DQ and cellular DP typing data. A limited family study demonstrates that DZ alpha polymorphism segregates with HLA-class II phenotypes.