Marius J. Giphart

Biotinylated DRB sequence-specific oligonucleotides : comparison to serologic HLA-DR typing of organ donors in Eurotransplant

A novel HLA-DR typing method was applied using PCR-amplified fragments and biotin-labeled oligonucleotides (PCR-biotin-SSO). The PCR-biotin-SSO method can be used efficiently to perform HLA-DR typing for a large number of individuals when time is not the limiting factor. The reliability of HLA typing of cadaveric organ donors is of vital importance for organ exchange organizations such as ET. Due to lack of time, these typings are usually performed by the complement-dependent cytotoxicity. The individual donor center typings are immediately reported to ET, where the recipient selection procedure is started. DNA isolated from donor spleen material, sent to the ETRL for retyping purposes, was subjected to PCR-biotin-SSO typing. The results were compared with the serological HLA-DR typings as reported to ET. The analysis of 1052 donor samples for the broad DR1-DR10 antigens revealed a concordance rate of over 90% between the donor center and the ETRL. The majority of the discrepancies involved specificities of the HLA-DR5, DR6, and DR8 cross-reacting group, with DR6 as the predominant discordant specificity. The results indicate (a) that PCR-biotin-SSO is a reliable technique for DNA-based HLA-DR typing and (b) that HLA-DR serology is still a useful technique when time is limited, such as for cadaveric donor typing.

Polymorphism of HLA-DRB, -DQA1, and -DQB1 in rheumatoid arthritis in Asian Indians: Association with DRB1 *0405 and DRB1 *1001

We investigated the DRB, DQA1, and DQB 1 polymorphism and haplotypes in sporadic and familial RA subjects of Asian Indian origin by PCR oligotyping using biotinylated SSOPs. Molecular subtyping of DRB 1*04 in RA patients showed strongest association with highest relative risk with DRB 1*0405, followed by DRBI*0401. A significant decreased frequency of DRBI*1502 was observed in patients compared to controls (chi 2 = 4.5). Among other alleles, DRBI*1001 was found to be significantly increased. A total of 73.3% of patients carried the shared sequence of the third HVR (67-74) of DRB1 domain compared to its presence in only 37.6% of controls. A significant number of patients carried DR4 haplotypes on DQBI*0302 (58%) as against DQBI*0301 which was present only on 10.5% of the haplotypes. When compared to controls, the difference was significant for the latter allele only. Few unique DRDQ haplotypes were observed in Asian Indians. Among DR-DQ haplotypes, DRB1*0401-DQB1*0302 gave the highest risk whereas DRB1*0403-DQB1*O301 was negatively associated. Alleles with negative charge at position 70 confer protection or are negatively associated with RA whereas among the associated alleles, glycine at position 86 resulted in higher risk than those with valine at this position. A heterogenous association of DQB1 alleles with DR4 subtypes, influencing susceptibility to RA, suggests the DQB locus is not primarily associated with RA and susceptibility lies in the sequence 67-74 of the DRB1 loci.

Tumor necrosis factor α-308 gene variants in relation to major histocompatibility complex alleles and Felty's syndrome

Abstract The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5′ regulatory region of the human TNFA gene a G ( TNFA −308G ) to A ( TNFA −308A ) transition polymorphism at position −308 was discovered. We have developed a simple PCR assay to facilitate the screening of the −308 polymorphism at the DNA level. In view of the possible linkage between the TNFA −308A allele and a certain MHC type, TNFA −308 genotypes in HLA-typed healthy individuals ( n = 88) were determined. A statistically significant association between the TNFA −308A allele and HLA-DR3, DQB1 ∗ 0201, DQA1 ∗ 0501, A1, B8, and the Ncol 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA −308A allele in patients with FS ( n = 13), an HLA-DR4-associated disease. In this study, no association was found of Feltys syndrome with the TNFA −308A allele, indicating that this allele does not appear to be a susceptibility factor for FS. Human Immunology 41, 259–266 (1994) .

Asian Indian HLA-DR2-, DR4-, and DR52-related DR-DQ genotypes analyzed by polymerase chain reaction based nonradioactive oligonucleotide typing unique haplotypes and a novel DR4 subtype

We have employed a PCR-based nonradioactive technique using biotinylated SSOPs to define HLA-DR2-, 4-, DR51-, and DR52-associated DR-DQ genotypes in Asian Indian families. In the DR2 group, most haplotypes described by us in a previous study were confirmed by family analysis. Evidence for one additional haplotype was available in this study. The classic DRB1*1501- and DRB1*1502-associated caucasoid haplotypes occurred with an appreciable frequency in Asian Indians, but two of the DRB1*1601-associated Caucasoid haplotypes were absent. At least six unique and unusual DR2-associated genotypes were encountered. In the DR52 group, the three most common alleles are DRB1*0301, DRB1*1404, and DRB1*1101. The DR6-associated alleles were DRB1*1301, 1302, 1401, and 1404. A few unique haplotypes occurred with low frequency in this group. In the DR4 group, at least three unusual patterns of hybridization were noticed by family analysis. One of these appears to be a novel DR4 subtype upon sequencing. These results demonstrate that, besides HLA-DR2, appreciable complexity occurs in the DR4- and DR52-associated alleles among Asian Indians. The presence of unique DR-DQ haplotypes in addition to those found characteristically among Western Caucasians suggests that the Indian population provides valuable source of many HLA class II haplotypes.

Distribution of HERV-LTR elements in the 5'-flanking region of HLA-DQB1 and association with autoimmunity.

Abstract. We established the detailed polymorphism of the 5′-flanking region and the first exon of the human leukocyte antigen (HLA)-DQB1 alleles. One hundred and forty-five Spanish rheumatoid arthritis (RA) patients and 200 healthy voluntary blood donors from southern Spain along with 42 B-cell lines were analyzed for the presence of the retrovirus-derived long terminal repeats (LTRs) LTR3, LTR5, and LTR13. LTR3 positivity was always associated with certain DQB1 alleles, i.e., *0302, *0402, *0601, *0202, and *0305. Sequencing analysis of the 5′-flanking region of DQB1*0301, *0303 and *0502 alleles in homozygous B-cell lines showed the absence of LTR3 and a massive deletion of 5635 base pairs. The undetected deletion in the flanking region of some DQB1 alleles and a lack of stratification for HLA typing explain previously reported associations of the LTR3 element with RA and type I diabetes (IDDM). LTR5 showed identical distribution to LTR3, consistent with a previously suggested LTR3-LTR5 tandem arrangment. LTR13 positivity was associated with DQB1*0302, *0303, and *0402 alleles. Distributions of the LTR elements in all B-cell lines, RA patients, and controls could be explained entirely by linkage disequilibrium with DQB1 alleles, independently of the haplotypes carrying them. LTR elements are known to regulate gene expression. Therefore, a possible involvement of LTR13 in the association of DQB1*0302, *0303, and *0402 with IDDM requires further investigation. The sequencing results of the DQB1 first exon demonstrated that DQB1*0601 was generated by a recombination event between a DR53 and a non-DR53 haplotype. Our results shed new light on the phylogeny of the HLA region and the possible contribution of DQB1 to susceptibility to autoimmunity.

Association of interferon-γ and interleukin 10 genotypes and serum levels with partial clinical remission in type 1 diabetes

We studied whether serum interferon (IFN)‐γ or interleukin (IL)‐10 levels and their corresponding functional polymorphic genotypes are associated with partial remission of type 1 diabetes (T1D). A multi‐centre study was undertaken in patients with newly diagnosed T1D and matched controls. T1D patients were followed for 3u2003months and characterized for remission status. Partial clinical remission was defined as a daily insulin doseu2003≤u20030·38 units/kg/24u2003h with an HbA1cu2003≤u20037·5%. Thirty‐three patients and 32 controls were phenotyped for serum concentrations of IFN‐γ and IL‐10 and genotyped for functional polymorphisms of the IFN‐γ and IL‐10 genes. Sixteen of 25 informative patients (63%) remitted. Serum IFN‐γ concentrations were significantly decreased in remitters but increased in non‐remitters compared to controls, and did not change over time in any group. IFN‐γ genotypes corresponded with serum levels in controls and non‐remitters, but not in remitters who displayed the lowest serum IFN‐γ levels despite more often carrying high‐producing IFN‐γ genotypes. Neither the frequency of IL‐10 genotypes nor serum IL‐10 concentration differed between patients and controls. The combination of high‐producing IFN‐γ genotype together with low serum IFN‐γ concentration at the time of diagnosis provided a strong positive predictive value for remission. Serum IFN‐γ concentrations predicted by genotype and observed serum levels were discordant in remitters, suggestive of regulation overruling genetic predisposition. Although high‐producing genotypes were less frequent in remitters, they were predictive of remission in combination with low serum IFN‐γ levels. These data imply that remission is partially immune‐mediated and involves regulation of IFN‐γ transcription.

Matching for TNF microsatellites is strongly associated with matching for other non-HLA MHC sequences in unrelated bone marrow donor-recipient pairs

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings. We have demonstrated previously that matching of unrelated donors and recipients for TNFa microsatellites is correlated with lower CTLp frequencies. Matching of unrelated donors and recipients for other non-HLA sequences in the major histocompatibility complex has been reported to result in less graft-versus-host disease and improved survival. It has been argued that matching for non-HLA sequences in the MHC in addition to the HLA genes themselves results in matching for the entire MHC and is therefore the equivalent of providing an HLA identical sibling donor. In order to test this hypothesis we have examined TNFa microsatellites of unrelated donor recipient pairs in whom matching for HLA loci, non-HLA sequences near HLA B (beta-block markers) and non-HLA sequences near DRB1 (delta-block markers) had been determined. All 17 patients who were matched for HLA and non-HLA markers were also matched for TNF microsatellites. This data supports the idea that matching for HLA genes and non-HLA markers results in matching at all other loci in the MHC.

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.

D6STNFa Microsatellite Locus Correlates with CTLp Frequency in Unrelated Bone Marrow Donor-Recipient Pairs

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings, primarily due to an increased rate of graft-versus-host-disease. HLA matching for donors and recipients is the most important factor influencing the outcome of BMT. However, unrelated donor selection generally relies on matching only for HLA antigens without considering potential incompatibility for other MHC loci. Cellular assays have been developed to predict incompatibility that cannot be detected by current typing methods. The CTLp frequencies correlate with the degree of incompatibility of patient/donor and the clinical grade of GVHD. Since the CTLp assay is expensive and time consuming, an alternative is wanted. We studied the means of matching for microsatellites in determining MHC identity and possible correlation with CTLp frequencies. Therefore, 26 recipient/donor pairs were analysed for eleven microsatellite loci within and around the MHC region. Our study provides evidence that the D6STNFa locus correlates with CTLp frequency. The D6STNFa locus provides an additional marker that may help to improve the matching of unrelated donors and bone marrow recipients.

Subtyping for TNFa microsatellite sequence variation.

Abstract. The microsatellite locus TNFa is frequently used as an additional genetic marker in studies of the major histocompatibility complex (MHC). Novel sequence variations at the TNFa locus have been described, and which may have implications for genetic analyses. In this study, we set up a nested polymerase chain reaction-sequence-specific primer (PCR-SSP) approach to type for these TNFa sequence variations. First, sequencing analysis of workshop B lymphoblastoid cell lines (n=13) showed the presence of three sequence variations upstream of the dinucleotide repeat at TNFa. Using nested PCR-SSP, we were able to detect these variations in a larger B lymphoblastoid cell line panel (n=34). Furthermore, we were able to show that TNFa alleles a7 and a10 are present in two distinct conformations leading to splitting of TNFa alleles exhibiting identical fragment lengths. To establish the frequency of the TNFa alleles and their variants, we performed microsatellite typing of a large panel of random individuals from the Dutch population (n=272). Subsequent nested PCR-SSP typing showed the presence of three previously described sequence variations in the Dutch population. Furthermore, the presence of a fourth subtype was established. The described variations of allele TNFa7 and TNFa10 are present in the random population with significant frequencies. Haplotyping analysis between HLA-DR, TNFa, and HLA-B showed that allele TNFa7.2 is present in an extended DR7-TNFa7.2-B13 haplotype. In this way, we were able to show that the additional sequence variations behave like distinct TNFa alleles.