HLA allele detection using molecular techniques.

There are now many molecular biological techniques available to define HLA class I and class II alleles. Some of these are also applicable to other human polymorphic genes, in particular to those non-HLA genes encoded within the Mhc. The range of techniques available allows laboratories to choose those most suited to their purpose. The routine laboratory supporting solid organ transplants will need to type large numbers of potential recipients over a period of time, probably using PCR-SSOP while donors will be typed singly and rapidly using PCR-SSP with HLA allele compatibility determined by heteroduplex analysis. Laboratories supporting bone marrow transplantation, where time is less pressing, can choose from the whole range of techniques to determine accurately donor recipient Mhc compatibility. For disease studies, techniques defining precise HLA allele sequence polymorphisms are needed and high sample numbers have to be accommodated. When an association is established allele sequencing has to be used. In the near future, the precise role of HLA alleles in transplantation and disease susceptibility is likely to be established unambiguously.

Interbreed variation of DLA-DRB1, DQA1 alleles and haplotypes in the dog.

Although 36 DLA-DRB1 and 10 DLA-DQA1 allele sequences have been published to date, no data on individual allele frequencies exists, either for specific breeds or cross breeds, and the full extent of the polymorphism at each of these loci is still not known. We have used sequence-specific oligonucleotide probing (SSOP) to characterise a series of 367 dogs for their DRB1 and DQA1 alleles. These included individual animals from over 60 different breeds, with numbers per breed ranging from 1 to 39. DLA types were generated from 218 dogs for DRB1 and from 330 dogs for DQA1, while 181 dogs were characterised for both these loci. The frequency of individual DRB1 and DQA1 alleles showed considerable interbreed variation, e.g. 83% of West Highland White Terriers were DRB1*01 as opposed to 9% of Collies. No breed had >9 of the 22 DRB1 types defined in this study; several breeds had only two DRB1 types. DLA-DQA1 showed less variation in allele numbers per breed, but also showed considerable interbreed frequency variation. Haplotype analysis revealed over 44 different DRB1/DQA1 combinations. Of these, 25 were in a number of animals, and also in an animal that was homozygous for one or both of these loci. Some DRB1 alleles could be found in combination with several different DQA1 alleles, while others were only present in one haplotypic combination. DLA allele frequency data in normal dogs will be critical for disease association studies. It may also be possible to use haplotype data to establish the genetic relationships between different dog breeds.

The IL-10 -1082G polymorphism is associated with clearance of HPV infection.

The role of cytokines in protecting against human papillomavirus (HPV) and HPV-associated disease is not fully understood. We compared the frequency of the interleukin (IL)-10 polymorphism (G allele) at position --1082 and the distribution of GG/GA/AA genotypes among 116 HPV-positive women, grouped according to their cervical cytological profiles, with 119 HPV-negative controls with normal smears. No difference was observed in genotype frequency between the groups. Among women in the HPV-positive, smear-normal group, who were re-tested for HPV after 12 months, there was a significant inverse association between presence of at least one variant G allele (high activity) and HPV persistence (OR per G allele = 0.082 [95% CI 0.009-0.73], P= 0.001; after controlling for ethnicity). This association remained significant after controlling for age, smoking and hormonal contraception (OR = 0.028 [95% CI 0.001-0.66], P= 0.001). This preliminary study suggests that higher levels of IL-10 may prevent cervical neoplasia through their role in eliminating HPV.

[A custom grooved impression tray]. / Le porte-empreinte individuel à logettes.

Individual compartment trays can be used for dental impression in fixed prosthodontics and represent an original method which alleviates the pitfalls of standard technics. This method is based upon a strict adaptation of the tray to both the preparations and the dental arcade using the primary impression. Impression pastes can be used in their optimal conditions using the I.C.T. (Individual Compartment Trays) and a higher definition of cervical limits can be obtained. The use of I.C.T. reduces the risks of interposing free marginal gingival and eliminates the inconvenients of intrasulcular leakage.

HLA: linkage with rheumatoid arthritis or seropositivity.

HLA haplotype sharing was compared in sibships from multicase families with rheumatoid arthritis (RA), subdivided by rheumatoid factor status, to investigate the claim that HLA is linked more to RA severity than susceptibility. Considerable deviation from expected (Mendelian) inheritance towards greater sharing of inherited parental haplotypes was observed in the sibships as a whole and when subdivided according to the serological status of the sibship's members. Further, there was no evidence that linkage was stronger in the seropositive concordant than in the other sibships. Linkage was also demonstrated between HLA and seropositivity even in sibships where not all members expressed clinical RA. These results, therefore, do not support the existence of genetic heterogeneity between seropositive and seronegative RA, a possibility previously suggested from population studies of antigen associations.

Rheumatoid factor detection in the unaffected first degree relatives in families with multicase rheumatoid arthritis.

Rheumatoid factor (RF) was assayed in 354 unaffected first degree relatives from 59 families with multicase rheumatoid arthritis (RA), using both the latex test and ELISA for different RF isotypes. The association between HLA and presence of RF was also analyzed. In all, 39% of the relatives were positive for at least one class of RF compared to 7% positive for latex. There were no differences between the sexes in these rates. There were significant excess rates of HLA-DR4 in RF positive when compared to the RF negative relatives. The HLA-DR4 frequency in these RF positive RA negative individuals is similar to that observed in series of RA positive patients. Thus HLA status did not usefully discriminate between RA positive and RA negative but RF positive individuals in these families.

Maternal exposure to paternal HLA does not explain the postpartum increase in rheumatoid arthritis.

The postpartum period, particularly after the first pregnancy, represents a time of increased risk for the development of rheumatoid arthritis (RA). The present study was undertaken to investigate whether this increase in risk may be due to maternal exposure to fetally inherited paternal HLA-DR antigens that were either 1) similar to their own or 2) had an increased likelihood of being one of the two specific types, HLA-DR1 and DR4, implicated in the etiology of RA. We recruited 94 families where the mother had developed RA within 12 months of a pregnancy, and HLA typed the mother, father, and relevant child of each family. Mothers were not more likely to share HLA-DR genes with their partners than would be expected, and children whose parents shared one HLA-DR gene were not more likely to inherit the shared gene from their father as opposed to the non-shared gene. Further, those children whose fathers were heterozygous for HLA-DR1 or DR4 were not more likely to inherit these genes as opposed to the non-DR1/DR4 gene. In conclusion, maternal exposure during pregnancy to either fetally inherited paternal HLA-DR1 and DR4 genes or to paternal DR genes similar to their own does not appear to contribute to postpartum maternal susceptibility of RA.

Incidence of rheumatoid arthritis in a genetically predisposed population.

A follow-up study (mean duration five years) was undertaken on 370 previously unaffected first degree relatives from multicase rheumatoid arthritis (RA) families. The objectives were to determine the incidence of RA in this group and the possible predictors of disease development. In all, 14 individuals developed RA, equivalent to an incidence of 8/1000 person-years of observation. There was no control group included in this study as the intensive-follow-up required substantial compliance from highly motivated families. Population-based estimates, however, from a number of sources would suggest an annual incidence below 0.5/1000 per year, substantially less than the rate obtained in this study. The small number of incident cases precluded definitive conclusions about risk factors within these families but there were no important effects of age or sex. Possession of HLA-DR1 or DR4 explained only some of the increased risk. The increased incidence observed in the previously unaffected relatives of such families would suggest that this familial clustering did not for the most part arise by chance and that other shared genetic or environmental influences are relevant.

Are both genetic and reproductive associations with rheumatoid arthritis linked to prolactin?

The risk of rheumatoid arthritis (RA) seems to be associated with reduced fecundity and with breastfeeding; these apparently contradictory risk factors can be explained by their association with high prolactin concentrations. The only consistent genetic association with RA is for genes encoded in the HLA complex, particularly HLA DR4. We have identified some data indicating that the effects of breastfeeding and nulliparity are modified by HLA DR4 status, suggesting an interaction between genetic and reproductive risk factors in the aetiology of RA. The prolactin gene is in close proximity to the HLA region on the short arm of chromosome six. We therefore propose the hypothesis that the associations between DR4 and reproductive risk factors in RA are due to linkage disequilibrium between DR4 and an abnormally regulated prolactin gene polymorphism.

HLA heterozygosity contributes to susceptibility to rheumatoid arthritis.

We have investigated the role of HLA-DR genotypes in 184 patients with severe rheumatoid arthritis (RA) and in 46 patients with Felty syndrome, to establish the relative contribution of the RA-associated subtypes of DR4 (Dw4, Dw14, and Dw15). There was an excess of DR4 homozygotes, particularly Dw4/Dw14 compound heterozygotes (relative risk [RR] 49). The risk associated with Dw4 depended on the other allele present--Dw4/DR1 (RR 21), Dw4/Dw4 (RR 15), and Dw4/DRX (RR 6). There was a significant risk from Dw4/Dw14 compared with Dw4/Dw4, both in those with severe RA (RR 2.9; P less than .02) and in those with Felty syndrome (RR 4.2; P less than .02). In contrast, in a further 63 known DR4 homozygotes with RA, not selected for severe disease, the excess of Dw4/Dw14 was much less striking (RR 1.4; not significant), suggesting that this genotype may be particularly associated with more severe disease. We also found four cases with the rare Dw4/Dw15 genotype (expected less than or equal to 0.5; P less than or equal to .02). Since the Dw4, Dw14, Dw15, and DR1 molecules have similar antigen-binding sites and since combinations of these alleles particularly predispose to severe RA, we suggest that synergistic mechanisms are involved. These could include an effect on T-cell repertoire selection.

Allelic markers close to prolactin are associated with HLA-DRB1 susceptibility alleles among women with rheumatoid arthritis and systemic lupus erythematosus.

OBJECTIVE: To investigate linkage disequilibrium between HLA-DRB1 disease susceptibility alleles and microsatellite markers close to the prolactin gene, among women with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and normal controls. METHODS: DNA from 89 women with RA, 76 women with SLE, and 94 controls was typed for HLA-DRB1 status and D6S422 and D6S285, 2 highly polymorphic microsatellite markers close to the prolactin gene. RA patients were stratified by DRB1*0401 status, and SLE patients were stratified by *0301 status. RESULTS: There was an excess frequency of D6S422*1 among SLE patients with DRB1*0301 (odds ratio [OR] 3.1). The frequency of this allele was also slightly in excess among RA patients with DRB1*0401 (OR 1.9). D6S285*5 was also in excess among female RA patients with DRB1*0401 (OR 3.5), and was slightly increased among female SLE patients with DRB1*0301. None of these alleles were found to be increased among *0401-positive or *0301-positive controls. CONCLUSION: These data indicate that there may be linkage disequilibrium between HLA-DRB1 alleles and microsatellite marker alleles close to the prolactin gene among women with RA and SLE. This suggests the possibility of extended haplotypes encoding for HLA-DRB1 susceptibility and high prolactin production, which contribute to susceptibility to both RA and SLE.

Quantifying the exact role of HLA-DRB1 alleles in susceptibility to inflammatory polyarthritis: results from a large, population-based study.

OBJECTIVE: To accurately determine the contributions of HLA-DRB1 alleles in explaining susceptibility to inflammatory polyarthritis in a large, true population-based cohort of new-onset cases. METHODS: A cohort of 680 consecutive patients with inflammatory polyarthritis, of whom 404 satisfied the American College of Rheumatology (ACR) criteria for rheumatoid arthritis (RA), was recruited from the population-based Norfolk Arthritis Register. All cases were compared with 286 local population controls. A standardized clinical assessment was performed on all patients. HLA-DRB1 phenotypes, including DR4 subtypes, were determined using a semiautomated, reverse dot-blot method. Results were expressed as odds ratios (OR) and 95% confidence intervals (95% CI). RESULTS: There was only a modest association (OR 1.8, 95% CI 1.4-2.4) between inflammatory polyarthritis and the presence of any shared epitope (SE) allele; the strongest individual risk was with DRB1*0404 (OR 3.5, 95% CI 1.8-6.8). Comparison of the genotypes demonstrated that the effect of being SE homozygous (OR 2.1, 95% CI 1.5-3.0) was only moderately greater than the effect of being SE heterozygous (OR 1.3, 95% CI 1.1-1.6). The exception to this was genotypic combinations that included HLA-DRB1*0404, which exhibited ORs ranging up to 18.0. There were no differences between either the phenotype or genotype data when the patients were stratified by RA status (defined by the ACR criteria). In contrast, the associations were substantially stronger in patients who were positive for rheumatoid factor. CONCLUSION: Previous studies had not been able to clarify whether the influence of HLA-DRB1 on RA was related to disease susceptibility or to disease severity and progression. These data on a unique population-based incident cohort suggest only weak effects on susceptibility, with the exception of the clearly distinct influence of HLA-DRB1*0404.

DLA-DRB1 polymorphisms in dogs defined by sequence-specific oligonucleotide probes (SSOP).

To date, DNA sequences for 29 dog DLA-DRB1 alleles have been reported. However, no data exists on the frequencies of these alleles within the general dog population, nor is there any indication of whether there is interbreed variation of allele distribution. We have addressed this by establishing a molecular based sequence-specific oligonucleotide probing (SSOP) method to identify all of the known broad DRB1 types and we have used this to type a random panel of dogs. A series of oligonucleotide probes were designed to detect known polymorphisms in the three DRB1 hypervariable regions, together with two distinctive motifs in other regions of exon 2. This set of probes enabled us to assign broad DRB1 types. Two hundred and eighteen dogs were SSOP typed for DRB1. All but 4 of the published DLA-DRB1 alleles were identified in these animals. Interbreed variation in both allele distributions and allele frequencies were observed, which may be useful in the study of genetic variation between breeds. This variation also has implications for the selection of control groups for studies aimed at identifying MHC associations with disease susceptibility in the dog.

The influence of HLA-DRB1 alleles and rheumatoid factor on disease outcome in an inception cohort of patients with early inflammatory arthritis.

OBJECTIVE: There are conflicting data concerning the role of HLA-DRB1 alleles in disease outcome in early rheumatoid arthritis. The exact role of these alleles in short-term outcome is determined in this large, prospective, population-based study. METHODS: We recruited 532 patients with inflammatory polyarthritis from the Norfolk Arthritis Register and typed their sera for HLA-DRB1 alleles using polymerase chain reaction-based methods. Disease outcome was assessed at 2 years in terms of persistent joint inflammation, functional disability, and radiologic erosions. Results are expressed as risk ratios (RR) with 95% confidence intervals (95% CI). RESULTS: There was no influence of HLA-DRB1 alleles, in any combination, on the likelihood of disease persistence, and only a modest effect on functional disability (Health Assessment Questionnaire score > or = 1). The most obvious effect was on the development of erosions (RR 1.9, 95% CI 1.4-2.6 for those who carried at least 1 DRB1 shared epitope [SE] allele), with slightly greater effects for those who were homozygous for SE-bearing alleles (RR 2.5, 95% CI 1.8-3.6). This effect of HLA-DRB1 was restricted to patients whose sera were negative for rheumatoid factor. Among patients with erosions, HLA-DRB1 had no influence on the severity of radiologic damage (defined as the number of eroded joints, or total Larsen score). CONCLUSION: These data do not support routine HLA-DRB1 screening of patients with early arthritis to identify those at risk for subsequent severe disease.

Identification of further DLA-DRB1 and DQA1 alleles in the dog.

Three novel DLA-DRB1 alleles and one novel DQA1 allele have been identified in a panel of 367 dogs. These were suggested by unusual reaction patterns found in sequence specific oligonucleotide probing (SSOP) data. Four new alleles were confirmed using DNA cloning and sequencing.