Validation of an RNA cell cycle progression score for predicting death from prostate cancer in a conservatively managed needle biopsy cohort.

BACKGROUND: The natural history of prostate cancer is highly variable and difficult to predict accurately. Better markers are needed to guide management and avoid unnecessary treatment. In this study, we validate the prognostic value of a cell cycle progression score (CCP score) independently and in a prespecified linear combination with standard clinical variables, that is, a clinical-cell-cycle-risk (CCR) score. METHODS: Paraffin sections from 761 men with clinically localized prostate cancer diagnosed by needle biopsy and managed conservatively in the United Kingdom, mostly between 2000 and 2003. The primary end point was prostate cancer death. Clinical variables consisted of centrally reviewed Gleason score, baseline PSA level, age, clinical stage, and extent of disease; these were combined into a single predefined risk assessment (CAPRA) score. Full data were available for 585 men who formed a fully independent validation cohort. RESULTS: In univariate analysis, the CCP score hazard ratio was 2.08 (95% CI (1.76, 2.46), P<10(-13)) for one unit change of the score. In multivariate analysis including CAPRA, the CCP score hazard ratio was 1.76 (95% CI (1.44, 2.14), P<10(-6)). The predefined CCR score was highly predictive, hazard ratio 2.17 (95% CI (1.83, 2.57), χ(2)=89.0, P<10(-20)) and captured virtually all available prognostic information. CONCLUSIONS: The CCP score provides significant pretreatment prognostic information that cannot be provided by clinical variables and is useful for determining which patients can be safely managed conservatively, avoiding radical treatment.

Prognostic value of PTEN loss in men with conservatively managed localised prostate cancer.

BACKGROUND: The natural history of prostate cancer is highly variable and difficult to predict. We report on the prognostic value of phosphatase and tensin homologue (PTEN) loss in a cohort of 675 men with conservatively managed prostate cancer diagnosed by transurethral resection of the prostate. METHODS: The PTEN status was assayed by immunohistochemistry (PTEN IHC) and fluorescent in situ hybridisation (PTEN FISH). The primary end point was death from prostate cancer. RESULTS: The PTEN IHC loss was observed in 18% cases. This was significantly associated with prostate cancer death in univariate analysis (hazard ratio (HR)=3.51; 95% CI 2.60-4.73; P=3.1 × 10(-14)). It was highly predictive of prostate cancer death in the 50% of patients with a low risk score based on Gleason score, PSA, Ki-67 and extent of disease (HR=7.4; 95% CI 2.2-24.6; P=0.012) ), but had no prognostic value in the higher risk patients. The PTEN FISH loss was only weakly associated with PTEN IHC loss (κ=0.5). Both PTEN FISH loss and amplification were univariately predictive of death from prostate cancer, but this was not maintained in the multivariate analyses. CONCLUSION: In low-risk patients, PTEN IHC loss adds prognostic value to Gleason score, PSA, Ki-67 and extent of disease.

Prognostic value of a cell cycle progression signature for prostate cancer death in a conservatively managed needle biopsy cohort.

BACKGROUND: The natural history of prostate cancer is highly variable and it is difficult to predict. We showed previously that a cell cycle progression (CCP) score was a robust predictor of outcome in a conservatively managed cohort diagnosed by transurethral resection of the prostate. A greater need is to predict outcome in patients diagnosed by needle biopsy. METHODS: Total RNA was extracted from paraffin specimens. A CCP score was calculated from expression levels of 31 genes. Clinical variables consisted of centrally re-reviewed Gleason score, baseline prostate-specific antigen level, age, clinical stage, and extent of disease. The primary endpoint was death from prostate cancer. RESULTS: In univariate analysis (n=349), the hazard ratio (HR) for death from prostate cancer was 2.02 (95% CI (1.62, 2.53), P<10(-9)) for a one-unit increase in CCP score. The CCP score was only weakly correlated with standard prognostic factors and in a multivariate analysis, CCP score dominated (HR for one-unit increase=1.65, 95% CI (1.31, 2.09), P=3 × 10(-5)), with Gleason score (P=5 × 10(-4)) and prostate-specific antigen (PSA) (P=0.017) providing significant additional contributions. CONCLUSION: For conservatively managed patients, the CCP score is the strongest independent predictor of cancer death outcome yet described and may prove valuable in managing clinically localised prostate cancer.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer.

BACKGROUND: Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH). METHODS: Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines. RESULTS: Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10(-11)). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10(-5) and 10(-29)), and identified breast and pancreatic cell lines with BRCA defects. CONCLUSION: The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Cellular immune mechanisms in rheumatoid arthritis and other inflammatory arthritides.

Advances in molecular genetics, cellular immunology and microbiology have offered promise in unravelling the aetiopathogenesis of inflammatory arthritides such as rheumatoid arthritis and reactive arthritis. Such insights are challenging the orthodox view that these diseases are primarily autoimmune in nature, and should lead to exciting and novel therapeutic approaches.

Allelic variation at the interleukin-1 receptor antagonist gene is associated with early postmenopausal bone loss at the spine.

Genetic factors play an important role in determining bone mineral density (BMD) in later life, with the genetic influence mediated through effects on both peak mass and on age- and menopause-related bone loss. At menopause there is an increase in the production and activity of various cytokines and growth factors within the bone microenvironment. The activity of interleukin-1 (IL-1), a powerful stimulant of osteoclastic bone resorption, is increased in estrogen-deficient states with increased production of IL-1 and inhibition of the IL-1 receptor antagonist (IL-1ra). Treatment with IL-1ra blocks the bone loss associated with ovariectomy in animals and the IL-1 receptor antagonist gene (IL-1RN) is therefore a potential candidate gene for the regulation of postmenopausal bone loss. We examined the relationship between annual rates of change in BMD over 5 years and an 86 bp variable number tandem-repeat polymorphism of the IL-1RN gene in 108 early postmenopausal women. All women were within 5 years of a natural menopause at the study's onset, healthy, and not on hormone replacement therapy or other medication known to affect bone metabolism. BMD was measured annually over the 5 year study period at the lumbar spine and femoral neck using dual-energy X-ray absorptiometry. Three alleles were identified (A1 = 4 repeats, A2 = 2 repeats, A3 = 5 repeats), with five genotypes observed: A1A1 (41.7%), A1A2 (45.4%), A2A2 (6.5%), A1A3 (2.8%), and A2A3 (3.7%). For analysis, alleles were collapsed into a biallelic system grouping the A1 and A3 alleles. There was no significant relationship between the IL-1RN genotypes and baseline bone mass at either the spine or hip. IL-1RN genotype was significantly associated with annual rates of change in spinal bone mass (p < 0.05), and this finding remained significant after adjustment for age, weight, and baseline BMD. Carriage of at least one copy of the A2 allele was associated with reduced bone loss at the spine (mean change in BMD +/- SD: -0.81 +/- 1.46%/year) when compared with noncarriage of the A2 allele (mean change -1.38 +/- 1.48%/year), p = 0.05. We therefore conclude that allelic variation at the IL-1RN locus is associated with differential rates of early postmenopausal bone loss at the spine. Further research will be required to clarify the mechanisms underlying these findings and to determine whether this association translates into a significant long-term effect on BMD and fracture in later life.

Strategy for controlling preferential amplification and avoiding false negatives in PCR typing.

The use of the PCR method for routine testing has increased dramatically during recent years. Most assays involve co-amplification either of an internal control, of several alleles at a given locus or of a variety of bands produced by low-stringency primer annealing. In such multiplex reactions, certain products will often amplify preferentially. Amplimers that are more sensitive can be outcompeted under suboptimal PCR conditions, leading to assignment of false negatives. Optimization of PCR parameters such as temperature steps, relative concentrations of primers and their annealing temperature do not alone ensure against false negatives when caused by stable double-stranded DNA (dsDNA) regions in the amplified sequence. A two-step strategy to solve this problem is presented in this paper: (i) titration of the PCR with NaCl as a model inhibitor to establish the critical range within which false negatives occur; (ii) titration of the PCR with a dsDNA-destabilizing additive under false-negative-inducing conditions until the relative amplification efficiencies of co-amplified fragments are adjusted. Betaine is introduced as a novel and efficient cosolute. These measures to achieve reliable PCR typing of a difficult target should be useful for many qualitative and quantitative multiplex PCR applications.

Plasma tumor necrosis factor alpha levels and the -238*A promoter polymorphism in patients with antiphospholipid syndrome.

OBJECTIVES: To explore the possible involvement of the proinflammatory and prothrombotic cytokine TNFalpha in APS by determining the plasma levels in patients and to test for association of TNFA promoter polymorphisms and HLA class II genotypes with both plasma TNFalpha and disease. PATIENTS AND METHOD: We studied 83 Caucasoid patients with APS and two groups of healthy controls. TNFalpha levels were determined in plasma from 35 patients' and 21 controls using a highly sensitive sandwich ELISA. The full patient group was genotyped together with 95 ethnically matched healthy controls. -308 and -238 TNFA promoter polymorphisms were assessed by ARMS-PCR. HLA-DQB1, DQA1 and DRB1 genotypes were determined by PCR using sequence specific primers. RESULTS: TNFalpha levels were significantly higher in patients with APS than healthy controls (median 2.95 pg/ml [range 0.51-10.75] vs. 0.95 pg/ml [0.51-1.6], respectively; p = 0.0001). Frequencies of TNFA-308*2 genotype did not differ between patients and controls. In contrast, TNFA-238*A positive genotype was more frequent in APS patients with arterial thrombosis and pregnancy loss than in controls (OR 3.7 [95% CI 1.37-10.1], p = 0.007 and OR 3.95 [95% CI 1.3-11.7], p = 0.01; respectively). DQB1*0303-DRB1*0701 haplotype was associated with TNFA-238*A in the control group (OR 96.0 [95% CI 9.6-959], p <0.0001) as well as in APS patient's group (OR 54.2 [95% CI 9.6-306.5], p <0.0001). CONCLUSIONS: Raised plasma TNFalpha levels were found in patients with APS. As a prothrombotic and proinflammatory cytokine, TNFalpha may be involved in the development of clinical features of APS. The lack of correlation between the TNFA-238 polymorphism and plasma levels associated with disease suggests that the TNF genetic marker may only indirectly relate to protein levels by virtue of allelic association with a functional marker which may reside in the HLA class II region.

Techniques used to define human MHC antigens: monoclonals, class I and class II biochemistry.

A variety of straightforward biochemical techniques have been applied to the analysis of HLA molecules. These techniques centre largely on two components: monoclonal antibodies (mAbs) and gel analysis systems. Although only the basic applications are discussed here, the techniques have broad applications. Monoclonal-gel systems may be used for the characterisation of polymorphisms in HLA class I alpha chains or class II alpha and beta chains. This requires the use of mAbs which are locus-specific and monomorphic. Alternatively, B lymphoblastoid cell lines (B-LCL) whose HLA antigen mobilities are known in advance may be used for screening uncharacterised monoclonals for locus specificity.

Healthy human T-cell receptor beta-chain repertoire. Quantitative analysis and evidence for J beta-related effects on CDR3 structure and diversity.

Analysis of TCR repertoire usage and clonality is of potential value in understanding the pathogenesis of a number of human immune-mediated diseases. In diseases that are likely to be dependent on antigen-driven T cells, it has been suggested that particular TCR junctional region or CDR3 sequences may be critical. Rigorous methods for TCR analysis which are both quantitative and qualitative are therefore required. Of those commonly available, only anchor and inverse PCR are capable of giving high-quality information on V, D, N, and J region usage, but it has not been established whether both methods are quantitatively or analytically equivalent. We show here that both methods detected considerable variability in the usage of V beta and J beta segments in the peripheral blood repertoire of a normal individual. No preferential V-J pairing could be demonstrated. An excess usage of J beta 2 family members was indicated by both methods, although the relative usage of different J beta 2 families differed between the two techniques. The predominantly used V beta usage showed that for some families, estimates of their frequency in the repertoire differed significantly between the anchor and inverse libraries. When sampling relatively few clones the variation between V beta families estimated using the two methods can be considerable. This is likely to be a result of sampling error from a large gene family. Large-scale screening of several thousand clones is recommended to confirm the absolute values obtained from sequencing. Variation in CDR3 length appeared to be normally distributed, suggesting that a statistically optimal junctional region length may have been selected for contact with antigen. CDR3 length distribution differs significantly between receptors, which have rearranged to J beta 1 versus J beta 2 families, with the J beta 2-associated CDR3 on average between 0.5 and 1.2 of an amino acid longer. Thus the TCR beta junctional region repertoire of humans is subject to structural constraints associated with J beta usage. It will be important to establish whether variation in CDR3 length and J beta usage exists between subsets of human T cells in order to interpret TCR repertoire data from disease and control tissues.

An integrated multiplex-PCR and PCR-RFLP typing system for markers associated with seronegative arthritides.

The system was designed with emphasis on the identification HLA-B alleles and genotypes associated or potentially associated with seronegative arthritides. By using a combination of multiplex SSP and PCR-RFLPs, the assays can be economically performed on a large range of sample sizes in diagnosis and epidemiology. 24 HLA-B alleles and subtypes can be discriminated, including options for PCR-RFLP or sequence specific amplification of the allele groups B27 and B60 (B*4001 and B*4007). In addition, the internal control carries central MHC polymorphisms, which can help to identify HLA extended halplotypes. False negatives, caused by preferential amplification of the internal control under suboptimal PCR conditions, were prevented by employing new, optimized PCR buffer. Four of the HLA-B primers were pooled into a multiplex reaction whose products were subtyped by digestion with seven restriction endonucleases. Specificity and sensitivity were verified in a panel of 68 homozygous cell lines and 200 heterozygous samples. An HLA-B*27-B*40 hybrid allele was observed in 3 out of 95 B*27-positive individuals from Berlin, Germany. Such an allele could be mistyped by some published assays as a B*27/B*40 heterozygote, a genotype reported to confer an increased risk for ankylosing spondylitis.

Structure and diversity of the T-cell receptor alpha chain in rhesus macaque and chimpanzee.

We cloned and sequenced cDNA for the TCRAC1 of a healthy rhesus monkey and chimpanzee. TCRAC1 from both nonhuman primates show extensive conservation compared to the human sequence and to other mammals. A possible primate-specific insertion near the hinge region of the TCRAC1 region is described. Characterization of 18 rhesus macaque and eight chimpanzee TCRA chain cDNA clones derived from inverse PCR revealed 12 different TCRAV and 16 different TCRAJ regions which corresponded closely to known human counterparts. One functional rhesus macaque TCRDV-TCRAJ rearrangement was detected, suggesting a genomic organization of the macaque TCRD locus which is similar to humans. At the genomic level, a single TCRAC1 gene segment was detected in rhesus macaque and chimpanzee. The close phylogenetic relationship between primates shown here for TCRA chain components supports the use of these species as animal models of human immune-mediated disease.

Celiac disease is associated with an extended HLA-DR3 haplotype which includes HLA-DPw1.

HLA-DP polymorphism was examined among celiac disease patients and controls. Restriction fragment length polymorphism (RFLP) genotyping showed a significant association of DPw1 in celiac disease (32/80) compared with controls (6/53, p = 0.0002). DPw1 typing by RFLP was verified using DPB1-amplified DNA and an oligonucleotide probe specific for the DPw1-associated DPB1 gene. The RFLP-assigned DPw1 genotype corresponded closely to the binding pattern of the sequence-specific oligonucleotide probe, although discrepancies did occur. The association between celiac disease and DPw1, however, remained. Oligonucleotide probe specificity was confirmed by sequencing DPB1-amplified DNA from four DPw1-genotyped celiacs. DPw1 is only present in celiacs who genotype DR3a-positive. Of DR3a controls 24% are DPw1-positive compared with 5% of non-DR3a controls (p = 0.03), suggesting that an extended DR3a, DPw1 haplotype occurs in the control population. This haplotype forms a large proportion of the DR3a haplotypes predisposing to celiac disease. Alternatively, DPw1 may represent an independent risk factor inherited in linkage with HLA-DR3 and -DQw2. Although predisposition to celiac disease is likely to be mediated by a specific DQ alpha/DQ beta heterodimer, a direct role for the DPw1 antigen cannot be discounted.

Sequence analysis of HLA-DR4B1 subtypes: additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing.

The stimulating capacity of HLA-DR4 variants in mixed leukocyte culture correlates with variation in the polymorphic regions of the first domains of their DR beta 1 chains. Variability between amino acids 67 and 86 appears largely to determine HLA-DR4,Dw type. We have used a combination of a DR4B1-specific primer set in the polymerase chain reaction and specific oligonucleotide probes to examine DR4,Dw-associated nucleotide polymorphisms. Phenotype and gene frequencies are reported among 44 normal DR4 Caucasoids. Oligonucleotide probes were selected which enabled definition of Dw4-, w14-, w10-, w13-, and w15-associated sequences. Unexpectedly, several subjects were positive for Dw15 sequences which are usually characteristic of Oriental populations. Dw15 assignment was confirmed by nucleotide sequencing of DR4B1 polymerase chain reaction products. A pair of oligonucleotides informative for the glycine or valine dimorphism at position 86 was used to identify two novel DR4B1 alleles, designated 13.2 and 14.2. Nucleotide sequencing shows that these represent recombinants between third hypervariable regions associated with Dw13 and Dw14 and a codon for glycine at position 86 which is usually found associated with Dw4 and Dw15 sequences.

Associations between alleles of the major histocompatibility complex-encoded ABC transporter gene TAP2, HLA class II alleles, and celiac disease susceptibility.

The study of celiac disease among Southern European populations has confirmed the hypothesis from Northern Europe that a close association exists between disease susceptibility and a combination of polymorphic alleles at the HLA-DQ loci (DQA1*0501-DQB1*0201) arranged either in a cis- or a trans-configuration. Attempts to identify additional genetic influences have been inconclusive, although many studies have raised possibilities of further major histocompatibility complex (MHC)-linked susceptibility genes. This study examines the disease associations of polymorphisms of a recently discovered gene located close to the DQ loci, TAP2, whose products are thought to be involved in the transport of antigen peptides across the endoplasmic reticulum for binding to HLA class I molecules. Like the products of the DQ loci, the product of TAP2 forms part of a heterodimeric molecule with products of a second genetic locus, TAP1, which is located centromerically to TAP2. The populations studied were central Italians from Rome and Ashkenazi Jews from Rehovot, Israel. HLA studies demonstrate that the Roman celiac group has a high proportion of people with DR3-negative celiac disease in whom the combination DR5/7 is frequently found. In Israel, 20% of celiac patients lack the DQ susceptibility markers but are DR4 positive. The polymorphic substitutions of TAP2 studied encode amino acid changes in the trans-membrane region (position 379) and the ATP-binding cassette region (positions 565 and 665) of the protein. No TAP2-specific disease associations were found in any HLA subgroup.(ABSTRACT TRUNCATED AT 250 WORDS)

Celiac disease among Ashkenazi Jews from Israel. A study of the HLA class II alleles and their associations with disease susceptibility.

The immunogenetics of celiac disease demonstrates a highly significant association with the HLA class II alleles DQA1*0501 DQB1*0201 encoded in either a cis- or trans-configuration. In Northern Europe, these alleles are found in linkage disequilibrium with DRB1*0301 while in Southern Europe an additional secondary association through linkage disequilibrium is seen with the combination DRB1*1101/0701. This study examines 34 Ashkenazi Jews with celiac disease and 36 ethnically matched controls to determine alleles at the DRB, DQA1, DQB1, and DPB1 loci using SSO probes in conjunction with gene amplification by the PCR. The results confirm a highly significant association with the DQA1*0501 DQB1*0201 allelic combination (71% celiac vs 8% control individuals; p = 0.00005; chi 2 = 21.4). Of celiac subjects, 29% were negative for the proposed DQ susceptibility alleles, the majority of whom were DRB1*0402 positive (20% overall celiac group). No additional susceptibility was associated at the DRB3 and DPB loci. This study confirms that the MHC-linked celiac disease susceptibility among Ashkenazi Jews is closely associated with the presence of the combination of alleles DQA1*0501 DQB1*0201. However, within this population of relatively high-prevalence celiac disease, 30% of celiac patients do not carry these alleles and are therefore not covered by a single "unifying" hypothesis.

HLA-DQ2 second-domain polymorphisms may explain increased trans-associated risk in celiac disease and dermatitis herpetiformis.

Sequence polymorphism in the DQ2 beta chain was investigated in 80 Caucasoid patients with CD, 23 patients with DH, and 64 healthy controls. A set of amplification primers were designed to amplify a 281-bp region between amino acids 95 and 135 encoding the second domain of the DQ beta chain gene. A polymorphism at amino acid 135 was shown to distinguish DR3 and DR7 haplotypes. Two SSO probes were designed to identify amino acid sequences (133-135) RND (DR3-DQ2) and RNG (DR7-DQ2). To establish whether polymorphism existed elsewhere in the second-domain sequence, which could explain the migratory characteristics of the CD-associated DR3-DQ2 beta-chain reported by Roep et al. DQB1 second-domain PCR products were sequenced from the genomic DNA of three CD patients. The results showed that the polymorphism at amino acid 135 distinguishing DR3 and DR7 haplotypes was present in CD, DH patients, or normal controls of the appropriate DR and DQ genotypes by oligonucleotide hybridization. Cloning and sequencing of DQB1 second domains of three CD patients (two DR3,3 and one DR3,7) gave normal sequences expected from their genotypes. No specific polymorphism of DQB1 second domains on CD-associated DR3 haplotypes distinguishes them from normal DR3 haplotypes. We conclude that individuals positive for the DR3,7 genotype have the potential to express a unique trans-encoded heterodimer with enhanced ability to predispose people to CD.

HLA-DRB, -DQA, and -DQB polymorphism in celiac disease and enteropathy-associated T-cell lymphoma. Common features and additional risk factors for malignancy.

CD is a gluten-sensitive enteropathy, strongly associated with expression of the DQA1*0501, DQB1*0201 genotype. CD patients have an increased risk of malignancy, particularly EATCL. However, it is controversial as to whether adults with EATCL represent a subgroup of patients with CD or should be regarded as a distinct entity. To investigate the genetic relationship between CD and EATCL, HLA class II DRB1, DQA1, and DQB1 typing of peripheral blood, frozen or paraffin-embedded biopsy tissue obtained from Caucasian patients with CD (n = 91) or EATCL (n = 47) was performed by PCR-SSOP typing. Genotype frequencies were compared with those observed in 151 unrelated control individuals. A total of 83 (91%) of 91 CD patients were of DQA1*0501, DQB1*0201 genotype (pc < 10(-6), RR = 522.2), compared with 40 (93%) of 43 EATCL patients (pc < 10(-6), RR = 44.2) with amplifiable DNA versus 35 (23%) of 151 controls. DRB1*03 frequencies were also elevated in both patient groups (79 of 91 in CD [87%; pc < 10(-6), RR = 24.5] and 38 of 40 in EATCL [95%; pc < 10(-6), RR = 70.7]) compared with controls (32 of 151, 21%). These results confirm previous studies of HLA associations in CD and also suggest that EATCL arises in individuals with the DQA1*0501, DQB1*0201 CD-predisposing genotype. However, the frequency of DRB1*03,04 heterozygotes was significantly increased in the EATCL group (16 of 40, 40%) compared with both control individuals (3 of 151, 2%; pc < 10(-6), RR = 32.9) and uncomplicated CD patients (6 of 91, 7%; pc = 0.04, RR = 9.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Strong primary selection for the Dw4 subtype of DR4 accounts for the HLA-DQw7 association with Felty's syndrome.

Felty's syndrome (FS) is a rare complication of rheumatoid arthritis (RA) previously shown to be strongly associated with HLA-DR4 and less significantly with HLA-DQw7. To map more precisely the HLA locus responsible for susceptibility to FS, we have examined HLA-DR4 and DQ beta-chain polymorphisms in FS patients and controls using restriction fragment length polymorphism analysis and polymerase chain reaction amplification in conjunction with oligonucleotide probing. The increased frequency of DR4 in FS (93% vs. 32% controls) was due almost entirely to enrichment for the Dw4 subtype (88% vs. 20% controls) with a secondary increase of the Dw14 subtype. Dw10 and Dw13 subtypes of DR4 were absent from the patient group. Increase in DQw7 frequency among DR4 FS patients could be accounted for by linkage disequilibrium between Dw4 and DQw7 alleles. Whereas susceptibility to RA is strongly associated with a conserved HLA-DR beta epitope associated with several DRB1 alleles, it is primarily the Dw4 allele which is associated with progression to Felty's syndrome. The finding that amino acid sequence variation at the DR4B1 locus rather than DQB1 is associated with development of FS will have important implications for the development of novel immunotherapies which are major histocompatibility complex allele-dependent.

HLA-DQ beta 3.1 allele is a determinant of susceptibility to DR4-associated rheumatoid arthritis.

Rheumatoid arthritis is associated with HLA-DR4 in several ethnic groups. Since DR4 haplotypes encode a diverse array of class II molecules, it is of interest to characterize the nature of the primary association. We have examined molecular polymorphisms of HLA class II gene products expressed by normals and rheumatoid arthritis patients using monoclonal antibodies and two-dimensional electrophoresis. Most homozygous DR4 rheumatoid arthritis patients express DR beta 1 molecules associated with Dw4 or Dw14 mixed lymphocyte culture determinants. In Caucasoids, two DR4-linked DQw3-associated beta-chain alleles are defined by two-dimensional electrophoresis. These variants, designated DQ beta 3.1 and 3.2, are associated with the serologic determinants DQw7 and DQw8, respectively. A panel of 40 DR4-positive normals was also examined for nucleotide sequence polymorphisms associated with DQB3.1 and 3.2 genes using the polymerase chain reaction and specific oligonucleotide probes. At the DQ beta level the rheumatoid arthritis panel was distinguished by enrichment for the DQ beta 3.1 allele with 100% of patients positive for DQw7. Results presented here suggest that specific DQ beta alleles may modify the effect of HLA-DR4 beta 1 alleles in conferring susceptibility to rheumatoid arthritis in a phenotype-specific fashion.