Differences in T cell receptor restriction fragment length polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE. The purpose of this study was to determine whether a T cell receptor (TCR) polymorphism, either by itself or in combination with particular HLA polymorphism, leads to susceptibility to rheumatoid arthritis (RA). METHODS. Eight restriction fragment length polymorphisms (RFLPs) detected with TCR gene segments were investigated in 46 individuals with RA and were compared with data from normal control subjects. RESULTS. A statistically significant difference in the genotype frequencies of a Taq I RFLP detected with the TCR alpha constant region (C alpha) gene was noted. In addition, when the DR4+ subpopulations were examined, the allelic frequency of a 2-kb Bam HI fragment detected with a V beta 8 gene was increased in the samples from RA patients (P less than 0.0086). CONCLUSION. The results of this study suggest that germline differences in the TCR repertoire may be associated with RA, and that there is a contributory effect of DR4+ haplotypes with certain TCR haplotypes in susceptibility to RA.     

Differences in t cell receptor restriction fragment length polymorphisms in patients with rheumatoid arthritis

Objective. The purpose of this study was to determine whether a T cell receptor (TCR) polymorphism, either by itself or in combination with particular HLA polymorphism, leads to susceptibility to rheumatoid arthritis (RA). Methods. Eight restriction fragment length polymorphisms (RFLPs) detected with TCR gene segments were investigated in 46 individuals with RA and were compared with data from normal control subjects. Results. A statistically significant difference in the genotype frequencies of a Taq I RFLP detected with the TCRα constant region (C_α) gene was noted. In addition, when the DR4+ subpopulations were examined, the allelic frequency of a 2-kb Bam HI fragment detected with a V_β8 gene was increased in the samples from RA patients (P < 0.0086). Conclusion. The results of this study suggest that germline differences in the TCR repertoire may be associated with RA, and that there is a contributory effect of DR4+ haplotypes with certain TCR haplotypes in susceptibility to RA.     

Novel genetic markers of rheumatoid arthritis in Chilean patients, by DR serotyping and restriction fragment length polymorphism analysis.

OBJECTIVE. The analysis of genetic markers of rheumatoid arthritis (RA) in a population in which the DR4 serotype is not strongly associated with the disease. METHODS. Chilean RA patients (56 seropositive and 22 seronegative) and 141 controls were studied by serotyping. Southern blot analysis of Bam HI restriction fragment length polymorphism (RFLP) was done in genomic DNA from 46 patients with seropositive RA, 17 patients with seronegative RA, and 45 controls, using a complementary DNA probe specific for DRB1 genes. RESULTS. The prevalence of the HLA-DR9 haplotype was strikingly higher in seropositive RA patients (21%) than in controls (3%) (Pcorr less than 0.0008, by Fisher's exact test; relative risk [RR] = 9.34). The prevalence of DR4 and DR1 haplotypes, although slightly increased, did not achieve a significant preponderance. The simultaneous presence of two Bam HI fragments (3.6 kb and 4.5 kb) was found with higher prevalence in seropositive patients (83%; RR = 9; Pcorr less than 0.00002) than in controls (36%), and seemed higher in seronegative RA patients as well (71%; RR = 4). Furthermore, its prevalence remained increased in comparisons of DR4 positive controls (36%) with DR4 positive seropositive patients (100%; RR = 67; Pcorr less than 0.0002) and DR4 positive seronegative patients (100%; RR = 36; Pcorr less than 0.006), even after excluding the DR9 positive individuals. A tendency toward higher association with DR1 seropositive RA patients (67%; RR = 12), a group with no DR4 or DR9 positive individuals, than in DR1 positive controls (14%), was also observed. CONCLUSION. The HLA-DR9 haplotype was definitively consolidated as a very strong genetic marker exclusively for seropositive RA in Chilean patients, as suggested by our previous observations. RFLP analysis showed that the simultaneous presence of 3.6-kb and 4.5-kb Bam HI fragments constituted a better RA marker than did any of the heretofore studied haplotypes. These fragments together would be linked to RA independently of the DR1, DR4, and DR9 haplotypes. The overall evidence indicates that Chilean seropositive RA patients display a genetic background that is different from that underlying RA susceptibility in other populations and suggests the existence of common, as well as distinct, genetic elements predisposing to seronegative and seropositive RA.     

Novel genetic markers of rheumatoid arthritis in chilean patients,by dr serotyping and restriction fragment length polymorphism analysis

Objective. The analysis of genetic markers of rheumatoid arthritis (RA) in a population in which the DR4 serotype is not strongly associated with the disease. Methods. Chilean RA patients (56 seropositive and 22 seronegative) and 141 controls were studied by serotyping. Southern blot analysis of Bam HI restriction fragment length polymorphism (RFLP) was done in genomic DNA from 46 patients with seropositive RA, 17 patients with seronegative RA, and 45 controls, using a complementary DNA probe specific for DRB1 genes. Results. The prevalence of the HLA—DR9 haplotype was strikingly higher in seropositive RA patients (21%) than in controls (3%) (P_corr < 0.0008, by Fisher's exact test; relative risk [RR] = 9.34). The prevalence of DR4 and DR1 haplotypes, although slightly increased, did not achieve a significant preponderance. The simultaneous presence of two Bam HI fragments (3.6 kb and 4.5 kb) was found with higher prevalence in seropositive patients (83%; RR = 9; P_corr < 0.00002) than in controls (36%), and seemed higher in seronegative RA patients as well (71%; RR = 4). Furthermore, its prevalence remained increased in comparisons of DR4 positive controls (36%) with DR4 positive seropositive patients (100%; RR = 67; P_corr < 0.0002) and DR4 positive seronegative patients (100%; RR = 36; P_corr < 0.006), even after excluding the DR9 positive individuals. A tendency toward higher association with DR1 seropositive RA patients (67%; RR = 12), a group with no DR4 or DR9 positive individuals, than in DR1 positive controls (14%), was also observed. Conclusion. The HLA—DR9 haplotype was definitively consolidated as a very strong genetic marker exclusively for seropositive RA in Chilean patients, as suggested by our previous observations. RFLP analysis showed that the simultaneous presence of 3.6-kb and 4.5-kb Bam HI fragments constituted a better RA marker than did any of the heretofore studied haplotypes. These fragments together would be linked to RA independently of the DR1, DR4, and DR9 haplotypes. The overall evidence indicates that Chilean seropositive RA patients display a genetic background that is different from that underlying RA susceptibility in other populations and suggests the existence of common, as well as distinct, genetic elements predisposing to seronegative and seropositive RA.     

Perivascular infiltration in normal skin of patients with rheumatoid arthritis: association with rheumatoid factors and HLA-DR antigens.

The relation between immunohistological findings in biopsy specimens of apparently normal skin, HLA antigens, and rheumatoid factors (RF) was studied in 120 patients with rheumatoid arthritis (RA), selected for treatment with D-penicillamine. Perivascular infiltration (PVI) of more than three mononuclear cells was present in 77 (68%) of 114 patients, accompanied usually by the presence of IgM or C3, or both, in immunofluorescence studies. The number of perivascular cells was associated significantly with the titre of circulating RF. A weak relation of both perivascular cellular infiltration and RF with HLA-DR3 and DR4 did not reach statistical significance. It is concluded that the histological presence of perivascular inflammation is associated mainly with deposition of RF. It is suggested that the first is merely an epiphenomenon of the latter. PVI was not prognostic for the occurrence of the clinical syndrome of rheumatoid vasculitis. For practical purposes skin biopsies do not appear to be useful in the evaluation of individual patients with RA.     

HLA class II DR, DQ, and DP restriction fragment length polymorphisms in rheumatoid arthritis.

HLA class II restriction fragment length polymorphisms (RFLPs) were studied in 43 individuals with established seropositive rheumatoid arthritis (RA) and in a group of healthy controls. All patients and controls were tissue typed for HLA-A, B, and DR antigens. Rapid, initial screening for RA associated RFLPs was conducted by pooling DNA samples from 11 HLA-DR4 positive patients with RA and comparing the RFLP patterns with those seen in a pool of DNA samples drawn from 11 HLA-DR4 positive healthy controls. Candidate RA associated RFLPs were examined in our full panel of patients with RA and controls. In most cases the RFLPs detected showed no significant association with RA. An exception was a 13.0 kb DraI DQ beta associated RFLP, which, when HLA-DR4 positive patients with RA and controls were considered alone, showed a weak positive association with susceptibility to RA. This RFLP was not associated with known DR, DQ, or Dw specificities. These results show a distinct paucity of class II RA associated RFLPs but may indicate a role for DQ beta genetic variation in the aetiology of RA.     

Linkage of HLA-DR beta specific restriction fragment length polymorphisms with Graves' disease

HLA-DR3 positive patients with Graves' disease (6 homozygotes, 7 heterozygotes, i.e. yielding 19 haplotypes) were studied by restriction fragment length polymorphism analysis using TaqI as restriction enzyme in order to look for polymorphisms in the HLA-DR3 allele of the human major histocompatibility complex. Polymorphic TaqI fragments of 11.6, 9.8 and 5.8 kb, each corresponding to HLA-DR beta sequences, were shown to differ in their prevalence in patients with Graves' disease and controls. The prevalence of DR3 polymorphisms in a total of 35 HLA-DR3-containing haplotypes was markedly different within patients with Graves' disease and Caucasian controls. Whereas a 11.6 kb fragment was rare in Graves' disease (2/19 haplotypes vs 8/16 in controls), the inverse ratio was found for a 9.8 kb fragment, with a prevalence of 17/19 and 8/16 haplotypes, respectively. A polymorphic fragment of 5.8 kb was exclusively seen in two DR3-containing haplotypes of patients with Graves' disease. Our data provide evidence that a DNA polymorphism of the HLA-DR beta genes, which is also reflected at the product level, is linked to Graves' disease.     

Inherited predisposition to iridocyclitis with juvenile rheumatoid arthritis: selectivity among HLA-DR5 haplotypes.

HLA-DR5 is associated with a chronic iridocyclitis and juvenile rheumatoid arthritis with onset in early childhood. Previously published data provided indirect evidence for selective linkage between two HLA-B alleles and HLA-DR5. To test this observation further, 38 families, the probands of which have chronic iridocyclitis and juvenile rheumatoid arthritis, were HLA typed so that haplotypes associated with disease could be established. HLA-DR5 was linked to HLA-Bw44 or to HLA-Bw35 and to HLA-Cw4 in the majority of haplotypes obtained in the probands. Both HLA-Bw44, -DR5 and HLA-Bw35, -Cw4, -DR5 occurred more commonly in the proband haplotypes than in the control haplotypes. The frequency of the haplotype HLA-Bw44, -DR5 was 0.133 compared with 0.007 (X2 = 27.04, P less than 10(-6] and for HLA-Bw35, -Cw4, -DR5, it was 0.093 compared with 0.11 (X2 = 13.83, P = 0.0002). The relative risks were 20.77 and 9.23, respectively, versus 3.52 for HLA-DR5 alone. HLA-Bw44 occurred much more commonly in proband HLA-DR5 haplotypes (0.455) compared with control HLA-DR5 haplotypes (0.067) (X2 = 8.26, P less than 0.01). Not all HLA-DR5-bearing haplotypes predisposed equally to chronic iridocyclitis and early-onset pauciarticular juvenile rheumatoid arthritis.     

Molecular analysis of HLA-DR beta and DQ beta polymorphism in Chinese with rheumatoid arthritis.

OBJECTIVES--Several studies have suggested that genetic predisposition to rheumatoid arthritis may be related to the presence of specific polymorphic HLA sequences that are often associated with HLA-DR4 haplotypes. This study was performed to determine if an association exists between Chinese with rheumatoid arthritis and a particular HLA-DR beta or DQ beta subtype. METHODS--This study used the polymerase chain reaction to amplify HLA-DR beta and DQ beta genes, and oligonucleotide probe hybridisation to examine the association of certain polymorphic sequences with rheumatoid arthritis in 23 Chinese patients from Shanghai. RESULTS--An HLA-DR4 associated sequence was significantly increased in the Chinese patients (43%) compared with healthy controls (14%) from the same location (relative risk = 4.6, 95% confidence limits 1.1 to 19.3). Analysis of the third hyperpolymorphic region of DR4 positive samples was performed to detect polymorphic sequences associated with Dw4, Dw10, Dw13, Dw14, Dw15, and KT2 cellular specificities. Examination of this region showed that 91% of patients had sequences encoding amino acids QRRAA (associated with Dw14 and Dw15) or QKRAA (associated with Dw4) compared with 64% of the DR4 positive controls. CONCLUSIONS--Rheumatoid arthritis in the Chinese is associated with HLA-DR4. There is a possible relationship between sequences within the third hyperpolymorphic region of the DRB allele and rheumatoid arthritis in the Chinese.     

Antibodies to DNA in patients with rheumatoid arthritis and juvenile rheumatoid arthritis.

Antibodies to double-stranded DNA (DSDNA) were found in 18 patients with RA, in 5 patients with JRA, and in 5 patients with undiagnosed connective tissue disease. Five patients had clinical features consistent with both RA and SLE, 11 with only RA, and 5 with only JRA. Based on these observations, the presence of serum anti-DSDNA antibodies should not be used as a sole criterion in the diagnosis of SLE.     

Analysis of LMP and TAP polymorphisms by polymerase chain reaction-restriction fragment length polymorphism in rheumatoid arthritis

OBJECTIVE—The aim of this study was to investigate the relation between the polymorphism of large molecular weight proteasome (LMP) (LMP2-LMP7) and transporter associated with antigen processing (TAP) (TAP1-TAP2) genes and rheumatoid arthritis (RA).
METHODS—Sixty RA patients and 102 ethnically matched unrelated healthy subjects were typed for LMP, TAP, and disease associated HLA-DRB1 alleles by using a new strategy based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with amplification created restriction sites.
RESULTS—The polymorphism of LMP (LMP2-LMP7) and TAP (TAP1-TAP2) genes was examined in shared epitope positive and negative RA patients and controls. No significant differences in the LMP or TAP allele frequencies were observed between the total patient and control groups or the patients and controls positive or negative for the shared epitope.
CONCLUSION—The data suggest that the polymorphisms of LMP and TAP genes do not have an important influence in the pathogenesis of RA, although larger studies will be needed to provide more conclusive evidence on the role of these genes in RA. A new, highly reliable strategy for typing LMP alleles is also described.

Keywords: large molecular weight proteasome; transporter assoicated with antigen processing; rheumatoid arthritis     

Mother-to-infant vertical transmission and cross-colonization of Streptococcus pyogenes confirmed by DNA restriction fragment length polymorphism analysis.

Restriction fragment length polymorphism (RFLP) analysis of total DNA and of ribosomal DNA (rDNA) regions (ribotyping) were used to document Streptococcus pyogenes vertical mother-to-infant transmission and to investigate the spread of S. pyogenes in an obstetric unit. Two isolates from a newborn, two isolates from his mother (patient 1), and two isolates from two other mothers (patients 2 and 3) were studied. RFLP of total DNA, both after HindIII and PvuII digestions and ethidium bromide staining, gave indistinguishable patterns for the strains isolated from the neonate, his mother, and patient 2. Strains from patient 3 and six unrelated strains studied for comparison showed different patterns. In our system, ribotyping was less discriminative than total DNA RFLP analysis. DNA RFLP analysis therefore provides a valuable molecular tool for studying S. pyogenes epidemiology.     

HLA-DR2, -DR5, and DRw6 associated Dw subtypes correlate with HLA-DR beta and -DQ beta restriction fragment length polymorphisms.

DNAs extracted from peripheral blood leukocytes of 24 individuals, selected for their HLA-DR types, -DR2, -DR5, and -DRw6, were analyzed with four restriction enzymes, BamHI, EcoRV, HindIII, and Taq I, using the Southern technique. This panel includes 16 individuals with homozygous typing cells and 8 heterozygous individuals who carry rare Dw subtypes or unusual DR-DQ associations. Eighty-five polymorphic fragments were detected and assigned to the DR or DQ gene families according to their hybridization signals. Thirty-eight fragments (DR or DQ) were found to correlate with single DR or Dw specificities or rare associations such as DRw14-DQw3. Forty-two fragments correlated with the association of immunologically defined specificities. In total, these 85 fragments constituted 44 different patterns, each comprising 1-9 fragments. For each homozygous typing cell a combination of patterns was observed. Fourteen different combinations of 10-20 patterns were found among the 16 individuals with homozygous typing cells, showing that Dw18, Dw19, Dw9, and Dw5 are heterogeneous at the genomic level whereas only the Dw2 individuals tested here are identical.     

HLA-DR antigens and HLA-DQ beta chain polymorphism in susceptibility to rheumatoid arthritis.

Forty four patients with rheumatoid arthritis (RA) were studied for HLA-DR antigens and for HLA-DQ beta chain gene restriction fragment length polymorphism using DNA hybridisation. A significant increase in the prevalence of the DR4 antigen and a tendency towards an increase of DR1 was found in patients with RA. No allelic form of HLA-DQ restriction fragment length polymorphism patterns was increased, but the prevalence of an allele characterised by a combination of 7.5 and 3 kb fragments was decreased among patients with RA. The DQw8 subtype represented by a 12 kb fragment was the most common DR4 associated allele, and a 3.7 kb fragment related to DQw7 was found in only 5/25 (20%) DR4 positive patients and 2/12 (17%) controls. The results support the hypothesis that RA susceptibility factors are primarily located within HLA-DR genes but HLA-DQ genes may have a role in protection from the disease.     

HLA-DR-DQ haplotypes and genotypes in Finnish patients with rheumatoid arthritis

OBJECTIVES: To elucidate the contribution of HLA-DR-DQ haplotypes and their genotypic combinations to susceptibility to rheumatoid arthritis, and to evaluate the various models for HLA associated risk for the disease in a series of Finnish patients. METHODS: 322 Finnish patients with rheumatoid arthritis were typed for common north European HLA-DR-DQ haplotypes and compared with a series of 1244 artificial family based control haplotypes. RESULTS: The association of the so called shared epitope (SE) haplotypes (DRB1*0401, *0404, *0408, and *01) with rheumatoid arthritis was confirmed. The DRB1*0401 haplotypes carried a far stronger risk for the disease than the (DRB1*01/10)-(DQA1*01)-DQB1*0501 haplotypes. Seven protective HLA haplotypes--(DRB1*15)-(DQA1*01)-DQB1*0602; (DRB1*08)-(DQA1*04)-DQB1*04; (DRB1*11/12)-DQA1*05-DQB1*0301; (DRB1*1301)-(DQA1*01)-DQB1*0603; (DRB1*1302)-(DQA1*01)-DQB1*0604; (DRB1*07)-DQA1*0201-DQB1*0303; and (DRB1*16)- (DQA1*01)-DQB1*0502--were identified. In accordance with the reshaped shared epitope hypothesis, all the protective DRB1 alleles in these haplotypes share either isoleucine at position 67 or aspartic acid at position 70 in their third hypervariable region motif. However, differences in the disease risk of haplotypes carrying the same DR but different DQ alleles were also found: (DRB1*07)-DQA1*0201-DQB1*0303 was protective, while (DRB1*07)-DQA1*0201-DQB1*02 was neutral. The same haplotypes carried different risks for rheumatoid arthritis depending on their combination in genotypes. CONCLUSIONS: When assessing the influence of HLA genes on the susceptibility to rheumatoid arthritis, not only should the HLA-DR or -DQ alleles or haplotypes be unravelled but also the genotype. The effect of HLA class II region genes is more complicated than any of the existing hypotheses can explain.     

Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors.

The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, we examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia.     

Polymorphism of major histocompatibility complex extended haplotypes bearing HLA-DR3 in patients with rheumatoid arthritis with gold induced thrombocytopenia or proteinuria.

The distribution of DR3 and of extended haplotypes bearing DR3 was studied in three groups of subjects: 35 patients with rheumatoid arthritis (RA) with gold induced thrombocytopenia or proteinuria, 185 patients with RA without these side effects, and 300 normal healthy controls. The extended haplotypes bearing DR3 were analysed with cDNA probes for DR alpha, DR beta, DQ alpha, and DQ beta genes. The data showed that the prevalence of DR3 was significantly higher in patients who developed gold induced thrombocytopenia or proteinuria than in normal controls or patients with RA without these side effects. Distribution of three extended haplotypes bearing DR3 (B8, DR3; B18,DR3; non-B8,non-B18,DR3) in patients with RA with thrombocytopenia or proteinuria was significantly different from that in normal controls, but not from that in patients with RA without these toxic reactions. Southern blot analysis of DR, DQ genes with cDNA probes showed that the extended haplotype bearing B8,DR3, which carries DQA2.1 and DQB2.1 genes, was present in a significantly higher proportion of patients with RA with gold induced thrombocytopenia or proteinuria (22/24, 92%) than in patients with RA without these side effects (32/45, 71%) or normal subjects (40/61, 66%). The data suggest that the genomic region on chromosome 6 involved in susceptibility to gold induced thrombocytopenia or proteinuria should be extended to the DQA2, DQB2 gene loci.