E Choi

RNA splicing generates a variant light chain from an aberrantly rearranged kappa gene.

Both Cκ regions in MPC 11 cells are rearranged into active transcription units, one producing a normal κ chain and the other an internally deleted κ fragment lacking a V region. The gene coding for the κ fragment mRNA is aberrantly rearranged and lacks a site for V → Cκ splicing. An alternative splicing event which deletes the V region from the nuclear RNA precursor generates the κ fragment mRNA.

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.

Uniparental isodisomy 6 associated with deficiency of the fourth component of complement.

We identified an extremely rare condition, isolated complete deficiency of the fourth component of complement, in a child with systemic lupus erythematosus. The genes for C4 are located within the major histocompatibility complex (MHC) on the short arm of chromosome 6. The patient expressed only paternal phenotypes for proteins encoded by the MHC (HLA and GLO), yet was 46XX with no detectable 6p deletion. Genomic DNA from patient, parents, and sibling was digested with restriction enzymes, and blots were probed for five chromosome 6 markers. At all loci, maternal and paternal RFLPs could be distinguished, and the patient showed only paternal bands. RFLP analysis of markers from four other chromosomes showed maternal and paternal contribution. The data are consistent with uniparental isodisomy 6 (inheritance of two identical chromosome 6 haplotypes from the father and none from the mother). Direct analysis of genetic material from both parents, as well as detection of multiple protein polymorphisms encoded on chromosome 6, clearly demonstrates this novel mechanism for the expression of a recessive genetic condition.

Comparative sequence analysis of the human T cell receptor β chain in juvenile rheumatoid arthritis and juvenile spondylarthropathies: Evidence for antigenic selection of T cells in the synovium

OBJECTIVE To identify features of the T cell receptors (TCRs) present on clonally expanded T cells in the joints of patients with similar types of childhood rheumatic disease. Vbeta8 and Vbeta20 TCRs were selected as prototypic for polyarticular juvenile rheumatoid arthritis (JRA) and pauciarticular/juvenile spondylarthropathy (SpA), respectively. METHODS The portion of the TCR beta chain involved in antigen recognition in the synovial tissue, synovial fluid, and peripheral blood from patients with JRA and juvenile SpA was cloned and sequenced. The frequency of expanded clonotypes, size of expansions, the Jbeta region, and sequence motifs were determined for >2,000 sequences. RESULTS The majority of Vbeta20 and Vbeta8 clonal expansions were found in the joint rather than the peripheral blood. While instances of both Vbeta8 and Vbeta20 clonal expansion were detected in all disease types, the features of these expanded clonotypes were specific for disease type and Vbeta family. For example, Vbeta20 clonal expansion was characterized by many small expanded clonotypes in samples from patients with pauciarticular JRA and juvenile SpA while single large Vbeta8-specific expansions were found only in patients with polyarticular disease. Motifs specific to individual patients were identified, and for Vbeta20 clonotypes, a motif was found in synovial tissue samples. CONCLUSION Identification of common TCR features in oligoclonal expansions within individual patients and between patients with the same type of JRA suggests the recognition of a common or limited group of antigens in these diseases.

HLA-DP/DR interaction in children with juvenile rheumatoid arthritis

Among the major histocompatibility complex class II genes, HLA-DP is least understood in terms of its contributions to the normal immune response and to autoimmunity. The few autoimmune diseases with HLA-DP associations can provide insight into the function of HLADP. Among these diseases is one form of juvenile rheumatoid arthritis (JRA) which has been associated with HLA-DPw2 (Hoffman et al. 1986; Odum et al. 1986). This particular type of JRA, early-onset pauciarticular disease (EOPA-JRA), has also been associated with other HLA class II genes -primari ly HLA-DR5 and HLA-DRw8. The two other HLA-DR genes carried on HLA-DRw52 haplotypes, HLA-DR3 and HLA-DRw6, have been associated with disease in certain study populations (Albert et al. 1989). In this study, HLA-DP typing by restriction fragment length polymorphism (RFLP) was used to analyze which subtype of HLA-DPw2 predisposes to EOPA-JRA. Second, the question was addressed as to whether the HLADPw2 association with EOPA-JRA is dependent on the combined presence of other disease-associated HLA-DR genes, and if so, whether all the disease-predisposing HLA-DR genes interact with HLA-DPw2 in a similar way. The 97 Caucasian patients studied met the Americn Rheumatism Association criteria for pauciarticular JRA (Cassidy et al. 1986) and had an onset of disease prior to the age of 6 years. The 130 controls were ethnically similar to the disease population; 65 % of the patients and 53 % of the controls originated from the British Isles and Germany. Most individuals had previously been serologically HLA-DR typed (Van Kerckhove et al. 1988). To HLA-

The CD4 molecule transmits biochemical information important in the regulation of T lymphocyte activity

The role of the CD4 molecule in the transmission and regulation of the biochemical signals involved in T cell activation was investigated using an anti-CD4 monoclonal antibody termed 6B10. 6B10 immunoprecipitated the 55-kDa CD4 molecule and detected an epitope of CD4 that overlapped with that detected by OKT4A, B, and D. 6B10, 6B10 Fab fragments and recombinant HIV envelope glycoprotein (gp120) induced calcium mobilization in PBMC. 6B10 stimulation also resulted in calcium mobilization in murine L cells expressing transfected CD4 gene products, indicating that CD4-mediated calcium mobilization occurred independently of the CD3/T cell receptor (TCR) complex. 6B10 induced a phosphatidylinositol response, but the response resulted in reduced inositol phosphate production compared to levels obtained using OKT3. Though 6B10 caused calcium mobilization and a phosphatidylinositol response, 6B10 did not induce DNA synthesis. The amount of inositol phosphates produced by 6B10 may be below the threshold necessary for cell cycle progression. We hypothesized that 6B10-mediated calcium mobilization is important in the regulation of T cell proliferation. 6B10, but not 6B10 Fab fragments, inhibited OKT3-induced DNA synthesis. Furthermore, 6B10 but not 6B10 Fab fragments inhibited OKT3-induced calcium mobilization, suggesting that crosslinking of CD4 may be an important factor determining whether signals result in both the up- and down-regulation of CD3/TCR complex function. The implication of this work is that signals generated via the CD4 molecule are important in the regulation of T cell function and that the signals generated as a result of HIV gp120 binding to CD4 can contribute to the mechanism by which HIV inhibits T cell function.

A distinct HLA-DRw8 haplotype characterizes patients with juvenile rheumatoid arthritis.

We studied the first domain of the HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci of 67 HLA-DRw8-positive Caucasians including 43 with early-onset pauciarticular juvenile rheumatoid arthritis (EOPA-JRA, alternatively known as early-onset pauciarticular juvenile chronic arthritis). Serology, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR) oligotyping revealed that 62, including all the EOPA-JRA patients, carried the HLA-DRB1*0801, DQA1*0401, DQB1*0402 genotype. Approximately onefifth of the controls carried atypical HLA-DRB1, HLA-DQA1, and/or HLA-DQB1 loci on their HLA-DRw8 haplotype confirmed by family studies. DNA sequences of HLA-DRB1, DQA1, and DQB1 alleles in patients and controls were identical to those previously reported. Disease association studies in 113 EOPA-JRA patients and 207 controls unselected for HLA-DRw8 revealed that the HLA-DRB1*0801, DQA1*0401, DQB1*0402 genotype was associated with a higher relative risk (RR) for disease (RR = 12.8, χ2 = 48.8, P < 10−4) than was the serologically defined presence of HLA-DRw8 (RR = 8, χ2 = 39, P < 10−4). Further analysis suggested that the DQ genes on HLA-DRw8 haplotypes are as likely as the DR genes to contribute to the pathogenesis of EOPA-JRA. This study increases to five the number of HLA-DR/DQ haplotypes identified in HLA-DRw8 Caucasians.

T-Cell receptor BV6S1 null alleles and HLA-DR1 haplotypes in polyarticular outcome juvenile rheumatoid arthritis

JRA is a complex of disease subtypes which are normally identified by clinical features such as age of onset and extent of joint involvement both at onset and during the course of the disease. We previously identified an association between TCR BV6S1 null allele and one subgroup of early-onset pauciarticular patients positive for HLA-DQA1*0101, an HLA haplotype predisposing to a polyarticular course of the disease. In this report we extend this observation by identifying an increased prevalence of this nonfunctional or null allele in the patients with a polyarticular disease course regardless of the mode of onset. This increase was most prominent in clinical subsets that have early onset of the disease and a polyarticular outcome. In one clinical group, stratification of patients by the HLA allele DQA1*0101 strengthened the association considerably. This implies that there is an increased genetic load defined by specific alleles of both MHC and TCR genes.

Polymorphic markers related to a single Tcrb-V6 gene segment

The central role of the antigen-specific alpha/beta T-cell receptor (Tcr) in immune recognition has led to a search for Tcr gene polymorphism relevant to autoimmune diseases. Previous reports primarily emphasized associations with constant (C)-region (Millward et al. 1987; Demaine et al. 1989; Freimark et al. 1987) restriction fragment lenght polymorphisms (RFLPs) as opposed to polymorphisms of variable (10-region genes, which determine the specificity of antigen-MHC recognition by the Tcr. Although Tcr-Vand C-region genes are linked, recent family studies have reported a lack of linkage disequilibrium between Vand C-region polymorphisms (Robinson and Kindt, 1987; Charmley et al. 1988; Charmley et al. 1990) indicating that C-region polymorphisms alone may be of limited value in studying Tcr disease association (Nivens et al. 1990; Charmley et al. 1990). Further study of disease associations has been hampered by the paucity of data regarding the extent of polymorphism of Tcr-V-region genes in normal Caucasian populations. In the present study, we evaluated RFLPs related to Tcrb-V genes in 100 normal, unrelated, Caucasoid individuals using five Tcrb-V gene specific cDNA probes, V4, V5, V6.1, V8.1 and V18 (Leiden and Strominger 1986; Yanagi et al. 1984). Southern blot analysis of genomic DNA digested with the restriction enzymes Bgl-II, Bam HI, Eco RI, and Taq I, was carried out as described (Southern 1975). Polymorphic restriction enzyme sites were detected by two restriction enzymes, Taq I and Bgl II, with the TcrbV6.1 cDNA probe. Each probe/enzyme combination defines a bi-allelic polymorphism. Hybridization of the Tcrb-V6.1 probe to blots containing Bgl II digested DNA revealed a variant band of 5.7 kilobases (kb) whose inten-

Variability in TRBV haplotype frequency and composition in Caucasian, African American, Western African and Chinese populations

The polymorphic T‐cell receptor Vβ (TRBV) genes encode much of the variable region of the T‐cell receptor β chain. Analysis of allele frequencies of three closely linked polymorphic TRBV genes, TRBV7‐3, TRBV9 and TRBV6‐4, was undertaken in several populations. The frequencies of these alleles are not significantly different in populations of Caucasians, African Americans and Western Africans. However, Chinese population is extremely homogenous at all three loci. The current study identifies the existence of haplotypic relationships between alleles of these genes in the Caucasian population. The ORF allele TRBV7‐3*A3 is found exclusively on chromosomes bearing TRBV9*A2 and TRBV6‐4*A2 in this cohort. In contrast, TRBV7‐3*A1 and the null allele TRBV7‐3*A2 are associated only with TRBV9*A1 and TRBV6‐4*A1. This pattern of linkage disequilibrium (LD) is altered in the African American and Western African populations. In these cohorts, there is a marked reduction in LD between alleles of TRBV7‐3 and TRBV9. This study is consistent with previous population genetic studies wherein African‐derived samples have a greater level of genetic diversity compared to Caucasians. These data also demonstrate that patterns of LD are not consistent across the entire TRBV locus.