Genome-wide association study identifies HLA 8.1 ancestral haplotype alleles as major genetic risk factors for myositis phenotypes.

Autoimmune muscle diseases (myositis) comprise a group of complex phenotypes influenced by genetic and environmental factors. To identify genetic risk factors in patients of European ancestry, we conducted a genome-wide association study (GWAS) of the major myositis phenotypes in a total of 1710 cases, which included 705 adult dermatomyositis, 473 juvenile dermatomyositis, 532 polymyositis and 202 adult dermatomyositis, juvenile dermatomyositis or polymyositis patients with anti-histidyl-tRNA synthetase (anti-Jo-1) autoantibodies, and compared them with 4724 controls. Single-nucleotide polymorphisms showing strong associations (P<5×10(-8)) in GWAS were identified in the major histocompatibility complex (MHC) region for all myositis phenotypes together, as well as for the four clinical and autoantibody phenotypes studied separately. Imputation and regression analyses found that alleles comprising the human leukocyte antigen (HLA) 8.1 ancestral haplotype (AH8.1) defined essentially all the genetic risk in the phenotypes studied. Although the HLA DRB1*03:01 allele showed slightly stronger associations with adult and juvenile dermatomyositis, and HLA B*08:01 with polymyositis and anti-Jo-1 autoantibody-positive myositis, multiple alleles of AH8.1 were required for the full risk effects. Our findings establish that alleles of the AH8.1 comprise the primary genetic risk factors associated with the major myositis phenotypes in geographically diverse Caucasian populations.

Restricted and shared patterns of TCR beta-chain gene expression in silicone breast implant capsules and remote sites of tissue inflammation.

Silicone breast implants (SBI) induce formation of a periprosthetic, often inflammatory, fibrovascular neo-tissue called a capsule. Histopathology of explanted capsules varies from densely fibrotic, acellular specimens to those showing intense inflammation with activated macrophages, multinucleated giant cells, and lymphocytic infiltrates. It has been proposed that capsule-infiltrating lymphocytes comprise a secondary, bystander component of an otherwise benign foreign body response in women with SBIs. In symptomatic women with SBIs, however, the relationship of capsular inflammation to inflammation in other remote tissues remains unclear. In the present study, we utilized a combination of TCR beta-chain CDR3 spectratyping and DNA sequence analysis to assess the clonal heterogeneity of T cells infiltrating SBI capsules and remote, inflammatory tissues. TCR CDR3 fragment analysis of 22 distinct beta variable (BV) gene families revealed heterogeneous patterns of T cell infiltration in patients' capsules. In some cases, however, TCR BV transcripts exhibiting restricted clonality with shared CDR3 lengths were detected in left and right SBI capsules and other inflammatory tissues. DNA sequence analysis of shared, size-restricted CDR3 fragments confirmed that certain TCR BV transcripts isolated from left and right SBI capsules and multiple, extracapsular tissues had identical amino acid sequences within the CDR3 antigen binding domain. These data suggest that shared, antigen-driven T cell responses may contribute to chronic inflammation in SBI capsules as well as systemic sites of tissue injury.

Genetic risk and protective factors for idiopathic inflammatory myopathy in Koreans and American whites: a tale of two loci.

OBJECTIVE: To better understand genetic contributions to autoimmunity, immunogenetic markers were studied in two racially discrete and geographically isolated populations of patients with idiopathic inflammatory myopathy (IIM). METHODS: Clinical characteristics, as well as clinical and autoantibody subsets, were defined in 151 American white patients and 50 Korean patients with IIM. HLA-DRB1 and DQA1 genotyping was performed on patients and racially matched controls by standard molecular techniques. Gm allotypes and phenotypes were determined by the hemagglutination-inhibition method. RESULTS: HLA-DRB1*0301, the linked allele DQA1*0501, and DRB1 alleles sharing the first hypervariable region motif 9EYSTS13 were major genetic risk factors for the development of myositis in whites (corrected P [Pcorr] < 0.0004, odds ratio [OR] 11.2, 4.5, and 3.1, respectively, for each factor versus controls). Although both the white and Korean patients had a similar distribution of clinical characteristics, autoantibody profiles, and clinical groups, no HLA-DRB1 nor DQA1 allele or motif was found to be a risk factor for IIM in the Korean patients. However, DRB1*14 was a protective factor in Korean patients without myositis-specific autoantibodies (Pcorr = 0.004, OR 0.046). In addition, although no Gm phenotype or allotype was identified as a risk factor in whites, Gm 21 was a protective factor for the development of IIM in Koreans (Pcorr = 0.024, OR 0.3). CONCLUSION: Although myositis patients in the US and Korea share similar clinical and serologic features, the immune response genes predisposing to and protecting from myositis in each of these ethnic groups differ at two chromosomal loci. These data suggest that multiple genetic loci should be studied to identify risk and protective factors for some autoimmune diseases in various ethnic populations.

Influence of T cell depletion method on circulating gammadelta T cell reconstitution and potential role in the graft-versus-leukemia effect.

BACKGROUND: Our laboratory previously reported that leukemia patients who developed > or = 10% gammadelta+ T cells during the first six months after receiving an anti-TCRalphabeta T-cell-depleted (TCD) graft from a partially mismatched related donor (PMRD) had a disease-free survival (DFS) advantage. These gammadelta+ T cells were V81+CD3+CD4-CD8-CD69+HLADR+ and are cytotoxic to K562 cells. METHODS: In order to determine whether the anti-alphabeta TCD regimen was associated with these findings, we compared the reconstitution of gammadelta+ T cells from patients who received TCD PMRD grafts using the anti-TCRc4 MAb TIOB9-1A31 (previously reported) with similar patients who received grafts using the anti-CD3 MAb OKT3. RESULTS: Increased cytotoxic Vdelta1+ T cells were seen in 10 of 43 T10B9 TCD patients compared to 7 of 100 in the OKT3 TCD group (23% versus 7%, p = 0.010). T10B9 patients with increased gammadelta+ T cells also exhibited a higher range of increased gammadelta+ T cells and the length of time the gammadelta+ T cells remained high was longer when compared to OKT3 patients. Patients with increased gammadelta+ T cells whose grafts were T-cell depleted with T10B9 showed a significant decrease in relapse (p = 0.038). Similar rates and reduction in relapse were seen in OKT3 TCD patients, although significance was not reached due to the small number of patients with increased gammadelta+ T cells. Estimated 3 year disease-free survival was significantly improved in T10B9 patients with increased gammadelta+ T cells (0.79 versus 0.31, p = 0.009), a trend also seen in OKT3 patients (p = 0.091). DISCUSSION: These observations indicate that Vdelta1+CD4-CD8-cytotoxic T cells are associated with lower relapse rates and improved survival, and thus may have a role in a graft-versus-leukemia effect.

Clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy.

OBJECTIVE: To describe the clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy (IIM) and to compare these with the features of sporadic IIM. METHODS: Clinical signs and symptoms, autoantibodies, HLA-DRB1 and DQA1 alleles, and GM/KM phenotypes were compared among 36 affected and 28 unaffected members of 16 unrelated families in which 2 or more blood relatives developed an IIM. In addition, findings in patients with familial IIM were compared with those in 181 patients with sporadic IIM. The families included 3 pairs of monozygotic twins with juvenile dermatomyositis, 11 families with other siblings or relatives with polymyositis or dermatomyositis, and 2 families with inclusion body myositis. RESULTS: The clinical features of familial IIM were similar to those of sporadic IIM, although the frequency of myositis-specific autoantibodies was lower in familial than in sporadic IIM. DRB1*0301 was a common genetic risk factor for familial and sporadic IIM, but contributed less to the genetic risk of familial IIM (etiologic fraction 0.35 versus 0.51 in sporadic IIM). Homozygosity at the HLA-DQA1 locus was found to be a genetic risk factor unique to familial IIM (57% versus 24% of controls; odds ratio 4.2, corrected P = 0.002). CONCLUSION: These findings emphasize that 1) familial muscle weakness is not always due to inherited metabolic defects or dystrophies, but may be the result of the development of IIM in several members of the same family, and 2) multiple genetic factors are likely important in the etiology and disease expression of familial IIM, as is also the case for sporadic myositis, but DQA1 homozygosity is a distinct risk factor for familial IIM.

T cell-mediated immune mechanisms in myositis.

Advances in molecular biologic techniques and the availability of novel immunologic reagents have allowed new approaches to understanding the pathogenesis of human autoimmune diseases, including the idiopathic inflammatory myopathies. Indirect evidence that autoreactive T cells mediate muscle inflammation in the human myositis syndromes has been strengthened by recent studies describing restricted T cell receptor gene expression in certain clinical and/or serologic groups of myositis patients. These findings are supported by other investigations documenting abnormal patterns of cytokine, adhesion molecule, and major histocompatibility complex antigen expression within inflammatory lesions. The major challenge of future studies is to identify the specific antigen(s) responsible for initiating and perpetuating these harmful immune responses.

Gamma delta T cell receptor gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies.

Autoreactive alpha beta T cells have been implicated as playing a primary pathogenic role in a group of diseases characterized by chronic muscle inflammation known as the idiopathic inflammatory myopathies (IIM). gamma delta T cells, a distinct and enigmatic class of T cells, play a less certain role in a variety of human autoimmune diseases including the IIM. In an attempt to understand the significance of gamma delta T cells in the IIM, we utilized a sensitive polymerase chain reaction (PCR) technique to evaluate gamma delta T cell receptor (TCR) gene expression in 45 muscle biopsies obtained from 42 IIM patients (17 polymyositis, 12 dermatomyositis, and 13 inclusion body myositis). gamma delta TCR gene expression was not detected in 36 specimens, the majority of muscle biopsies surveyed. gamma delta TCR gene expression by muscle-infiltrating lymphocytes was detected among nine clinically heterogeneous patients. We further analysed the junctional sequence composition of the V gamma 3 and V delta 1 transcripts, whose expression was prominent among gamma delta positive patients. DNA sequence analysis of V gamma 3 amplification products from two patients revealed the presence of several productively rearranged transcripts with amino acid sequence similarities within the V gamma 3-N-J gamma junctional domain. No amino acid sequence similarities were evident within the V delta-N-D delta-N-J delta region of V delta 1 transcripts amplified from four patients, although a distinct and dominant clonotype was detected from each patient. Our cumulative data suggest that unlike alpha beta T cells, gamma delta T cells do not play a prominent pathologic role in the IIM. In fact, the sporadic nature of gamma delta TCR gene expression detected among these patients implies that gamma delta T cell infiltration, when it occurs, is a secondary event perhaps resulting from non-specific inflammatory processes.

NIH conference. Myositis: immunologic contributions to understanding cause, pathogenesis, and therapy.

The myositis syndromes, the most common forms of which are polymyositis and dermatomyositis, are defined by idiopathic chronic inflammation in skeletal muscle. Although initially described more than a century ago, these diseases are so rare and heterogeneous that we have only a limited understanding of their causes and treatment. Recently, autoimmune responses to nuclear and cytoplasmic autoantigens that are unique to patients with myositis, the myositis-specific autoantibodies, have proved clinically useful in helping predict signs and symptoms of myositis, immunogenetics, responses to therapy, and prognosis. We summarize this new information on the variety and nature of these autoantibodies, their target epitopes, and their possible use in identifying causes, pathogenetic mechanisms, and better therapies for these increasingly recognized disorders.

A novel gene oriented in a head-to-head configuration with the human histidyl-tRNA synthetase (HRS) gene encodes an mRNA that predicts a polypeptide homologous to HRS.

The human histidyl-tRNA synthetase (HRS) gene encodes an enzyme that catalyzes the esterification of histidine to its cognate tRNA as an early step in protein biosynthesis. Previous reports have described a bidirectional promoter element which coordinates the transcription of both HRS and an unknown mRNA whose gene is oriented in a head-to-head configuration with HRS. We have isolated and characterized a human genomic DNA clone that encodes portions of these oppositely transcribed mRNAs and a putatively full-length cDNA clone (HO3) corresponding to the gene mapping immediately 5' of HRS. The largest open reading frame within HO3 (1518 bp) shares approximately 75% nucleotide sequence identity with human HRS (1527 bp) and predicts a polypeptide with extensive amino acid sequence homology with the HRS protein (72%). Moreover, amino acid sequence motifs characteristic of class II aminoacyl-tRNA synthetases are conserved within HO3. Despite their similarity, HRS and HO3 have divergent amino-terminal domains which correspond to the first two exons of each gene. RNA blot analysis revealed that HRS (2.0 kb) and HO3 (2.5 kb) exhibit distinct patterns of steady-state mRNA expression among multiple human tissues.

The alpha beta T-cell receptor repertoire in inclusion body myositis: diverse patterns of gene expression by muscle-infiltrating lymphocytes.

Inclusion body myositis (IBM) is one member of a group of disorders known as idiopathic inflammatory myopathies (IIM) in which autoreactive T cells directed against muscle are thought to play a primary role in disease pathogenesis. We have utilized the polymerase chain reaction to determine the pattern of alpha beta T-cell receptor (TCR) variable (V) gene expression in muscle biopsies from 13 IBM patients. In the majority of biopsies, we detected oligoclonal patterns of TCR V gene expression by muscle-infiltrating lymphocytes; an average of six out of the 22 TCR V alpha gene families surveyed and seven out of 24 TCR V beta gene families surveyed were detected per biopsy. While no TCR V alpha gene families were over-represented in our survey, TCR V beta 3 and V beta 6 gene usage was a prominent feature of IBM muscle biopsies. TCR gene expression was characterized further by analysing the junctional sequence composition of both V beta 3 and V beta 6 clones from muscle biopsies of the IBM patients. A large number of structurally diverse V beta 3 and V beta 6 clonotypes were identified from these patients demonstrating a polyclonal pattern of T cell infiltration. These data, while describing prominent TCR V beta 3 and V beta 6 gene detection, do not suggest that a common antigen-driven T-cell response promotes chronic inflammation in muscle of IBM patients.

Predominant TCR-alpha beta variable and joining gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies.

The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of diseases in which autoreactive T cells are thought to play a pathogenetic role. We have determined the pattern of TCR-alpha beta gene expression by muscle-infiltrating lymphocytes within clinically and serologically defined groups of IIM patients. We utilized the PCR to study TCR V gene expression in muscle biopsies from nine polymyositis (PM) and eight dermatomyositis (DM) patients, all of whom had autoantibodies directed against histidyl-transfer RNA synthetase (anti-Jo-1 autoantibodies). While the TCR repertoire in DM patients was generally polyclonal, an oligoclonal profile characterized PM patients. Certain V gene families were predominantly expressed; V alpha 1 and V beta 6 gene families were detected in 82 and 91% of PM biopsies, respectively. TCR expression was characterized further by analyzing J gene usage from four PM patients expressing the V beta 6 gene. Sequence analysis of 40 independent recombinants (10 per patient) identified only seven V beta 6 clonotypes and restricted usage of the related J beta 2.1, -2.3, and -2.7 genes. These data, describing predominant TCR V and J gene usage by muscle-infiltrating lymphocytes in myositis patients, suggest that Ag-driven T cell responses may play a primary role in mediating some forms of the IIM.

Analysis of kidney mRNAs expressed from the rat beta-galactoside alpha 2,6-sialyltransferase gene.

beta-Galactoside alpha 2,6-sialyltransferase (SiaT-1), like other glycosyltransferases, is differentially expressed in rat tissues. Two distinct size classes of SiaT-1 mRNAs expressed in rat kidney are comprised of at least three SiaT-1 transcripts. One mRNA, RKE, represents the larger transcript class (4.7 kb) and predicts a polypeptide identical to the hepatic SiaT-1. In transfected Chinese hamster ovary (CHO) cells, RKE polypeptides exhibit hemi-perinuclear staining with a SiaT-1 antibody (Ab-267) that is consistent with Golgi localization. RKE transfectants display cell-surface alpha 2,6-sialic acid linkages as determined by lectin affinity staining. Two other mRNAs, RKA and RKB, are members of a smaller size class (3.6 kb) that comprise predominant SiaT-1 transcripts in rat kidney. Both RKA and RKB encode polypeptides that are missing the amino-terminal 232 residues, but retain 171 amino acids of RKE carboxy-terminal sequence information. A short, leucine-rich peptide present in the divergent amino-terminus of RKA has sequence similarity to the secretory signal domain of several eukaryotic secretory and cell-surface proteins. In transfected CHO cells, both RKA and RKB polypeptides display an immunostaining pattern that is distinct from that of the Golgi-associated SiaT-1 protein (RKE). Furthermore, RKA or RKB transfectants do not display alpha 2,6-sialic acid linkages on cell-surface glycoconjugates. Consistent with the expression of divergent SiaT-1 mRNAs in rat kidney, protein blot analysis of rat tissue homogenates with Ab-267 reveals that in addition to protein that co-migrates with hepatic SiaT-1, rat kidney expresses a unique size class of SiaT-1 proteins.

Rat beta-galactoside alpha 2,6-sialyltransferase genomic organization: alternate promoters direct the synthesis of liver and kidney transcripts.

The rat beta-galactoside alpha 2,6-sialytransferase gene is differentially utilized by liver and kidney in the generation of mRNAs that predict substantially divergent polypeptides. In order to determine the biosynthetic relationship between these sialyltransferase mRNA isoforms, genomic sequences were isolated and analysed. Five exons that span at least 40 kb of DNA carry the coding information for the liver beta-galactoside alpha 2,6-sialyltransferase protein. An additional exon contains only sequences for the 5'-untranslated leader of the liver mRNA. In contrast, the predominant kidney mRNAs from this gene share only three coding exons that specify the carboxyl terminal 42% of the liver sialyltransferase protein sequence. In addition, these kidney mRNAs contain information from two other exons that comprise the 5' divergent region of these transcripts. Primer extension and S1 nuclease protection analysis demonstrate that the hepatic and kidney specific mRNAs are transcriptionally initiated at different sites within the sialyltransferase gene. While the hepatic sialyltransferase mRNAs are transcribed from the first exon, the kidney transcripts are initiated from a site within the third intron. Genomic regions upstream of both transcriptional initiation sites can regulate expression of the bacterial chloramphenicol acetyltransferase gene in transiently transfected L cells. Together, the data implicate multiple promoters as a principle mechanism in the generation of kidney and liver gene product diversity in sialyltransferase expression.

Tissue-specific expression of beta-galactoside alpha-2,6-sialyltransferase. Transcript heterogeneity predicts a divergent polypeptide.

The beta-galactoside alpha-2,6-sialyltransferase represents a member of a family of sialyltransferases which catalyze the terminal addition of sialic acid to maturing carbohydrate chains. We surveyed rat tissues using cDNA probes complementary to coding and noncoding domains of the rat liver alpha-2,6-sialyltransferase. In addition to the expected differences in the level of sialyltransferase mRNA among the tissues, there were dramatic qualitative differences as well. Hepatic sialyltransferase probes hybridize to mRNAs of varying size on Northern blots. A tissue-dependent pattern of expression of these transcripts is documented. Evidence is presented that the multiple transcripts are generated from a common gene sequence. At least one instance of alternate splicing in the generation of the kidney sialyltransferase transcripts is predicted by S1 nuclease analysis. We report the isolation of a rat kidney cDNA clone, RKA, that substantiates this tissue-specific alternate splicing event. The RKA insert, although less than full-length, apparently encodes a polypeptide divergent from the reported hepatic alpha-2,6-sialyltransferase (1). RNA blot analysis indicates that the RKA-type transcripts represent a significant proportion of sialyltransferase RNA in rat kidney. Another class of kidney cDNA clones, RKE, is colinear with the hepatic sialyltransferase sequence. RNA blots probed for the divergent and common regions suggest that complex processing pathways are operative in the tissue-specific expression of sialyltransferase mRNA.

Regulation of beta-galactoside alpha 2,6-sialyltransferase gene expression by dexamethasone.

The hepatic acute phase response is accompanied by increased levels of Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase activity in liver and in circulation. Previous studies suggested that cytokines and glucocorticoids mediate the induction of this sialyltransferase activity. In this study the regulation of sialyltransferase expression by dexamethasone in H35 rat hepatoma cells is assessed by Northern hybridization and enzyme activity assays. Exposure of H35 cells to 1 microM dexamethasone for 24 h causes a 3-4-fold enrichment of sialyltransferase mRNA and a corresponding increase in enzymatic activity. The induction of sialyltransferase mRNA begins within 3 h of dexamethasone treatment and reaches a plateau within 24 h. Sialyltransferase mRNA induction is dose dependent; the minimum concentration of dexamethasone necessary for induction is 10(-8) M, and induction was maximal at 10(-6) M. Induction is sensitive to actinomycin D, suggesting that regulation may be exerted by altering the rate of mRNA synthesis. Puromycin and cycloheximide are ineffective in blocking induction, suggesting that de novo protein synthesis is not required for induction. Finally, dexamethasone alone is sufficient for maximum induction of sialyltransferase mRNA. In contrast, maximal induction of alpha 1-acid glycoprotein, a well studied hepatic acute phase reactant, requires both dexamethasone and cytokines, implying that different pathways exist for the induction of participants in the acute phase response.