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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.amjmed.2005.01.055 Byline: Shervin Assassi (a), Maureen D. Mayes (a), Terry McNearney (b), Michael Fischbach (c), John D. Reveille (a), Frank C. Arnett (a), Filemon K. Tan (a) Author Affiliation: (a) Division of Rheumatology and Clinical Immunogenetics, University of Texas Health Science Center at Houston (b) Division of Rheumatology, University of Texas Medical Branch (c) Division of Rheumatology, University of Texas Health Science Center at San Antonio Article Note: (footnote) Supported by NIAMS Specialized Center of Research (SCOR) in scleroderma grant (P50AR4448) and University Clinical Research Center grants M01-RR02558 (UTH-HSC), M01-RR00073 (UTMB), M01-RR01346 (UT-SA).

OBJECTIVE: To identify differentially expressed genes in peripheral blood cells (PBC) of patients with ankylosing spondylitis (AS) relative to healthy controls and controls with systemic inflammation. METHODS: We investigated PBC samples of 16 patients with AS and 14 matched controls, in addition to systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) samples utilizing Illumina Human Ref-8 BeadChips. Candidate genes were confirmed using quantitative PCR. Subsequently, these genes were also validated in a separate sample of 27 patients with AS [before and after anti-tumor necrosis factor (anti-TNF) treatment] and 27 matched controls. RESULTS: We identified 83 differentially expressed transcripts between AS patients and controls. This gene list was filtered through the lists of differentially expressed transcripts in SLE and SSc, which resulted in identification of 52 uniquely dysregulated transcripts in AS. Many of the differentially expressed genes belonged to Toll-like receptor (TLR) and related pathways. TLR4 and TLR5 were the only dysregulated TLR subtypes among AS patients. We confirmed the overexpression of TLR4 and TLR5 in AS patients in comparison to controls (p = 0.012 and p = 0.006, respectively) and SLE (p = 0.002, p = 0.008) using quantitative PCR in the same sample. Similarly, TLR4 (p = 0.007) and TLR5 (p = 0.012) were significantly upregulated among the AS patients before anti-TNF treatment in the confirmatory sample. TLR4 (p = 0.002) and TLR5 (p = 0.025) decreased significantly after anti-TNF treatment. CONCLUSION: PBC gene expression profiling in AS shows an upregulation of TLR4 and TLR5. This supports the importance of TLR subtypes in the pathogenesis of AS that are responsible for the immune response to Gram-negative bacteria.

ObjectiveBirth order has been valuable in revealing the role of environmental influences on the risk of developing certain diseases such as allergy and atopy. In addition, pregnancy has profound effects on the immune system such as shortterm effects that permit fetal survival as well as longerterm effects that could influence lateonset diseases. In order to better evaluate these influences, we studied the association of birth order and gravidityparity as risk factors for systemic sclerosis SSc; scleroderma.MethodsData regarding SSc cases and their unaffected sibling controls were obtained from the Scleroderma Family Registry and DNA Repository. The casesibling design was used to minimize confounding due to differences in age, race, ethnicity, or calendar time. The gravidityparity analysis was based on sibships with at least one SScaffected and one unaffected sister.ResultsBirth order was examined in 974 sibships, comparing SSc cases n 987 with their unaffected siblings n 3,088. The risk of scleroderma increased with increasing birth order odds ratio OR 1.25, 95 confidence interval 95 CI 1.06–1.50 for birth order 2–5; OR 2.22, 95 CI 1.57–3.15 for birth order 6–9; and OR 3.53, 95 CI 1.68–7.45 for birth order 10–15. Gravidityparity was analyzed in 168 sibships 256 unaffected sisters, 172 SSc cases. We found an association between a history of one or more pregnancies and SSc OR 2.8.ConclusionBirth order and pregnancy were independently associated with a higher risk of developing SSc. These findings suggest that immune development in early childhood andor pregnancyassociated events, including but not limited to microchimerism, plays a role in SSc susceptibility.

OBJECTIVE: IL23R has been identified as a susceptibility gene for development of multiple autoimmune diseases. We investigated the possible association of IL23R with systemic sclerosis (SSc), an autoimmune disease that leads to the development of cutaneous and visceral fibrosis. METHODS: We tested 9 single-nucleotide polymorphisms (SNP) in IL23R for association with SSc in a cohort of 1402 SSc cases and 1038 controls. IL23R SNP tested were previously identified as SNP showing associations with inflammatory bowel disease. RESULTS: Case-control comparisons revealed no statistically significant differences between patients and healthy controls with any of the IL23R polymorphisms. Analyses of subsets of SSc patients showed that rs11209026 (Arg381Gln variant) was associated with anti-topoisomerase I antibody (ATA)-positive SSc (p = 0.001)) and rs11465804 SNP was associated with diffuse and ATA-positive SSc (p = 0.0001, p = 0.0026, respectively). These associations remained significant after accounting for multiple comparisons using the false discovery rate method. Wild-type genotype at both rs11209026 and rs11465804 showed significant protection against the presence of pulmonary hypertension (PHT). (p = 3x10–5, p = 1x10–5, respectively). CONCLUSION: Polymorphisms in IL23R are associated with susceptibility to ATA-positive SSc and protective against development of PHT in patients with SSc.

To determine the relative contributions of genetic, clinical, serologic, sociodemographic, and behavioral/psychological variables to early pulmonary involvement in the Genetics versus Environment in Scleroderma Outcome Study cohort.At the baseline visit (V0), 203 patients with systemic sclerosis (SSc) were examined (104 whites, 39 African Americans, and 60 Hispanics). We obtained sociodemographic, behavioral/psychological (illness behavior, social support, learned helplessness, smoking, drinking), clinical, serologic (autoantibodies), and genetic (HLA class II and FBN1 genotypes) factors; pulmonary function test results; electrocardiograms; and chest radiographs. Data analysis included Fisher's exact test, chi‐square test, Student's t‐test, analysis of variance, and stepwise linear and logistic regression methods.Significant pulmonary involvement was seen in 25% of patients within 2.8 years of SSc diagnosis. At V0, pulmonary fibrosis was significantly higher in African Americans compared with whites or Hispanics. African Americans had significantly lower percent predicted forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) compared with whites and significantly lower percent predicted diffusing capacity for carbon monoxide (DLCO) compared with whites and Hispanics. Significant, independent associations impacting early pulmonary involvement included African American ethnicity, skin score, serum creatinine and creatine phosphokinase values, hypothyroidism, and cardiac involvement. Anticentromere antibody seropositivity was a significant, independent, protective factor for restrictive lung disease and FVC or DLCO values. African Americans had significantly increased frequencies of anti–topoisomerase I, fibrillarin, and RNP autoantibodies compared with whites. African Americans scored significantly lower on the Interpersonal Support Evaluation List and significantly higher on the Illness Behavior Questionnaire.Early pulmonary involvement in SSc appears to be influenced by several factors delineated by ethnicity, including racial, socioeconomic, behavioral, and serologic determinants.

Recent family, twin, and genetic association studies suggest a genetic basis for the susceptibility to systemic sclerosis or scleroderma. The purpose of this review is to summarize the results of genetic association and gene expression profiling studies from January 2004 to May 2005.

Systemic sclerosis, or scleroderma, is an uncommon autoimmune connective tissue disease that results in systemic fibrosis. Its etiologic basis remains unclear. The pathogenesis of systemic sclerosis involves a proliferative and obliterative vasculopathy resulting from endothelial cell dysfunction, extensive fibrosis secondary to fibroblast activation, and autoimmunity as demonstrated by the presence of disease-specific autoantibodies. Although there is no clear and convincing evidence for an environmental trigger in most cases, accumulating data emphasize the role of genetic factors in systemic sclerosis. As in other complex human diseases, multiple genes likely contribute to disease susceptibility and the clinical manifestations of systemic sclerosis. This review will cover the application of genomics to the complex genetics of systemic sclerosis.

Fibroblasts from patients with systemic sclerosis (SSc) have an activated phenotype characterized by increased synthesis of extracellular matrix (ECM) components. SPARC (secreted protein, acidic and rich in cysteine) regulates the deposition or assembly of ECM components. The aim of this study was to investigate the role of SPARC in SSc susceptibility by functional and genetic association studies. Complementary DNA (cDNA) microarrays were used to obtain gene expression data on cultured dermal fibroblasts from SSc patients. SPARC protein levels were assessed by Western blotting. Five polymorphic microsatellite markers within 5 cM of the SPARC gene (chromosome 5q31-32) were genotyped in Choctaw Indians, a population previously shown to have a high prevalence of SSc. Discovery of single-nucleotide polymorphisms (SNPs) was accomplished by sequencing the SPARC cDNA. These SNPs were then genotyped in a multi-ethnic cohort of SSc patients to determine potential associations with disease susceptibility in a broader population of SSc patients, as well as with various clinical and immunologic features of SSc. The functional relevance of these SNPs with regard to transcript stability of SPARC was also assessed. Microarrays demonstrated increased expression of SPARC, along with other ECM genes, in SSc patients compared with normal controls. SSc fibroblasts also had increased SPARC protein levels. Three of 5 microsatellite markers near SPARC showed significant associations with SSc in the Choctaw SSc patients. Sequencing of SPARC cDNA revealed 3 novel SNPs in the 3'-untranslated region at +998 (C→G), +1551 (C→G), and +1922 (T→G). Homozygosity for the C allele at SNP +998 was significantly increased in SSc patients across ethnic lines. SPARC SNPs +1551 and +1922 demonstrated correlations with Raynaud's phenomenon and pulmonary fibrosis, respectively. Functional studies of SPARC SNP +998 in normal fibroblast cultures suggested a correlation between the SNP +998 C allele polymorphism and an increased messenger RNA half-life. This study is the first to show that polymorphisms of the SPARC gene are associated with susceptibility to, and clinical manifestations of, SSc and that they may also be functionally important in influencing SPARC expression in skin fibroblasts.

To determine if there are abnormalities in fibrillin 1–containing microfibrils in the extracellular matrix (ECM) of primary dermal fibroblasts explanted from patients with systemic sclerosis (SSc). Explanted fibroblasts from unaffected skin of 12 SSc patients were used to examine fibrillin 1–containing microfibrils by immunofluorescence (IF) using a monoclonal antibody (mAb) to fibrillin 1. Metabolic labeling of the fibroblast cultures was used to study the synthesis, secretion, and processing of fibrillin 1, as well as to observe microfibril formation and stability. Microfibrils elaborated by the SSc cells were analyzed by electron microscopy for ultrastructural abnormalities, and the results were confirmed by immunoblotting. Control and SSc fibroblasts displayed a prominent meshwork of fibrillin 1–containing microfibrils when visualized by IF using a fibrillin 1 mAb. Paradoxically, metabolic studies indicated a paucity of fibrillin 1 in the ECM in the majority of the SSc fibroblast strains. Subsequent rotary-shadowed electron microscopy revealed reduced amounts of and ultrastructural abnormalities in the microfibrils elaborated by all strains of SSc cells. Immunoblots confirmed the lack of the high molecular weight form of fibrillin 1 in the SSc fibroblasts of Choctaw American Indians. Finally, in vitro studies indicated that the amount of fibrillin 1 in the ECM of SSc cells diminished at a faster rate than the amount of fibrillin 1 in the ECM of control cells with time. Although SSc fibroblasts assemble microfibrils, these microfibrils are unstable, suggesting that an inherent defect of fibrillin 1–containing microfibrils may play a role in the pathogenesis of SSc.

Mutations in FBN1 cause the autosomal dominant condition, Marfan syndrome. A single-base mutation that results in a skipping of exon 2 of FBN1 was found in a Marfan patient. By sequencing this proband's entire FBN1 gene and comparing the mutated DNA sequence with proband's unaffected family numbers, we confirmed this alteration was the causative mutation. The skipping of exon 2 creates a frameshift and premature termination codon, and forms a truncated fibrillin-1 composed only of 55 amino acids of N-terminus plus 45 nonsense amino acids. The mRNA transcription levels of the mutated FBN1 allele and the deposition of fibrillin-1 into extracellular matrix in fibroblast cells culture were assessed. © 2001 Wiley-Liss, Inc.

Previously, we demonstrated with the use of microsatellite markers that a 2‐cM haplotype on chromosome 15q containing the fibrillin 1 gene (FBN1) was strongly associated with systemic sclerosis (SSc) in the Choctaw, a population with high SSc prevalence. In this study, all 69 known FBN1 exons were sequenced to ascertain the presence of changes that might show associations with SSc in the Choctaw and Japanese SSc patients and controls.Screening of FBN1 exons was accomplished by polymerase chain reaction–based fluorescence sequencing of genomic DNA using single‐nucleotide polymorphism (SNP) haplotypes, and their frequencies were determined with a new algorithm that recognizes past recombination events between sites. Haplotype phylogenies were inferred using the median‐joining network analysis.Five SNPs were identified in FBN1. They are located in the 5′‐untranslated region (SNP‐1), exon 15 (SNP‐2), intron 17 (SNP‐3), exon 27 (SNP‐4), and intron 27 (SNP‐5). Only SNP‐1 (T→C) demonstrated an association with SSc in the Choctaw. Eleven FBN1 SNP haplotypes were ascertained in the Choctaw population, 2 of which (SNPs 5 and 6) were found only in the SSc patients. These same FBN1 SNP haplotypes were associated with SSc in the Japanese.A SNP in the 5′‐untranslated region of FBN1 (SNP‐1, C allele) was strongly associated with SSc in the Choctaw. Furthermore, this polymorphism is present on 2 unique FBN1 haplotypes found only in Choctaw SSc patients. The same 2 haplotypes demonstrate associations with SSc in the Japanese. These data extend the earlier microsatellite studies and are consistent with the hypothesis that FBN1 or a nearby gene on chromosome 15q is involved in SSc susceptibility in the Choctaw and the Japanese.

There is increasing evidence that genetic factors play important roles in susceptibility to and expression of systemic sclerosis (SSc), as well as primary Raynaud phenomenon. Familial aggregation for SSc, although infrequent (1.2–1.5 of SSc families), has now been established, and when compared with population prevalence represents a significant risk factor for the disease and lays a firmer foundation for genetics in etiopathogenesis. Major histocompatibility complex class II alleles increase disease risk in some populations but are more strongly correlated with specific autoantibody profiles. Microchimerism influenced by human leukocyte antigen also remains an intriguing hypothesis. A variety of extracellular matrix genes, including fibrillin-1, have become additional candidates for contributing to what is likely a complex genetic disease. Reviewed here is evidence relating to these concepts, especially new data reported over the last year.

We previously reported the presence of autoantibodies to the extracellular matrix protein, fibrillin 1, in sera from patients with systemic sclerosis (SSc). These autoantibodies appeared to be highly disease-specific but had significantly different frequencies among ethnic groups. The aims of this study were 3-fold: 1) to determine whether sera from SSc patients of different ethnic backgrounds recognized different antigenic epitopes of fibrillin 1, 2) to determine whether sera from patients with polymyositis/dermatomyositis (PM/DM) with or without interstitial lung disease (ILD) also produced these antibodies, and 3) to determine any correlation of anti–fibrillin 1 antibodies with specific clinical features of SSc, other autoantibodies, or HLA class II alleles in a prospectively studied cohort of SSc patients with early (<5 years' duration) disease (the Genetics versus Environment In Scleroderma Outcome Study [GENISOS] cohort). Three recombinant peptides accounting for the N-terminal end, proline-rich C region, and epidermal growth factor–like calcium-binding (EGF-cb) domains of fibrillin 1 were used in a radioimmunoassay to screen sera from a large group of SSc and PM/DM patients and ethnically matched controls. The majority of Choctaw American Indians, Japanese, and African Americans with SSc produced IgM and/or IgG autoantibodies to one or more recombinant fibrillin 1 proteins, while <50% of Caucasians with SSc showed seroreactivity. There were striking ethnic differences in fibrillin 1 antigenic epitope recognition among these ethnic groups. African American SSc sera recognized primarily the N-terminal end, and Caucasian sera mostly recognized the EGF-cb repeats and the proline-rich C region. In contrast, most Choctaw American Indian and Japanese SSc sera appeared to recognize 2 or 3 epitopes, respectively. PM/DM patient sera did not recognize any of the fibrillin 1 epitopes regardless of the presence of ILD. In the prospective, multiethnic GENISOS cohort, the presence of anti–fibrillin 1 antibodies did not correlate with any major clinical manifestations, other autoantibodies, or HLA class II alleles. There are striking ethnic differences in antigenic epitope specificity of anti–fibrillin 1 antibodies in patients with SSc, and the majority of SSc patients, except for Caucasians, produce antibodies to fibrillin 1. The antifibrillin response thus far remains specific for scleroderma syndromes, but it does not correlate with any major clinical features, other autoantibodies, or HLA class II alleles.

Abnormalities of transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) α and β and/or their receptors have been demonstrated in systemic sclerosis (SSc). This study aimed to determine whether genetic polymorphisms in or near the TGFβ and PDGF gene families were associated with susceptibility to SSc in a Native American population with a high disease prevalence. Genotyping of 5 intragenic polymorphisms within the TGFβ1 gene and mapping of 35 microsatellites near the genes for TGFβ1, latent TGFβ1 binding protein (LTBP1), TGFβ receptors I and II, PDGFα, PDGFβ, PDGF receptor α, and PDGF receptor β was performed in 19 SSc patients, 76 controls, and 42 family members. Allele distributions and frequencies were examined between SSc patients and controls, and marker haplotypes were examined in families when allele frequencies appeared to be different between patients and controls. Although 1 polymorphism within the TGFβ1 gene (TGFB1) was modestly increased in the SSc patients, this did not maintain statistical significance after correction. Similarly, 1 microsatellite (D9S120) near the TGFβ receptor I gene (TGFBR1) showed a significant disturbance of allele frequencies between patients and controls; however, it did not form a disease-associated haplotype with other nearby markers. Weak disturbances of markers near PDGFα (PDGFA) and PDGFβ (PDGFB) also failed to maintain significance after correction. Both PDGF receptor genes (PDGFRA and PDGFRB) also showed no disease associations. The results of these preliminary analyses suggest that genetic anomalies of the TGFβ1 and PDGF gene families are not likely to explain the dysregulation seen in SSc or to account for the susceptibility to SSc in this population.

Serum autoantibodies to fibrillin 1, the major component of microfibrils in the extracellular matrix, recently have been reported to occur in the tight skin mouse and in patients with systemic sclerosis, but not in patients with other connective tissue diseases. This study was undertaken to determine whether anti–fibrillin 1 antibodies could be detected in patients with localized forms of scleroderma. Sera from 50 patients with localized scleroderma (27 with linear scleroderma and 23 with morphea) and 51 normal controls were tested for IgG and IgM antifibrillin 1 autoantibodies, using a radioimmunoassay (RIA) and a human recombinant fibrillin 1 protein (rFbn-1). Both in patients with linear scleroderma and in those with morphea, mean levels of IgM and IgG binding to rFbn-1 were significantly higher than in controls. Eight patients with linear scleroderma (30%) and 6 patients with morphea (26%) had IgG autoantibodies to fibrillin 1 (rFbn-1) by RIA, compared with 3 controls (6%) (P = 0.006 and P = 0.022, respectively). No correlations between antifibrillin 1 antibodies and active skin disease or antinuclear antibody positivity were found. Autoantibodies to fibrillin 1 occur in patients with both forms of localized scleroderma (linear scleroderma and morphea). The clinical and pathogenetic significance of this autoimmune response remains to be determined.

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