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Objective 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. Methods 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. Results 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 (CG), +1551 (CG), and +1922 (TG). 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. Conclusion 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.

Objective 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. Methods 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. Results 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 (TC) 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. Conclusion 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.

Objective 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). Methods 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. Results 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. Conclusion 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.

Objective To localize disease genes for scleroderma, or systemic sclerosis (SSc), in a population of Choctaw Native Americans with a high prevalence of SSc, in which there is evidence of a possible founder effect. Methods A candidate gene approach was used in which microsatellite alleles on human chromosomes 15q and 2q, homologous to the murine tight skin 1 (tsk1) and tsk2 loci, respectively, were analyzed in Choctaw SSc cases and race-matched normal controls for possible disease association. Genotyping first-degree relatives of the cases identified potential disease haplotypes, and haplotype frequencies were obtained by expectation-maximization and maximum-likelihood estimation methods. Simultaneously, the ancestral origins of contemporary Choctaw SSc cases were ascertained using census and historical records. Results A multilocus 2-cM haplotype was identified on human chromosome 15q homologous to the murine tsk1 region, which showed a significantly increased frequency in SSc cases compared with controls. This haplotype contains 2 intragenic markers for the fibrillin 1 (FBN1) gene. Genealogical studies demonstrated that the SSc cases were distantly related, and their ancestry could be traced back to 5 founding families in the mid-eighteenth century. The probability that the SSc cases share this haplotype due to familial aggregation effects alone was calculated and found to be very low. There was no evidence of any microsatellite allele disturbances on chromosome 2q in the region homologous to the tsk2 locus or the region containing the interleukin-1 family. Conclusion A 2-cM haplotype on chromosome 15q that contains FBN1 is associated with scleroderma in Choctaw Native Americans from Oklahoma. This haplotype may have been inherited from common founders about 10 generations ago and may contribute to the high prevalence of SSc that is now seen.