Zhou Xiaodong, Tan Filemon K, Reveille John D, Wallis Debra, Milewicz Dianna M, Ahn Chul, Wang Andrew, Arnett Frank C
Division of Rheumatology and Clinical Immunogenetics, University of Texas-Houston Medical School, 6431 Fannin, MSB 5.270, Houston, TX 77030, USA.
Arthritis Rheum. 2002 Nov;46(11):2990-9. doi: 10.1002/art.10601.
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.
系统性硬化症（SSc）患者的成纤维细胞具有活化表型，其特征为细胞外基质（ECM）成分合成增加。富含半胱氨酸的酸性分泌蛋白（SPARC）可调节ECM成分的沉积或组装。本研究旨在通过功能和基因关联研究探讨SPARC在SSc易感性中的作用。
利用互补DNA（cDNA）微阵列获取SSc患者培养的真皮成纤维细胞的基因表达数据。通过蛋白质印迹法评估SPARC蛋白水平。在乔克托印第安人中对SPARC基因（5号染色体q31 - 32）5 cM范围内的5个多态性微卫星标记进行基因分型，该人群先前显示SSc患病率较高。通过对SPARC cDNA测序发现单核苷酸多态性（SNP）。然后在多民族SSc患者队列中对这些SNP进行基因分型，以确定在更广泛的SSc患者群体中与疾病易感性以及与SSc的各种临床和免疫特征的潜在关联。还评估了这些SNP与SPARC转录稳定性的功能相关性。
与正常对照相比，微阵列显示SSc患者中SPARC以及其他ECM基因的表达增加。SSc成纤维细胞的SPARC蛋白水平也升高。在乔克托SSc患者中，SPARC附近的5个微卫星标记中有3个与SSc存在显著关联。SPARC cDNA测序在3'非翻译区发现3个新的SNP，分别位于+998（C→G）、+1551（C→G）和+1922（T→G）。在不同种族的SSc患者中，SNP +998处C等位基因的纯合性显著增加。SPARC SNP +1551和+1922分别与雷诺现象和肺纤维化相关。在正常成纤维细胞培养物中对SPARC SNP +998进行的功能研究表明，SNP +998 C等位基因多态性与信使RNA半衰期延长相关。
本研究首次表明，SPARC基因多态性与SSc的易感性和临床表现相关，并且它们在影响皮肤成纤维细胞中SPARC表达方面可能也具有重要功能。
