Anti-CENPI autoantibodies in scleroderma patients with features of autoimmune liver diseases

doi:10.1016/j.cca.2011.08.024

Clinica Chimica Acta

Volume 412, Issues 23–24, 20 November 2011, Pages 2267-2271

https://doi.org/10.1016/j.cca.2011.08.024 Get rights and content

Abstract

Background

Anticentromere autoantibodies have been reported to be associated with scleroderma and serve as a marker in different rheumatic diseases in humans. Major centromere autoantigens described so far include constitutive kinetochore proteins such as CENPA, CENPB, CENPC and CENPH and facultative proteins such as CENPE, CENPF and INCENP. We examined the inner kinetochore component CENPI as a new putative centromere autoantigen in scleroderma patients.

Methods

To test for the presence of CENPI centromere autoantibodies, 72 sera from patients with systemic lupus erythematosus and systemic sclerosis were assayed by immunofluorescence and further tested by immunoblots with an Nt-CENPI recombinant protein.

Results

8 out of 31 (25.8%) patients diagnosed of scleroderma or Undifferentiated Connective Tissue Disease (UCTD) produced anti-CENPI autoantibodies. Epitopes were demonstrated to be located mainly but not exclusively in the N-terminal domain of the human CENPI protein. Five of the 8 (62.5%) CENPI positive sera also had other autoantibodies related to primary biliary cirrhosis. Further, two patients (25%) with anti-CENPI autoantibodies had concurrent diagnosis of primary biliary cirrhosis.

Conclusions

This study demonstrates that CENPI, a centromere protein that localizes to the inner kinetochore structure, is a human autoantigen. The significance of anti-CENPI autoantibodies could be relevant in scleroderma patients as a marker for concurrent autoimmune liver disease.

Highlights

►In this study we examine the presence of anti-CENPI antibodies in scleroderma patients. ►We found CENPI autoepitopes on several region of the autoantigen. ►We demonstrated that CENPI is a new human autoantoantigen in scleroderma patients.

Introduction

In 1980 Moroi et al. first identified anticentromere autoantibodies (ACA) in a scleroderma (SSc) patient by indirect immunofluorescence (IF) analysis [1]. Since then, ACA have been shown to be highly specific for SSc particularly for the limited cutaneous form of the disease (lcSSc). These autoantibodies (autoAb) can also be detected in sera of patients with Raynaud's phenomenon without defined rheumatic disorder and in other rheumatic autoimmune diseases such as Sjögren's syndrome, rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC) [2], [3]. The major centromere proteins detected by ACA are CENPA, CENPB and CENPC [4]. Occasionally, some human ACA positive sera contain autoAb directed against CENPD, CENPE, CENPF and CENPG [5], [6]. Recently, another protein localized at the kinetochore throughout the cell cycle, CENPH, was described as a novel centromere autoantigen in patients with Sjögren's syndrome [7]. In addition, CENPO, a new component of the kinetochore, has been described as a novel antigenic target in SSc [8]. Finally, autoAb against the facultative centromeric protein INCENP has also been detected in a patient with Graham Little-Piccardi-Lassueur syndrome [9].

Antinuclear Ab (ANA) are present in more than 95% of patients suffering from SSc, although the pathogenic role of these autoAb is unclear [10]. To better understand the ANA response in SSc, we studied new antigenic targets by analyzing the immune response against the centromere protein CENPI [11]. The potential significance of anti-CENPI autoAb in patients with scleroderma is discussed.

Section snippets

Patients

Sera from 72 patients with different rheumatic diseases (69 women and 3 men; mean age 53.3 ± 14.2 years, age range 27–81) were collected over a period of two years. Patients were clinically diagnosed in the Unit of Rheumatology at the Hospital General of Jerez, Cadiz, Spain. Among them, 31 were diagnosed as SLE, 24 had lcSSc, 5 had SSc diffuse form (dcSSc), 8 were diagnosed with Sjögren's syndrome, 2 had overlap syndromes and 2 were diagnosed with undifferentiated connective tissue disease (UCTD).

Expression and characterization of human Nt-CENPI

The Nt-CENPB and Nt-CENPI human recombinant polypeptides used in this study contained a 6× histidine tag to facilitate the identification and eventual isolation by a nickel affinity column. The centromere antigens highly expressed in E. coli cells were isolated from inclusion bodies and both migrated at the predicted molecular weight for Nt-CENPB (42 kDa) and Nt-CENPI (32 kDa), as shown in Fig. 1A (lanes 1, 2). A rabbit anti-histidine serum served to recognize both antigens by immunoblotting as

Discussion

To date, more than 60 proteins have been identified as putative components of human centromeres, and a few of these were identified as autoantigens in a number of clinical studies [18]. In scleroderma patients, virtually all ACA-positive sera react against CENPB [19] and a solid-phase assay (blot) using a cloned CENPB antigen has been established for clinical studies with adequate sensitivity and specificity [20]. The contribution of other kinetochore proteins to the immune response in patients

Conflict of interest

All authors have declared no conflicts of interest.

Acknowledgments

This work was supported by a grant (SAF2007-61863) from the Ministerio de Educación y Ciencia to MMV. Financial support for research group CTS-579 was obtained from Plan Andaluz de Investigación, Junta de Andalucía. We thanks the English technical advice of Neil Thompson.

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Proteomics characterization of CENP-B epitope in Moroccan scleroderma patients with anti-centromere autoantibodies
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However, none of these two patients was diagnosed with PBC or had some clinical symptoms for this disease. These findings were similar to those reported by Hamdouch et al. in Spanish population [30]. They identified the presence of anti-Nt-CENPB auto-antibodies in patients with scleroderma and AMA positive.
Anti-centromere auto-antibodies (ACA) have been described as a marker in Systemic sclerosis (SSc) disease. CENP-B is the major centromere auto-antigen recognized by SSc patients with positive ACA. Our aim was to characterize the major epitope involved in the anti-CENP-B immune response of Moroccan SSc patients.
For identification of SSc biomarkers, 80 sera from patients with SSc and systemic lupus erythematosus (SLE) were screened by indirect immunofluorescence test (IIF) to assess the presence of ANA reactivity. Immunoblotting analysis was performed for 11 sera with positive ACA using the N-terminal and C-terminal region of CENP-B protein as antigens.
29 out of 30 (96, 66 %) patients with SSc had positive ANA. 11 out of 30 (36, 67 %) patients were ACA positive and 6 of them produced auto-antibodies against Nt-CENPB antigen. Two of these 6 Nt-CENPB positive sera produced also other auto-antibodies associated to primary biliary cirrhosis. None of all sera tested showed reactivity against Ct-CENPB.
Our data showed, for the first time in Morocco, that the Nt-CENPB contains a major epitope for Moroccan SSc patients. These findings could provide additional information that would contribute to improving the diagnosis and management of these patients.
CENPI is overexpressed in colorectal cancer and regulates cell migration and invasion
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Centromere protein I (CENPI) plays an important role in accurate chromosome alignment and segregation and it ensures proper mitotic progression (Okada et al., 2006; Cheeseman et al., 2008). CENPI was originally suggested to associate with autoimmune diseases (Hamdouch et al., 2011; Bien and Bauer, 2013; Chang et al., 2014). Recently, genetic mutations in CENPI were found in several types of human cancer such as breast carcinoma, large intestine adenocarcinoma, lymphoid neoplasm, melanoma and squamous cell carcinoma (Kumar et al., 2013).
Centromere protein I (CENPI),an important member of centromere protein family, has been suggest to serve as a oncogene in breast cancer, but the clinical significance and biological function of CENPI in colorectal cancer (CRC) is still unclear. In our results, we found CENPI was overexpressed in CRC tissues and cells, and associated with clinical stage, tumor depth, lymph node metastasis, distant metastasis and differentiation in CRC patients. However, there was no significant association between CENPI protein expression and overall survival time in colon cancer patients and rectal cancer patients through analyzing TCGA survival data. Moreover, CENPI mRNA and protein were increased in metastatic lymph nodes compared with primary CRC tissues. Down-regulation of CENPI expression suppresses CRC cell migration, invasion and epithelial mesenchymal transition process. In conclusion, CENPI is overexpressed in CRC and functions as oncogene in modulating CRC cell migration, invasion and EMT process.
Diagnostic and clinical significance of anti-centromere antibodies in primary biliary cirrhosis
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Citation Excerpt :
Third, several commercial ELISA and blotting kits have been developed over the years for ACA testing, and have been incorporated into routine laboratories, where their use has led to a better recognition of ACA positivity in autoimmune liver diseases, including PBC. ACA are highly prevalent of SSc, but are also reported in other autoimmune diseases including PBC [153–155]. Indeed, some 9 to 30% of patients with PBC are positive for ACA [156–159].
Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver characterised by biochemical evidence of cholestasis, elevated alkaline phosphatase levels and the presence of the highly disease specific anti-mitochondrial autoantibodies. Extra-hepatic autoimmune manifestations are common, including rheumatic disorders, such as systemic sclerosis (SSc). Notably, PBC is the most frequent autoimmune liver disease in SSc patients. Based on skin lesion extension, two major SSc disease subgroups are recognised: limited cutaneous SSc (lSSc) and diffuse cutaneous SSc. Anti-centromere antibody (ACA) positivity is highly characteristic of SSc, with up to 90% prevalence in lSSc patients. ACA has also been found in up to 30% of PBC patients and 80% of patients with a PBC/SSc overlap syndrome. The diagnostic and clinical significance of ACA positivity in patients with PBC without SSc has recently been under investigation, with several studies highlighting links to severe bile duct injury and portal hypertension. This review discusses the diagnostic and clinical relevance of ACA in patients with PBC, with or without SSc.
Liver abnormalities in connective tissue diseases
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SSc overlap syndromes with AIH and PSC have been reported only occasionally [56–58]. Recently, it has been suggested that antibodies against centromeric protein I (CENPI, a protein localized in the inner kinetochore structure) may be a marker of concurrent autoimmune liver disease in SSC patients [59]. Idiopathic inflammatory myopathies – polymyositis (PM) and dermatomyositis (DM) – are systemic autoimmune diseases characterized by skeletal muscle inflammation, but other organs are frequently involved such as skin in DM and lungs and heart in both PM and DM [60].
The liver is a lymphoid organ involved in the immune response and in the maintenance of tolerance to self molecules, but it is also a target of autoimmune reactions, as observed in primary liver autoimmune diseases (AILD) such as autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis. Further, the liver is frequently involved in connective tissue diseases (CTD), most commonly in the form of liver function test biochemical changes with predominant cholestatic or hepatocellular patterns. CTD commonly affecting the liver include systemic lupus erythematosus, antiphospholypid syndrome, primary Sjögren's syndrome, systemic sclerosis, dermatomyositis, polimyositis, and anti-synthetase syndrome, while overlap syndromes between AILD and CTD may also be diagnosed. Although liver cirrhosis and failure are extremely rare in patients with CTD, unusual liver conditions such as nodular regenerative hyperplasia or Budd–Chiari syndrome have been reported with increasing frequency in patients with CTD. Acute or progressing liver involvement is generally related to viral hepatitis reactivation or to a concomitant AILD, so it appears to be fundamental to screen patients for HBV and HCV infection, in order to provide the ideal therapeutic regimen and avoid life-threatening reactivations. Finally, it is important to remember that the main cause of biochemical liver abnormalities in patients with CTD is a drug-induced alteration or coexisting viral hepatitis. The present article will provide a general overview of the liver involvement in CTD to allow rheumatologists to discriminate the most common clinical scenarios.
Autoantibodies in systemic sclerosis
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Besides antibodies directed to CENP-A and CENP-B, other CENP targets have been described (reviewed in [79]). For example, anti-CENP-C has been associated with Sjögren's syndrome [103,104] and a recent study identified antibodies to CENP-I that may be a biomarker for concurrent autoimmune liver disease [105]. Autoantibodies to RNAP I and RNAP III (anti-RNAP I and III) often coexist and this pattern of autoantibody expression are highly specific for SSc [106,107].
Autoantibodies directed against a variety of nuclear, cytoplasmic and extracellular autoantigens are a serological hallmark of systemic sclerosis. This review provides an overview of the history and clinical association of many of the autoantibodies identified in SSc sera to date. Some of these autoantibodies predate the clinical diagnosis of SSc, some are pathogenic while others have no apparent role in pathogenesis. It was once thought that the autoantibody spectrum of individual SSc sera were less complex than other systemic autoimmune rheumatic diseases with respect to heterogeneous B cell responses reflected in circulating autoantibodies. However, with the advent of array technologies, there is now an unprecedented capability to detect multiple autoantibodies in an individual serum and this long held tenet of clinical diagnostic immunology is being reexamined.
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Anti-CENPI autoantibodies in scleroderma patients with features of autoimmune liver diseases

Abstract

Background

Methods

Results

Conclusions

Highlights

Introduction

Section snippets

Patients

Expression and characterization of human Nt-CENPI

Discussion

Conflict of interest

Acknowledgments

Rheum Dis Clin North Am

Clin Gastroenterol Hepatol

Dig Liver Dis

Autoantibody to centromere (kinetochore) in scleroderma sera

Proc Natl Acad Sci U S A

The clinical significance of antinuclear antibodies in connective tissue diseases

Semin Clin Immunol

Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma

Chromosoma

CENPF is a ca 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization

Cell Motil Cytoskeleton

The centromere kinesin-like protein CENPE. An autoantigen in systemic sclerosis

Arthritis Rheum

Anti CENPH antibodies in patients with Sjogren's syndrome

Rheumatol Int

CENPO a protein localized at the centromere throughout the cell cycle is a novel target antigen in systemic sclerosis

J Rheumatol

Autoantibodies against the chromosomal passenger protein INCENP found in a patient with Graham Little-Piccardi-Lassueur syndrome

J Autoimmun Dis

The role of autoantibodies in autoimmune disease

Annu Rev Immunol