Abstract
Objective. Systemic sclerosis (SSc) is a fibrotic immune-mediated disease of unknown etiology. Among its clinical manifestations, pulmonary involvement is the leading cause of mortality in patients with SSc. However, the genetic factors involved in lung complication are not well defined. We aimed to review the association of the MIF gene, which encodes a cytokine implicated in idiopathic pulmonary hypertension among other diseases, with the susceptibility and clinical expression of SSc, in addition to testing the association of this polymorphism with SSc-related pulmonary involvement.
Methods. A total of 4392 patients with SSc and 16,591 unaffected controls from 6 cohorts of European origin were genotyped for the MIF promoter variant rs755622. An inverse variance method was used to metaanalyze the data.
Results. A statistically significant increase of the MIF rs755622*C allele frequency compared with controls was observed in the subgroups of patients with diffuse cutaneous SSc (dcSSc) and with pulmonary arterial hypertension (PAH) independently (dcSSc: p = 3.20E–2, OR 1.13; PAH: p = 2.19E–02, OR 1.32). However, our data revealed a stronger effect size with the subset of patients with SSc showing both clinical manifestations (dcSSc with PAH: p = 6.91E–3, OR 2.05).
Conclusion. We reviewed the association of the MIF rs755622*C allele with SSc and described a phenotype-specific association of this variant with the susceptibility to develop PAH in patients with dcSSc.
Systemic sclerosis (SSc) is a complex disease of unknown etiology influenced by both genetic and environmental factors. It is characterized by a progressive tissue fibrosis along with vascular anomalies and dysfunction, and the presence of autoantibodies directed to different cellular structures, mainly antitopoisomerase (anti-topo I) and anticentromere antibodies (ACA). Depending on the extent of the skin implication, SSc is classified as diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Among the clinical manifestations, pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH) are the leading causes of death in patients with SSc1. These pulmonary complications can be present separately as interstitial lung disease (ILD) or isolated PAH, or combined2.
In recent years, great advances have been made in the determination of the genetic component of SSc3,4. Nevertheless, we are still far from its complete understanding, especially regarding pulmonary involvement, in which only a few associated genes have been described3,4.
Macrophage migration inhibitory factor (MIF) gene encodes a constitutively expressed protein that seems to have an important role in autoimmune and inflammatory processes. Infections, proinflammatory cytokines, and antigen-specific activation can also lead to an increased expression of MIF5,6. The promoter region of this gene contains a single-nucleotide polymorphism (SNP) at position −173 (rs755622) that has been associated with several immune-mediated diseases, including SSc and systemic lupus erythematosus7,8,9. This SNP is in high linkage disequilibrium (LD) with a functional polymorphism, a −794 CATT5,6,7,8 microsatellite repeat (rs5844572) also located at the promoter region of the gene10. Interestingly, increased MIF protein levels have been reported in patients with idiopathic pulmonary fibrosis, pulmonary hypertension, and in individuals affected by SSc-associated PAH11,12,13.
In our present study, we have analyzed for the first time, to our knowledge, the possible involvement of the MIF rs755622 polymorphism in the susceptibility to develop PF and PAH in patients with SSc.
MATERIALS AND METHODS
Samples
Overall, 4392 patients with SSc and 16,591 unaffected controls from Spain, Germany, the Netherlands, Italy, the United Kingdom, and Norway were included in our present study. The cohorts included in our study were partially overlapping with the cohorts in Bossini-Castillo, et al7, based on the presence of pulmonary involvement clinical data. In total, the overlap between our global study cohort and the one included in Bossini-Castillo, et al’s study7 was 30%. All patients were classified as having dcSSc or lcSSc, following the criteria described by LeRoy, et al14. Patients were additionally classified accordingly with the presence or absence of ACA and anti-topo I. PF was diagnosed by the presence of interstitial abnormalities in high-resolution computed tomography (HRCT) and forced vital capacity (FVC) lower than 60%. Pulmonary hypertension was considered PAH if FVC was higher than 60% or there was no moderate-severe extent of ILD in HRCT. Thus, patients were defined as PAH+ if they showed a mean resting pulmonary artery pressure ≥ 25 mmHg at the time of a right heart catheterization, pulmonary artery wedge pressure ≤ 15 mmHg, and FVC > 60%15,16. The control population consisted of unrelated healthy individuals recruited in the same geographic regions as the patients with SSc.
The local ethical committees from all the contributing centers approved the project in compliance with the Helsinki Agreement (Valle de Hebron Hospital, 12 de Octubre University Hospital, San Cecilio University Hospital, de la Santa Creu i Sant Pau Hospital, Carlos Haya Hospital, San Carlos Hospital, Bellvitge University Hospital, Virgen del Rocio Hospital, San Jorge General Hospital, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinic Foundation, Università degli Studi di Verona University, Spedali Civili Hospital, Oslo University Hospital, Josefs-Hospital, University of Lübeck, Hannover Medical School, University of Cologne, Radboud University Nijmegen Medical Centre, VU University Medical Center, Leiden University Medical Center, Glasgow Biomedical Research Centre, Manchester University, and University Medical Center Utrecht). Patients and controls gave written informed consent for their participation in the present study.
Genotyping
DNA extraction was performed using standard methods. MIF rs755622 was genotyped using a TaqMan SNP genotyping assay (assay ID: C_2213785_10) in a Light Cycler 480 Real-Time PCR System (Roche Applied Science). We also used available MIF rs755622 genotyping data from a previously published study based on the Immunochip17, a genotyping platform that was specifically designed for the study of the genetic component of immune-mediated diseases. Supplementary Table 1 (available with the online version of this article) shows the number of samples genotyped in each platform. The genotyping call rate (the fraction of called samples per SNP over the total number of samples in the dataset) was 97% for the TaqMan assay and 99% for the Immunochip data. The genotype-genotype concordance was evaluated in 1253 samples with genotyping data from both platforms. A 100% of concordance was observed between both genotyping platforms.
Statistical methods
Using the Power Calculator for Association Studies (CaTS) software 2006 (Center for Statistical Genetics, The University of Michigan), and assuming an effect size of OR 1.2, we estimated that the statistical power of the study was 99% for the overall analysis of the whole cohort (SSc cases vs controls), 98% for the analysis of dcSSc and PF phenotypes, 61% for the analysis of patients who were PAH+, and 5% for the analysis with patients with dcSSc who have PAH. Because the statistical power depends on the OR of the SNP, we also calculated the statistical power for this last comparison assuming the observed effect size in our study, obtaining a 97% of statistical power. Additionally, no deviation from Hardy-Weinberg equilibrium was detected in our datasets.
All the statistical analyses were carried out with PLINK v1.07 (pngu.mgh.harvard.edu/purcell/plink). Association statistics for the 6 cohorts were calculated in each population by 2 × 2 contingency tables and the chi-square test. P values lower than 0.05 were considered statistically significant. The inverse-variance–weighted fixed-effects metaanalysis method was used for the pooled analyses.
RESULTS
We first investigated whether there were significant differences between the rs755622 allele frequencies of the whole SSc group or the different stratified groups (by clinical/auto-antibody phenotypes) and those of the control set. Supplementary Table 2 (available with the online version of this article) summarizes the results for the analyses of the independent cohorts and Table 1 shows the results of the pooled analysis. As observed in Table 1, a significant p value was obtained when the dcSSc group was compared against the control set in the metaanalysis (p = 3.20E–2, OR 1.13, 95% CI 1.01–1.26). Moreover, there was a significant increase of the minor allele frequency in the group of patients with SSc affected by PAH compared with controls (p = 2.19E–02, OR 1.32, 95% CI 1.04–1.67). However, no association was observed between rs755622 and the PF-positive SSc group (PF+ vs controls: p = 0.257; PF+ vs PF−: p = 0.737).
Pooled analyses of MIF rs755622 in patients with SSc, SSc phenotypic and serological subgroups, and unaffected controls.
To dissect the genetic association of rs755622*C with the SSc clinical phenotypes, we classified the patients into 4 additional subgroups based on the SSc subtype and the presence/absence of PAH. Then we carried out a pooled analysis of the 6 cohorts by comparing these new subsets against controls (Table 2; see Supplementary Table 3 for the results of each cohort independently, available with the online version of this article). A considerably significant difference in the rs755622 frequencies between controls and the subgroup of patients with both dcSSc and PAH phenotypes was observed (dcSSc with PAH vs controls: p = 6.91E–03, OR 2.05, 95% CI 1.30–4.05). However, no association was evident in the analysis of the other subgroups against controls (lcSSc with PAH vs controls: p = 0.178; dcSSc without PAH vs controls: p = 0.086; lcSSc without PAH vs controls: p = 0.436). We also did not find evidence of association between MIF rs755622 and SSc-related PF and dcSSc-related PF (data not shown).
Pooled analyses of MIF rs755622 with the SSc subtype and PAH status.
DISCUSSION
Our study, which consists of the largest cohort of patients with SSc-associated PAH analyzed to date, clearly suggests that the MIF rs755622*C variant is a firm genetic risk factor for the susceptibility to PAH in patients with dcSSc. Previous reports described an association of this SNP with the diffuse form of the disease7,8. Consistent with this, we also observed a significant increase in the MIF rs755622*C frequency in the dcSSc subgroup compared with the control set. However, subphenotype analyses indicated that the significant associations detected in dcSSc and the PAH+ subgroups may rely on the presence of subjects with both phenotypes in each dataset. Therefore, those carrying the MIF rs755622*C allele are at a higher risk of having dcSSc with PAH rather than PAH or dcSSc independently. The strongest effect size of MIF rs755622*C was observed in the subset of patients having both dcSSc and PAH (OR 2.05), which clearly supports this idea. Therefore, our findings may suggest MIF rs755622*C as a marker for patients with dcSSc at risk of developing PAH.
In our set, the percentages of PAH in the different phenotype subsets, dcSSc or lcSSc, were 2.55% and 6.41%, respectively. That is, 14.78% of PAH+ cases were patients with dcSSc and 85.22% corresponded to lcSSc cases, in line with previous reports in SSc patient cohorts18,19. However, as mentioned above, we found that the association observed between MIF rs755622 and the PAH+ group remained significant only under a dcSSc phenotype. In this regard, gene expression patterns specific for SSc, SSc-related PF, and SSc-related PAH have been described20, thus indicating that each SSc phenotype may develop under a distinct molecular environment. The high specificity of the MIF rs755622 association with a particular subset of patients with SSc may be related to this fact.
MIF rs755622 is a promoter polymorphism that has been linked to the upregulation of the MIF expression in immune-mediated diseases5. As mentioned, this SNP is in high LD with the functional CATT5-8 polymorphism, and it is still not established whether rs755622 influences MIF promoter activity by itself or whether the observed effect may be linkage to CATT5-8. Interestingly, elevated protein levels of this gene have been detected in the sera of patients with dcSSc affected by PAH13,21. Moreover, Le Hiress, et al showed through in vitro studies that the MIF receptor (CD74) is overexpressed in individuals with idiopathic PAH22. These authors also provided evidence for the effect of an MIF antagonist and anti-CD74–neutralizing antibodies on the reversion of PH in a rat model22. MIF is a pleotropic protein expressed in several human cell types that can act as a cytokine, hormonal, and immune modulatory factor. As a cytokine, MIF induces the expression and secretion of several immune mediators, including interleukin 6 (IL-6)23. Increased levels of IL-6 have been described in serum and lungs of patients with idiopathic PAH and those with dcSSc compared with lcSSc24,25, and IL-6 has a proved effect on the generation and development of PAH in vivo24. Moreover, allelic combinations of SNP in the IL-6 gene have been suggested as susceptibility factors for SSc26. Based on the above, we speculate that MIF rs755622 could be influencing PAH development in dcSSc-affected individuals by promoting IL-6 secretion, which could contribute to the obstruction of small pulmonary vessels that leads to hypoxia, and eventually to PAH.
Here we provide novel insights into the genetic background of SSc-related PAH. We have shown that the MIF allele rs755622*C is associated with higher risk of being affected by this severe condition in patients with dcSSc, and our results may be helpful for a better evaluation of the prognosis in SSc and for the development of more effective personalized treatments.
ONLINE SUPPLEMENT
Supplementary material accompanies the online version of this article.
Acknowledgment
We thank Sofia Vargas, Sonia García, and Gema Robledo for their excellent technical assistance, and all the patients and control donors for their essential collaboration. We thank Banco Nacional de ADN (University of Salamanca, Spain), and the Norwegian Bone Marrow Donor Registry, which supplied part of the control DNA samples. We are also thankful to the European League Against Rheumatism Scleroderma Trials and Research group (EUSTAR) and the German Network of Systemic Sclerosis for the facilitation of this project.
APPENDIX 1.
List of study collaborators. Spanish Scleroderma Group: Norberto Ortego-Centeno, and Raquel Ríos and Unidad de Enfermedades Sistémicas Autoinmunes, Department of Internal Medicine, Hospital Clínico Universitario San Cecilio, Granada; José Antonio Vargas Hitos, Department of Internal Medicine, Hospital Virgen de las Nieves, Granada; Rosa García Portales, Department of Rheumatology, Hospital Virgen de la Victoria, Málaga; María Teresa Camps, Department of Internal Medicine, Hospital Carlos Haya, Málaga; María F. González-Escribano, Department of Immunology, Hospital Virgen del Rocío, Sevilla; Julio Sánchez-Román, and Mª Jesús Castillo, Department of Internal Medicine, Hospital Virgen del Rocío, Seville; Mª Ángeles Aguirre and Inmaculada Gómez-Gracia, Department of Rheumatology, Hospital Reina Sofía/IMIBIC, Córdoba; Benjamín Fernández-Gutiérrez Department of Rheumatology, Hospital Clínico San Carlos, Madrid; Esther Vicente, Department of Rheumatology, Hospital La Princesa, Madrid; José Luis Andreu and Mónica Fernández de Castro, Department of Rheumatology, Hospital Puerta de Hierro Majadahonda, Madrid; Paloma García de la Peña, Department of Rheumatology, Hospital Madrid Norte Sanchinarro, Madrid; Francisco Javier López-Longo and Lina Martínez, Department of Rheumatology, Hospital General Universitario Gregorio Marañón, Madrid; Vicente Fonollosa and Alfredo Guillén, Department of Internal Medicine, Hospital Valle de Hebrón, Barcelona; Gerard Espinosa, Department of Internal Medicine, Hospital Clinic, Barcelona; Carlos Tolosa, Department of Internal Medicine, Hospital Parc Tauli, Sabadell; Anna Pros, Department of Rheumatology, Hospital Del Mar, Barcelona; Mónica Rodríguez Carballeira, Department of Internal Medicine, Hospital Universitari Mútua Terrassa, Barcelona; Francisco Javier Narváez, Department of Rheumatology, Hospital Universitari de Bellvitge, Barcelona; Vera Ortiz-Santamaría, Department of Rheumatology, Hospital General de Granollers, Granollers; Miguel Ángel González-Gay, Department of Rheumatology, Hospital Universitario Marqués de Valdecilla, IFIMAV, Santander; Bernardino Díaz and Luis Trapiella, Department of Internal Medicine, Hospital Central de Asturias, Oviedo; Mayka Freire and Adrián Sousa, Unidad de Trombosis y Vasculitis, Department of Internal Medicine, Hospital Xeral-Complexo Hospitalario Universitario de Vigo, Vigo; María Victoria Egurbide, Department of Internal Medicine, Hospital Universitario Cruces, Barakaldo; Patricia Fanlo Mateo, Department of Internal Medicine Hospital Virgen del Camino, Pamplona; Luis Sáez-Comet, Unidad de Enfermedades Autoinmunes Sistémicas, Department of Internal Medicine, Hospital Universitario Miguel Servet, Zaragoza; Federico Díaz and Vanesa Hernández, Department of Rheumatology, Hospital Universitario de Canarias, Tenerife; Emma Beltrán, Department of Rheumatology, Hospital General Universitario de Valencia, Valencia; Elena Grau, Department of Rheumatology, Hospital Universitario y Politécnico La Fe, Valencia; José Andrés Román-Ivorra, Department of Rheumatology, Hospital Universitari i Politécnico La Fe, Valencia; Juan José Alegre Sancho, Department of Rheumatology, Hospital del Doctor Peset, Valencia; Francisco J. Blanco García and Natividad Oreiro, Department of Rheumatology, INIBIC-Hospital Universitario A Coruña, La Coruña.
Footnotes
Supported by the following grants: JM was funded by SAF2015-66761-P from the Spanish Ministry of Economy and Competitiveness. This study was also funded by PI-0590-2010, from Consejería de Salud y Bienestar Social, Junta de Andalucía, Spain. ELI was supported by Ministerio de Educación, Cultura y Deporte through the program FPU.
- Accepted for publication May 5, 2017.
REFERENCES
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