Abstract
Identification of genetic predisposition to cardiac sarcoidosis could play a critical role in the detection of sub-clinical forms of the disease. The aim of this study was to investigate the possible correlations between the emergence of cardiac sarcoidosis and the -1.031T/C, -857C/T, -308G/A, and -238G/A Tumor Necrosis Factor-α (TNFA) polymorphisms in a well-defined Greek cohort. One-hundred and seventy-three patients of Greek origin with sarcoidosis were recruited in the present study. Cardiac sarcoidosis was determined according to established criteria. Blood samples were collected and the TNFA polymorphisms were genotyped. No significant difference was noted between the patients with cardiac involvement and those without, concerning the -1.031T/C and -238G/A TNFA polymorphisms. Regarding the -857C/T polymorphism, the TT genotype and the T allele were found to be over-represented in patients with cardiac involvement (p=0.02 and 0.012, respectively). AA genotype of the -308G/A as well as the A allele were also found significantly more frequently in patients with cardiac sarcoidosis (p=0.014 and 0.012 respectively). From the investigated TNFA promoter polymorphisms, we were able to deduce nine main haplotypes. Haplotypes 3 and 5, including A nucleotide at position -308, and T nucleotide at position -857 respectively, were significantly over-represented in the group with cardiac involvement. We detected an increased presence of genetic polymorphisms in the TNFA gene of patients with cardiac involvement. However, the role and the clinical application of these findings need further exploration.
Sarcoidosis is a multi-systematic disease of unknown etiology characterized by the presence of non-caseating granulomas in the involved organs, predominantly the lung parenchyma and the mediastinal lymph nodes (1). Recent advances in the immunopathogenesis of sarcoidosis have shown that environmental antigen stimulus and genetic predisposition interact in the course of the development of the disease (2). No single antigen or gene polymorphism has been identified as initiating and maintaining this disease. To date, the HLA class II histocompatibility antigen, DRB1-9 beta chain (HLA-DRB1) and major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) alleles are considered as having strong genetic associations with sarcoidosis, while interactions between the emergence of HLA-DQB1 and exposure to high humidity may be considered possible causes of sarcoidosis (3).
The initial lesion within the pulmonary system is CD4+ (cluster of differentiation 4) T-cell alveolitis, followed by the development of noncaseating granulomas. The granuloma formation is based on the accumulation and interaction of T-lymphocytes, mononuclear phagocytes, B-lymphocytes and dendritic cells, and is thought to result from an exaggerated cell-mediated immune response to one or more unidentified antigens (4). The interactions between these cells are regulated by cytokines, mainly interleukin-1 and tumor necrosis factor-α (TNFα). The induction of Th1 response and suppression of Th2 response in parallel is considered as predominant in granuloma formation and strongly associated with TNF production (5). Studies on the role of cytokine gene polymorphisms have shown that polymorphisms in the promoter region of the TNFA gene affect the levels of TNFα production, resulting in high and low TNFA producers (6). Moreover, it has been reported that the frequency of the rare TNFA -308 allele was significantly higher in Japanese patients with cardiac sarcoidosis (7).
Cardiac sarcoidosis is the leading cause of morbidity and mortality in the course of this disease. Although clinical evidence of myocardial involvement is present in approximately 5% of patients with sarcoidosis, autopsy studies have revealed sub-clinical cardiac involvement to be present in 20-30% of cases (8).
Determining the genetic predisposition to cardiac sarcoidosis could play a critical role in the identification of underlying sub-clinical forms of the disease. The present study was conducted to investigate possible correlations between the emergence of cardiac sarcoidosis and the -1.031T/C, -857C/T, -308G/A, and -238G/A TNFA polymorphisms in a well-defined Greek cohort.
Materials and Methods
Patients. One-hundred and seventy-three patients with sarcoidosis out of a total of 415 (42 with cardiac involvement and 131 with pulmonary sarcoidosis at a ratio of 1 cardiac vs. 3 non-cardiac) of Greek origin were recruited in the present study. For all patients, the diagnosis of sarcoidosis was established when clinicoradiological findings were supported by histological evidence of noncaseating granulomas on transbronchial biopsies, and after exclusion of other known causes of granulomatosis (9). Twenty-one were newly-diagnosed sarcoid cases. A complete clinical, detailed cardiac and pulmonary evaluation was performed for all patients including standard electrocardiography, transthoracic echocardiography, 24-h Holter monitoring, cardiac magnetic resonance imaging, chest X-ray and pulmonary function testing. All consecutive patients with sarcoidosis were evaluated for the presence of cardiac involvement based on the modified criteria of the Japanese Ministry of Health and Welfare (10). Before commencement of the study, the Ethical Committee at the participating centers approved the recruitment protocols. All participants were informed regarding the study, and their written consent was provided.
Genotyping. DNA was isolated from peripheral blood with the NucleoSpin Blood Kit (Macherey-Nagel, Germany). The -1031(T/C) polymorphism of TNFA was amplified by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) method with a set of primers: 5’-TATGTGATGG ACTCACCAGG-3’ and 5’-CCT CTACATGGCCCTGTCTT-3’. The 35 cycles of PCR were performed at 96°C for 5 min, 94°C for 30 s, 63°C for 40 s, and 72°C for 1 min and final cycle of 72°C for 10 min. Amplified PCR products were digested with BbsI (New England BioLabs, Beverly, MA, USA) for 14 h at 37°C. Fragments of 251 and 13bp revealed homozygosity for the T allele and 180, 71 and 13 bp fragments indicated homozygosity for the C allele.
The promoter polymorphism -857(C/T) of TNFA was identified with the allele-specific reverse primers 5’-CTACATGGCCCTGTC TTCG-’ and 5’-TCTACATGGCCCTGTCTTCA-3’ in combination with the consensus forward primer 5’-AAGGATAAGGGCT CAGAGAG-3’ in two separate PCR reactions, with an expected PCR product size of 270 bp. The 3’ ends of the forward primers were able to anneal to regions that differ between the two alleles. The cycling parameters for the were 96°C for 1 min, followed by five cycles of 96°C for 25 s, 70°C for 45 s, and 72°C for 25 s, 21 cycles of 96°C for 25 s, 65°C for 50 s, 72°C for 30 s; and four cycles of 96°C for 30 s, 55°C for 60 s, and 72°C for 90 s.
Clinical characteristics of patients with sarcoidosis.
The -308 (G/A) and the -238 (G/A) polymorphisms of TNFA were genotyped as previously described (11).
Statistical analysis. Genotypic frequencies were compared with the Chi-square test with Yate's correction using S-Plus (v. 6.2; Insightful, Seattle, WA USA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00; GraphPad Software, San Diego, CA, USA). The p-values are all two-sided. p-Values of less than 0.05 were considered to be significant. Hardy–Weinberg equilibrium was verified by calculating the expected frequencies and numbers and was tested separately in patients and in controls using the goodness-of-fit χ2 test.
Results
Table I summarizes the clinicopathological characteristics of the studied population. In our population, we observed a significant frequency of cardiac involvement in women patients compared to males.
The frequencies of each genotype of the -1.031(T/C), - 857(C/T), -308(G/A), and -237(G/A) TNFA polymorphisms are shown in Table II, and conformed to Hardy–Weinberg equilibrium (p>0.05). There was no significant difference between the panel of patients with cardiac sarcoidosis and those without cardiac involvement concerning the -1.031(T/C) and -238(G/A) TNFA polymorphisms. Regarding the -857 (C/T) polymorphism, the TT genotype and the T allele were found to be over-represented in patients with cardiac involvement (p=0.02 and 0.012, respectively). AA genotype of the -308 (G/A) polymorphism as well as the A allele were also found significantly more frequently in patients with cardiac sarcoidosis (p=0.014 and 0.012, respectively).
From the investigated TNFA promoter polymorphisms, we were able to deduce nine main haplotypes, which are shown in Table III. Haplotypes 3 and 5, including A nucleotide at position -308, and T nucleotide at position -857 respectively, were significantly over-represented in the sarcoidosis group with cardiac involvement, whereas no differences were found in the frequencies of the other haplotypes between the two groups.
Genotypic frequencies of the -1.031(T/C), -857(C/T), -308(G/A), and -238 (G/A) Tumor Necrosis Factor-α (TNFA) polymorphisms in patients with sarcoidosis.
Tumor Necrosis Factor-α (TNFA) promoter haplotype frequency in patients with sarcoidosis.
Finally, there was no statistically significant relationship between the existence of the TNFA polymorphisms tested and the presence of late gadolinium enhancement in cardiac magnetic resonance imaging or lung staging.
Discussion
It has been widely accepted that TNFα plays a central role in granulomatous inflammation in sarcoidosis (12). Several studies have supported the contribution of inter-individual variations in TNFα production to the immune response, thus promoter polymorphism of TNFA are of great immunogenetic interest (13). In British and Dutch patients with sarcoidosis, Grutters et al. demonstrated a clearly significant increase in the rarer TNFA -857T allele in comparison to othe the control groups (14). Medica et al. in a meta-analysis demonstrated a significant association for the -308 TNFA gene polymorphism and sarcoidosis under the dominant genetic model (15).
In this study, in order to address the possibility that TNFA gene also confers susceptibility to cardiac sarcoidosis, we investigated the association of four TNFA promoter polymorphisms with cardiac involvement in a well-defined cohort of Greek patients with sarcoidosis. Our results indicated that the -857TT and -308AA genotypes, as well as the rare alleles -857T and -308A, were significantly over-represented in patients with cardiac sarcoidosis.
To our knowledge, there are limited data regarding the association of specific gene polymorphisms with cardiac sarcoidosis. Our findings are in agreement with those of Takashige et al. who showed that the -308A allele of TNFA controlled the genetic susceptibility to cardiac sarcoidosis in a Japanese population (16). The same polymorphism has been implicated in genetic susceptibility to idiopathic dilative cardiomyopathy which resembles sarcoidosis and has a similar clinical phenotype (17). Liang et al. also stated that the presence of this particular polymorphism increases the risk for dilative cardiomyopathy (18).
It has been suggested that TNFA polymorphism could be involved in the clinical course of sarcoidosis, but these suggestions are contradictory (6, 19, 20). In our Greek sarcoidosis group, no relationship between TNFA polymorphisms and the existence of gadolinium enhancement or radiographic staging of the patients was found. Moreover, although several studies support an association of the TNFA - 308A allele with the presence of Löfgren's syndrome (21, 22), our data do not support these findings.
To date, we still do not know what predisposes certain patients with sarcoidosis to have cardiac involvement. In the present study, we have established that TNFA -857T and -308A variants are associated with cardiac involvement in Greek patients with sarcoidosis. However, further investigations performed on a larger number of patients with a different ethnical background would additionally highlight the importance of this type of polymorphism for the clinical presentation of sarcoidosis.
Footnotes
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Conflicts of Interest
None.
- Received August 5, 2014.
- Revision received September 14, 2014.
- Accepted September 19, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
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