|Year : 2018 | Volume
| Issue : 1 | Page : 306-310
Association of macrophage migration inhibitory factor - 173 G/C polymorphism with dilated cardiomyopathy in children
Rawhia H El-Edel1, Rasha I N. El-Din1, Rania S El-Zayat2, Hanan Abd El-Aziz Mohamed El-Bagoury1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebein El-Kom, Egypt
2 Department of Pediatrics, Faculty of Medicine, Menoufia University, Shebein El-Kom, Egypt
|Date of Submission||09-Jun-2015|
|Date of Acceptance||02-Aug-2015|
|Date of Web Publication||14-Jun-2018|
Hanan Abd El-Aziz Mohamed El-Bagoury
Shebein El-Kom, Menoufia Governorate
Source of Support: None, Conflict of Interest: None
The objective of this study was to detect the relation between macrophage migration inhibitory factor (MIF) −173 G/C gene polymorphism and dilated cardiomyopathy (DCM) in children.
DCM is the most common cardiomyopathy. Myocardial inflammation is one of the commonest mechanisms in cardiomyopathy. MIF plays an important role in the control of innate immune responses and promotes proinflammatory biological activities; hence, MIF gene polymorphisms may predispose affected hosts to severe inflammatory or infectious disease.
Patients and methods
This study was conducted on 50 patients, 30 patients with DCM and 20 age-matched and sex-matched healthy controls. All patients were subjected to history taking, clinical examination, echocardiography, and laboratory measurement of serum sodium and uric acid. Patients and controls were subjected to PCR-restriction fragment length polymorphism to determine MIF 173 G/C polymorphism.
Our study revealed a significant statistical difference in the distribution of the genotypes and allele frequencies between patients and controls. Studying the relation between different genotypes and echocardiographic parameters (ejection fraction, fractional shortening, left ventricular end-diastolic diameter and left ventricular end-systolic diameter) revealed a highly significant difference between different genotypes among patients. With regard to laboratory characteristics, there was a significantly higher serum sodium level in the CC genotype, while the uric acid level showed no significant difference between different genotypes among patients.
In our study, we conclude that the CC genotype of MIF gene may be a risk factor for cardiomyopathic patients and that the C allele is associated with severe clinical condition.
Keywords: children, dilated cardiomyopathy, macrophage migration inhibitory factor, polymorphism
|How to cite this article:|
El-Edel RH, N. El-Din RI, El-Zayat RS, Mohamed El-Bagoury HA. Association of macrophage migration inhibitory factor - 173 G/C polymorphism with dilated cardiomyopathy in children. Menoufia Med J 2018;31:306-10
|How to cite this URL:|
El-Edel RH, N. El-Din RI, El-Zayat RS, Mohamed El-Bagoury HA. Association of macrophage migration inhibitory factor - 173 G/C polymorphism with dilated cardiomyopathy in children. Menoufia Med J [serial online] 2018 [cited 2019 Aug 23];31:306-10. Available from: http://www.mmj.eg.net/text.asp?2018/31/1/306/234209
| Introduction|| |
Dilated cardiomyopathy (DCM) is the most common cardiomyopathy, accounting for ∼55% of cardiomyopathies . DCM is characterized by left ventricular dilatation and global systolic dysfunction [ejection fraction (EF)<50%], in the absence of coronary artery disease or other identifiable causes (such as systemic hypertension, valve disease, drugs, inflammatory heart diseases), capable of causing that magnitude of impairment .
The annual incidence is 2–8/100 000 with an estimated prevalence of 1/2500 population. The percentage of cases with a genetic etiology is ∼30–50% . Three major factors have been implicated in the pathogenesis of myocardial damage in DCM: preceding viral myocarditis, autoimmunity, and underlying genetic predisposition. However, in the majority of patients, no specific etiology is demonstrable (i.e., idiopathic DCM) . DCM is manifested by heart failure, thromboembolism, and sudden cardiac death , and it is the cause of significant morbidity and mortality as well as a common indication for cardiac transplantation .
Although the pathogenesis of CMP is not fully understood, cellular as well as humoral autoimmune responses are critically associated with the pathogenesis and progression of the disease, and myocardial inflammation is one of the commonest mechanisms in cardiomyopathy .
Macrophage migration inhibitory factor (MIF) is known to be a proinflammatory cytokine and plays a role in cell proliferation and differentiation . MIF was shown to contribute to neuroendocrine modulation, as a pituitary gland-derived hormone, inflammation, atherosclerosis, cancer development, and cancer progression . It plays an important role in the control of innate immune responses and promotes proinflammatory biological activities; hence, MIF gene polymorphisms would predispose affected hosts to severe inflammatory or infectious disease .
The MIF gene is located on the long arm (q) of chromosome 22 (22q11.2) . It has three exons of 205, 173 and 183 bp. These are separated by two introns of 189 and 95 bp  and encodes a protein of 115 amino acids and of molecular weight 12.5 kDa .
Human MIF gene has four reported polymorphisms: three single-nucleotide polymorphisms (SNPs) repeat at positions −173 (−173 G/C), +254 (+254 T/C), and + 656 (+656 C/G) and a 5–8-CATT tetranucleotide repeat at position −794 [−794 CATT (5–8)]. The + 254 and + 656 SNPs are positioned in introns and do not affect the coding sequence of the gene. Thus, MIF genotyping studies have focused mainly on the -173 G/C and the -794 CATT polymorphisms .
| Patients and Methods|| |
Study population and selection of patients
This study was conducted at the Clinical Pathology Department, Faculty of Medicine, Menoufia University, in Collaboration with Pediatric Department, Faculty of Medicine, Menoufia University, during the period spanning from May 2013 and March 2015.
This study was conducted on 50 (28 boys and 22 girls) individuals; their ages ranged between 1 and 18 years.
The studied individuals were divided into two groups:
- Group I included 30 patients having DCM (16 boys, 14 girls). Their ages ranged between 1 and 18 years
- Group II included 20 apparently healthy, age-matched and sex-matched individuals as a control group (12 boys, eight girls); their ages ranged between 2 and 18 years.
Inclusion criterion for patients was age below 18 years.
Patients having valvular or congenital heart diseases were excluded.
The study protocol was approved by the Local Ethics Committee of the Menoufia University. Informed consents were taken from both the patients and the control group participants before the beginning of the study.
All the patients were subjected to the following:
- History taking: personal history, medication (patients were on medication such as Frusemide, Captopril and Digoxin). Moreover, family history of DCM was taken
- Clinical examination
- Laboratory tests including serum uric acid and serum sodium (Na)
- Molecular testing for MIF 173 G/C gene polymorphism was performed by PCR on the basis of restricted fragment length polymorphism (RFLP).
All the controls were subjected to the following:
Molecular testing for MIF 173 G/C gene polymorphism was performed by PCR on the basis of RFLP.
Sample collection and preparation
A volume of 5 ml of blood sample was collected from each individual under complete aseptic condition by clean venipuncture. The sample was divided as follows:
A volume of 2 ml of whole blood was added to an EDTA-contained sterile tube for immediate extraction of genomic DNA.
A volume of 3 ml of whole blood was added to a sterile plain tube and allowed to clot at 37°C. Serum was separated by centrifugation and used for immediate assay of serum uric acid and serum Na.
- Estimation of serum uric acid:
The estimation was carried out by an enzymatic method on Au 480 autoanalyzer
- Estimation of serum Na:
Serum Na was estimated by ion-selective electrodes on Au 480 autoanalyzer
- Determination of MIF − 173 SNP.
Determination of MIF − 173 SNP was carried out by RFLP-PCR:
Total DNA was extracted from the EDTA-treated blood sample using Thermo Scientific Gene JET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
The 366 bp fragment was amplified using the amplification mix, which consisted of the following:
- 1 μl of each of primer (forward: 5′-ACTAAGAAAGACCCGAGGC-3′ and reverse: 5′-GGGGCACGTTGGTGTTTAC-3′)
- 25 μl of Dream Taq Green PCR master mix (#K1081; Thermo Scientific, Burlington, Ontario, Canada)
- 18 μl of nuclease-free water
- Thereafter, 5 μl of extracted DNA was added to the corresponding reaction tube.
PCR cycling conditions
The PCR amplification was performed on pre programmed thermal cycler (GeneAmp PCR System 2400 Thermal Cycler; PerkinElmer, Waltham, Massachusetts, USA) under the following conditions: an initial denaturation step at 94°C for 3 min, followed by 40 cycles, each cycle consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min. Final extension at 72°C for 5 min was carried out.
As a negative control, PCR mix without DNA sample was used to ensure contamination-free PCR product.
Confirmation of successful PCR amplification was carried out by 2.5% agarose gel electrophoresis. A volume of 5 μl normal range (50 bp) ladder and 5 μl of PCR product were applied. Thereafter, electrophoresis was performed for 15 min at 100 V then for 30 min at 80 V [Figure 1].
|Figure 1: A representative agarose gel picture showing migration inhibitory factor gene amplification bands that correspond to ladder band size of 366 bp (lanes 2–7), lane 1 and 8: 50 bp DNA ladder.|
Click here to view
Identification of MIF 173 G/C gene polymorphism using a specific restriction enzyme
After PCR, the products are digested with a specific restriction enzyme, FastDigest Alu1; hence, the following reaction components were mixed gently at room temperature:
- 5 μl nuclease-free Water
- 2 μl 10× FastDigest Green Buffer
- 1 μl FastDigest Alu1 enzyme (#FD0014, Lot: 00147479; Thermo Fisher Scientific)
- 12 μl DNA amplification product.
The mixture was incubated at 37°C in a heat block for 3 h.
The reaction mixture was loaded directly and electrophoresed on 3% agarose gel containing ethidium bromide and visualized under UV illumination. PCR products with GG genotype were digested into two fragments, 268 and 98 bp, whereas those with CC genotype were digested into three fragments, 205, 98 and 63 bp. PCR products with GC genotype were digested into four fragments, 268, 205, 98 and 63 bp.
Results were collected, tabulated and statistically analyzed by SPSS, version 20 (Armonk, NY: IBM Corp).
| Results|| |
Considering MIF 173 G/C gene polymorphism, our study showed a significant difference in distribution of the genotypes and allele frequencies between patients and controls (P < 0.05).
Odds ratio showed that GC genotype is more risky than GG 'reference group' by 1.02 (95% confidence interval: 0.30–3.44). The CC genotype has no odds ratio, as the CC genotype in controls was zero. The C allele is more risky than the G allele by 2.31 (95% confidence interval: 0.98–5.45) [Table 1] and [Table 2].
|Table 1: Comparison between patients and control groups with regard to genotype distribution|
Click here to view
|Table 2: Comparison between patients and control groups with regard to alleles|
Click here to view
We also found that there was a highly significant difference with regard to echocardiography parameters (EF, fractional shortening, left ventricular end-diastolic diameter and left ventricular end-systolic diameter) between different genotypes among patients (P < 0.001) [Table 3].
|Table 3: Comparison between the different genotypes among patients with regard to echo parameters|
Click here to view
With regard to serum Na level in patients, the CC genotype showed higher statistically significant differences compared with GC (P > 0.001) [Table 4].
|Table 4: Comparison between CC and GC genotypes among patients with regard to serum sodium|
Click here to view
With regard to uric acid level, there was no statistically significant difference in the uric acid level between different genotypes among the patients (P > 0.05) [Table 5].
|Table 5: Comparison between the different alleles among patients with regard to uric acid|
Click here to view
| Discussion|| |
This work aimed to study MIF 173 G/C SNP in Egyptian children with DCM.
Our study showed a significant difference in distribution of the genotypes and allele frequencies between patients and control.
Similarly, Col-Araz et al. , found a significant difference in the distribution of the genotypes and allele frequencies between patients with cardiomyopathy and controls, and the CC genotype was significantly higher in the patient group.
In patients with rheumatic fever, Col-Araz et al. , found that the CC genotype was significantly higher in the patient group.
In patients with inflammatory coronary heart disease, Ji et al. , found a significant difference in the distribution of different genotypes between patients and controls.
Our study found that there was a highly significant difference, with regard to echocardiography parameters (EF, fractional shortening, left ventricular end-diastolic diameter and left ventricular end-systolic diameter) between different genotypes among patients, suggesting that the MIF 173 C allele indicates poor prognosis and a more severe disease condition.
Unlike these results, Col-Araz et al. , showed that there was no relationship found between MIF genotypes and echocardiography parameters (cardiac functions). However, in patients with juvenile idiopathic arthritis, De Benedetti et al. , showed that the MIF − 173 C allele had a significantly greater number of joints with active arthritis and was associated with a poor response to glucocorticoids; moreover, Berdeli et al. , showed that the MIF − 173 C allele was a poor outcome predictor in juvenile rheumatoid arthritis.
With regard to serum Na level in this study, we found that there were significant differences between CC genotype and GC genotype, and serum Na levels were higher in patients with MIF CC genotype. Similarly Col-Araz et al. , found that serum Na levels were higher in patients with MIF CC genotype.
With regard to uric acid level, our study found no difference in uric acid level betweendifferent genotypes among patients. Gullu et al. , measured uric acid levels in idiopathic DCM, and they observed that serum uric acid levels are significantly higher in the lower coronary flow reverse group than in the higher coronary flow reverse group.
Col-Araz et al. , showed that serum uric acid levels were higher in patients with AA genotype of mannose-binding lectin gene in patients with DCM.
| Conclusion|| |
The CC genotype of the MIF gene may be a risk factor for cardiomyopathic patients, and C allele was associated with severe clinical condition.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jeffrey A, Towbin MD. Inherited cardiomyopathies. Circ J 2014; 78
Cecchi F, Tomberli B, Olivotto I. Clinical & molecular classification of cardiomyopathies. Global Cardiol Sci Pract 2012; 2012
Pahl E, Sleeper LA, Canter CE. Incidence of and risk factors for sudden cardiac death in children with dilated cardiomyopathy a report from the pediatric cardiomyopathy registry. J Am Coll Cardiol 2012; 59
Robert S, Stephanie W. Disease of the myocardium and pericardium. In: Kliegman RM, Bewhrman E, Jenson HB, Santon F, editors. 19th
ed. Philadilphia, PA: Saunders Elsevier; 2011.
Col-Araz N, Oguzkan-Balci S, Baspinar O, Sever T, Balat A, Pehlivan S. Mannose binding lectin and macrophage migration inhibitory factor gene polymorphisms in Turkish children with cardiomyopathy: no association with MBL2 codon 54 A/B genotype, but an association between MIF −173 CC genotype. Int J Med Sci 2012; 9
Bucala R, Donnelly SC. Macrophage migration inhibitory factor: a probable link between inflammation and cancer. Immunity 2007; 26
Bifulco C, McDaniel K, Leng L, Bucala R. Tumor growth-promoting properties of macrophage migration inhibitory factor. Curr Pharm Des 2008; 14
Elsby LM, Donn R, Alourfi Z, Green LM, Beaulieu E, Ray DW. Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expression. Arthritis Rheum 2009; 60
Renner P, Roger T, Calandra T. Macrophage migration inhibitory factor gene polymorphisms and suspectibility to inflammatory diseases. Clin Infect Dis 2005; 41
Babu SN, Chetal G, Kumar S. Macrophage migration inhibitory factor: a potential marker for cancer diagnosis and therapy. Asian Pac J Cancer Prev 2012; 13
Col-Araz N, Pehlivan S, Baspinar O, Sever T, Oguzkan-Balci S, Balat A. Association of macrophage migration inhibitory factor and mannose-binding lectin-2 gene polymorphisms in acute rheumatic fever. Cardiol Young 2013; 23
Ji K, Wang X, Li J, Lu Q, Wang G, Xue Y, et al
. Macrophage migration inhibitory factor polymorphism is associated with susceptibility toinflammatory coronary heart disease. Biomed Res Int 2015; 2015
De Benedetti F, Meazza C, Vivarelli M, Rossi F, Pistorio A, Lamb R, et al
. Functional and prognostic relevance of the − 173 polymorphism of the macrophage migration inhibitory factor gene in systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2003; 48
Berdeli A, Ozyürek AR, Ulger Z, Gürses D, Levent E, Salar K, et al
. Association of macrophage migration inhibitory factor gene −173 G/C polymorphism with prognosis in Turkish children with juvenile rheumatoid arthritis. Rheumatol Int 2006; 26
Gullu H, Erdogan D, Caliskan M, Tok D, Kulaksizoglu S, Yildirir A, et al
. Elevated serum uric acid levels impair coronary microvascular function in patients with idiopathic dilated cardiomyopathy. Eur J Heart Fail 2007; 9
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]