- Email: [email protected]
Role of polymorphism (rs1024611) in monocyte chemoattractant protein-1 gene in diabetic foot ulcer of Iraqi patients
Gene Reports 17 (2019) 100502
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Role of polymorphism (rs1024611) in monocyte chemoattractant protein-1 gene in diabetic foot ulcer of Iraqi patients
T
Mariam Riyadh Obieda, , Fadhil Jawad Al-Tu'maa, Hammed Hussein Al-jameelb ⁎
a b
Department of Biochemistry, College of Medicine, University of Kerbala, Iraq Department of Internal Medicine, College of Medicine, University of Kerbala, Iraq
ARTICLE INFO
ABSTRACT
Keywords: DFU T2DM A/G alleles MCP-1
Background: Diabetic foot ulcer (DFU) is one of the frequent diabetic problems, and the main reason for hospitalization and lower extremity amputation, among patients with type 2 diabetes mellitus (T2DM). The factors correlated with vascular functions and angiogenesis may be involved in the development and prevalence of DFU. Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory cytokine, playing a principal role in the inflammatory process. The influence of MCP-1 –2518A/G polymorphism and its association with the MCP-1 expression level had hardly been reported among patients with DFU. Objective: Our objective was to study the risk of genetic variation of MCP-1 -2518A/G in DFU and its association with the MCP-1 expression level and some biomarkers in serum of DFU patients. Subjects and methods: A cross-sectional study was performed on 235 subjects, including 135 individuals with DFU and another 100 with T2DM but without foot ulcer. Phenotypic data, including serum MCP-1 level, lipid profile, blood glucose, and HOMA-IR was investigated. DNA was extracted from the whole blood and the genotyping of the rs1024611 was determined, using tetra amplification refractory mutational system-polymerase chain reaction (tetra ARMS-PCR), followed by electrophoresis on 1.5% agarose gel. Results: The amplification of the MCP-1 gene gives three genotypes as indicated by (255 bp 379 bp) bands for those with homozygous wild type (AA), homozygous mutant (GG) genotypes and heterozygous (255 bp, and 379 bp) respectively, in addition to control with 594 bp band. Genotype frequencies of the rs1024611 polymorphism were consistent with Hardy–Weinberg equilibrium (HWE). Allele frequencies were 17%, 60% and 22% for AA, AG and GG, respectively, in DFU group. While the frequencies in the non-DFU group were 36%, 52% and 12% for AA, AG and GG, respectively. The homozygous genotype (GG) significantly increased the risk of DFU with respect to the AA wild type (OR = 3.35, CI 95% = 1.3–8.62, P = 0.01). The AG genotype significantly raised the risk of DFU (OR = 2.8, CI 95% = 1.42–5.7, P = 0.002). Co-dominant genotypes of the rs1024611 polymorphism exhibited significant association with high serum MCP-1 level, insulin resistance and lipid profile, in DFU group. Conclusion: Accordingly, we conclude that the rs1024611 polymorphism was significantly correlated with increased DFU susceptibility in the Iraqi Patients, and the frequency of AG and GG genotypes was higher in DFU, compared to T2DM without DFU.
1. Introduction Diabetes mellitus is a chronic, metabolic disease and known to
affect every vital organ in the human body (Madmoli et al., 2019). The global prevalence of this condition continues to rise and it is projected to affect 439 million people by the year 2030 (Jain et al., 2018). Type 2
Abbreviation: T-ARMS-PCR, Tetra amplification refractory mutational system-polymerase chain reaction; bp, Base pair; BMI, Body Mass Index; CCL2, C-C Motif Chemokine Ligand 2; DFU, Diabetes foot ulcer; DM, Diabetes mellitus; DNA, Deoxyribonucleic acid; EDTA, Ethylene diamine tetra acetic acid; ELISA, Enzyme-linked immunosorbent assay; HbA1c, Glycated hemoglobin A1c; HDL, High density lipoprotein; HOMA-IR, Homeostatic Model Assessment of Insulin Resistance; HWE, Hardy–Weinberg equilibrium; LDL-C, Low density lipoprotein cholesterol; MCP-1, Monocyte chemoattractant protein; SNPs, Single nucleotide polymorphisms; T2DM, Type 2 diabetes mellitus; TC, Total cholesterol; TG, Triglyceride; VLDL, Very low density lipoprotein; χ2, Chi-square test; FO, Forward outer primer; RO, Reverse outer primer; FI, Forward inner primer; RI, Reverse inner primer ⁎ Corresponding author. E-mail address: [email protected] (M.R. Obied). https://doi.org/10.1016/j.genrep.2019.100502 Received 22 August 2019; Accepted 5 September 2019 Available online 10 September 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 17 (2019) 100502
M.R. Obied, et al.
Table 1 Primer sequences for alleles of MCP-1 gene. Alleles of MCP-1 gene External primers Inner primers
Primer sequences Forward outer primer (FO) Reverse outer primer (RO) Forward inner primer (FI) (A allele) Reverse inner primer (RI) (G allele)
diabetes mellitus (T2DM) is one of the most common metabolic disorders and its occurrence shows an upward trend in the past years (Piwernetz et al., 1993; Al-Tu'ma et al., 2018; Khodaeian et al., 2015) Diabetic foot ulcer (DFU) is one of the typical diabetic problems, and primary reason for amputation and hospitalization among patients affected by T2DM (Uçkay et al., 2016). DFUs are the major reason for lower extremity amputation; 15% of diabetic foot patients develop foot problems during their life and a significant number of individuals with diabetic foot required a lower extremity amputation (Yasir and Mahdi, 2019). DFUs normally developed by nervous and vascular lesion, and infection in the lower limbs of patients affected by DM. DFU could affect the blood vessel, nerve, tendon, skin, and surprisingly bone. DM occurrence is increasing, and the morbidity of DFUs demonstrates an upward trend (Su et al., 2018). In terms of cost, the DFU represents 12–15% of the overall cost of health care and up to 40% in developing countries, including Iraq (Solan et al., 2017). Recent studies have shown multiple risk factors are correlated with the progress of DFU, such as lifespan of diabetes > 10 years, gender, old age of patients and high Body Mass Index (BMI), and other co-morbidities, including diabetic peripheral neuropathy, glycated hemoglobin level, retinopathy, foot deformity, peripheral vascular disease, inappropriate foot self-care habits, high plantar pressure, and infections (Wang et al., 2019; Pilla et al., 2019; Mishra et al., 2017). Monocyte chemoattractant protein-1 (MCP-1), also called chemokine (C-C motif) ligand 2 (CCL2), is a chemokine, which could activate lymphocytes, macrophages, and monocytes (Su et al., 2018; Kasiewicz and Whitehead, 2016; Li, 2018). The gene encoding the protein is situated on chromosome 17, q11.2; human MCP-1 is 13 kDa in size and made of 76 amino acids (He et al., 2017). Atypical expression of MCP-1 has been found in numerous diseases, including coronary artery disease, cerebral ischemic stroke and clear-cell renal cell carcinoma (Yang et al., 2016). Hyperglycemia can induce the expression of MCP-1 in vascular endothelial cells, and its atypical expression may cause problems related to vascular functions and angiogenesis in T2DM patients. Lately, developing studies have shown that the polymorphisms of MCP1 (–2518A/G) may affect the expression of MCP-1 (Afarideh et al., 2016). Van Asten et al. recommended that the MCP-1 expression level was elevated in patients with diabetic foot osteomyelitis (Van Asten et al., 2014). Serum MCP-1 level was significantly associated with DFUs (He et al., 2017). However, the effects of MCP-1 –2518A/G polymorphism and its relationship with MCP-1 level had been hardly described in DFU patient. The aim of the current study was to examine the association of the rs1024611 polymorphism with DFU susceptibility, and the serum protein level of MCP-1 and some biomarkers in the Iraqi patients.
5′-TAACTGAGGATTCTGGACAG-3′ 5′-TTATCTGATAAAGCCACAATC-3′ 5′-GTGGGAGGCAGACAGATA-3′ 5′-AGAAAGTCTTCTGGAAAGTTAC-3′
similar age range, and including 56% males and 44% females. Blood sample (5 ml) was collected from each DFU and non-DFU patient, 2 ml added to an EDTA tube for molecular studies, the remaining sample, 3 ml was used for serum separation at 3000 ×g and utilized for various biochemical investigations, including lipid profile, blood glucose and MCP-1 level. The MCP-1 protein level was measured by ELISA, the other biomarkers were assessed, using the Roche COBAS c311, and HOMA-IR by equation. The DNA was extracted from frozen whole blood of both DFU and non-DFU groups, using the Geneaid kit and stored at −20 °C. The amplification refractory mutation system polymerase chain reaction (ARMS-PCR) is a rapid and simple technique to detect a SNP (Hashemi et al., 2011; AL-Koofee and Mobarak, 2018). A tetra primer ARMS-PCR was used for the detection of –2581 A/G polymorphism of MCP-1 (rs1024611). Sequences of primers are presented in Table 1. The PCR products were analyzed by agarose gel electrophoresis, using 1.5% agarose gel, and visualized by staining with ethidium bromide (Promega, USA). PCR cycling conditions were as follows (Table 2). The data were expressed as mean ± SD, the student t-test was used for calculating the possibility. The PAST version 3.09 used for calculating the probability value (P value), chi- square (X2), odds ratio (OR) and confidence interval 95% (CI 95%), and to express a significance between the study groups (polymorphisms and biochemical parameters). In all statistical analysis, the P = 0.05 was considered significant and P˂0.01 as highly significant. 3. Results The patient characteristics such as age, gender, and biochemical parameters such as HDL and blood glucose had no significant difference among DFU and non-DFU groups (Table 3, P > 0.05). Excepting the MCP-1 level, other characteristics were significantly different among the study groups (Table 3, P < 0.05). Genotype frequencies of the MCP-1 gene were not consistent with the Hardy–Weinberg equilibrium (HWE) in DFU group. While, with the non-DFU group were consistent with HWE (Table 4). The genetic power found to be 56.9%. Comparing the DFU with the non-DFU group, the rs1024611 GG and AG genotypes were significantly related to the increased susceptibility to DFUs (Table 4, AG vs AA, P = 0.002, OR = 2.8, 95%CI = 1.42–5.7; GG vs AA, P = 0.01, OR = 3.35, 95%CI = 1.3–8.62). The association was examined between G allele and susceptibility to DFU (P = 0.06, OR = 1.6, 95% CI = 0.9–2.9). Table 2 PCR condition for the SNP analysis of rs1024611.
2. Materials and methods This study was a cross-sectional study. The total number of DFU patients was 135 with an age range of 35–74 years, of whom 61% were males and 38% females, recruited from the Al-Hassan Center for Endocrinology at the Al-Hussein Medical City/Kerbala Health Directorates/Kerbala, Iraq, from Nov 2018 to July 2019. The exclusion criterion was T2DM patients with no DFUs, but with other clinical complications. Subsequently, 100 patients with T2DM with no other problems were randomly chosen, as the non-DFU group, having the 2
Type of cycle
Temperature (°C)
Time
No. of cycles
Initial denaturation Denaturation Annealing Extension Final extension Hold Total time: 1 h and 20 min
95 95 60 72 72 4
5 min 30 s 30 s 60 s 5 min Infinite
1 cycle 35 cycles 1 cycle
Gene Reports 17 (2019) 100502
M.R. Obied, et al.
Table 3 Biochemical parameters of study subjects.
Table 5 Biochemical parameters compared in different MCP-1 genotypes.
Characteristics
DFU (N, %) Total N = 135
Non-DFU (N, %) Total N = 100
P-value
Gender
83(61%) 52(38%) 5(3%) 35 (25%) 56(41%) 39(28%) 236.2 ± 99.94 196.98 ± 37.93 205.14 ± 96.95 136.74 ± 32.53 39.39 ± 10.64 247.8 ± 88.67 13.69 ± 6.6
56(56%) 44(44%) 1(1%) 36(36%) 35(35%) 28(28%) 193.2 ± 85.28 162.4 ± 37.15 173.1 ± 55.6 106.8 ± 39.4 37.07 ± 7.5 239.9 ± 90.2 9.23 ± 3.9
0.38
Male Female Age (Years) 35–44 45–54 55–64 65–74 MCP-1 level (pg/mL) Total cholesterol (mg/dL) Triglycerides (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) Blood glucose (mg/dL) HOMA-IR ⁎
Parameters
MCP-1 (pg/mL)
0.17
Blood glucose (mg/ dL)
0.001 0.001⁎ 0.005⁎ 0.001⁎ 0.09 0.53 0.001⁎ ⁎
Triglycerides (mg/ dL) Total-cholesterol (mg/dL) HDL-C (mg/dL)
Significant P-value˂0.05.
LDL-C (mg/dL)
Table 4 MCP-1 genotypes correlated with DFU and non-DFU groups. Genotype
DFU, (N, %) Total N = 135
Non-DFU, (N, %) Total N = 100
χ2
OR
95% CI
P-value
AA AG GG X2 P-HWE A G
24(17%) 81(60%) 30(22%) 5.53 0.01 0.49 0.51
36 (36%) 52 (52%) 12 (12%) 1.07 0.3 0.62 0.38
10.3 4.05 1.41 – – – –
– 2.8 3.35 – – – 1.6
– 1.42–5.7 1.3–8.62 – – – 0.9–2.9
– 0.002⁎ 0.01⁎ – – – 0.06
⁎
HOMA-IR
⁎
MCP-1 gene, Total N = 235, (Mean ± SD)
AA AG GG AA AG GG AA AG GG AA AG GG AA AG GG AA AG GG AA AG GG
DFU
Non-DFU
267.8 ± 106.7 221.41 ± 94.5 262.3 ± 101.12 219.1 ± 115.8 247.2 ± 79.24 275.6 ± 84.82 154.06 ± 77.76 216.6 ± 106.9 208.6 ± 41.29 155.2 ± 38.74 209.1 ± 20.87 189.7 ± 54.89 35.61 ± 6.99 38.48 ± 10.4 46.11 ± 11.36 99.13 ± 33.69 147.8 ± 18.22 129.7 ± 44.19 10 ± 5.09 14.71 ± 6.26 13.22 ± 7.78
192.3 ± 76.05 202.6 ± 94.13 155.5 ± 51.67 228 ± 84.78 258.4 ± 94.82 195.9 ± 54.83 213.1 ± 51.36 140.2 ± 39.02 208.7 ± 26.37 152.9 ± 31.84 165.4 ± 41.59 174.8 ± 18.1 86.09 ± 35.57 124.8 ± 36.9 90.5 ± 11.87 37.36 ± 9.02 38.55 ± 5.89 29.8 ± 2.82 9.38 ± 4.49 8.96 ± 3.14 10.01 ± 4.94
P-value
0.02⁎ 0.28 ˂0.001⁎ 0.79 0.49 0.004⁎ 0.018⁎ ˂0.001⁎ 0.99 0.7 ˂0.001⁎ 0.29 0.46 0.96 ˂0.001⁎ 0.22 ˂0.001⁎ 0.001⁎ 0.53 ˂0.001⁎ 0.17
Significant P-value˂0.05.
population. As a result, the individuals harboring GG and AG genotypes had a higher chance of having DFU. Moreover, DFU and non-DFU groups had high levels of MCP-1 protein. Our data demonstrated the important role of MCP-1 polymorphism in the etiology of DFU. Whereas, Li (2018) has reported that patients carrying GG genotype demonstrated a higher MCP-1 expression level than those with the AA genotype. No significant difference was found between AA and AG genotype. The population of diabetic women in many studies is greater than men, suggesting that this may be attributed to their gender characteristics, but men develop more diabetes complications than women (Madmoli et al., 2019). In the study of Li, 71 men and 50 women were studied (Li, 2018), while in the study of Frykberg et al., 90.3% of the population were male (Frykberg et al., 2017), and in the study of Tan and colleagues, the number of men and women was equal (Tan et al., 2015) The relationship between TG, cholesterol and LDL and DFU group was significant (P-value < 0.05). However, there was no significant difference in HDL in DFU group (P-value > 0.05), which is in the line with the results of studies (Madmoli et al., 2019; Parial et al., 2013; Hussian et al., 2013).
Significant P-value˂0.05.
The amplification of MCP-1–2581A/G allele resulted in 379 bp for G allele, 255 bp for A allele, and 594 bp for two outer primers (control band) (Fig. 1). Data analysis demonstrated that patients, harboring GG genotype showed a higher significant MCP-1 expression level, blood glucose, LDL-C and HDL-C, compared to those with the AA genotype (P˂0.05). No significant difference of parameters was found in AG genotype (p˃0.05). Expect TG, HOMA-IR and total cholesterol, there was a significant difference in other biological parameters in AG genotype among patients (Table 5, P = 0.05). 4. Discussion In the current study, the genetic association of the MCP-1 rs1024611 polymorphism with DFU susceptibility was investigated in the Iraqi
Fig. 1. Electrophoresis pattern of T-ARMS-PCR for detection of the SNP (–2581 A/G) in MCP-1. M = DNA marker (100 bp), line 1–4 patient 1/heterozygote and line 5–8 patient 2/homozygote genotype. Line 1 (G allele 379 bp) (FO + RI), line 2 and 5 (A allele 255 bp) (FI + RO) line 3 and 7 (control bands 594 bp)(FO + RO), line 4 and 8 (Tetra ARMS) four primers (FO + RO + FO + RI).
3
Gene Reports 17 (2019) 100502
M.R. Obied, et al.
References
It was a significant association between the blood glucose and DFU susceptibility in study groups, meaning that people who had higher blood sugar, had more severe leg ulcers (Raisifar et al., 2018; Dekker et al., 2016). MCP-1 gene is situated on chromosome 17q12, harboring three exons. The evidences have shown that the polymorphisms in MCP-1 gene may affect the genetic predisposition to the problems associated with DM (Wang et al., 2019). The rs1024611 is a commonly investigated polymorphism in the promoter region of MCP-1 gene, which has been involved in numerous inflammatory and immune diseases (Lee and Bae, 2016). He et al. found that the G allele of the rs1024611 SNP was associated with the high MCP-1 expression (He et al., 2017). Therefore, we speculated that the rs1024611 SNP might be contributed to the progression of DFUs. The homozygous wild type genotype AA was found to increase the risk of DFU two folds, with respect to those of the heterozygous genotype AG. On the other hand, the homozygous variation in GG genotype statistically and significantly increased the risk of DFU by 3.35 folds, compared to the wild type AA genotype. Our data show that a high significant association was found between the rs1024611 G allele and high susceptibility to DFUs, as about 1.6 times in total patients. The rs1024611 SNP was positively related to the increased susceptibility of diabetic retinopathy in patients with T2DM, based on a recent meta-analysis study (Wang et al., 2019). Furthermore, in patients with T2DM, the rs1024611 SNP was markedly related to the end stage renal disease (Raina et al., 2015). Increased levels of MCP-1 have been observed in the patients with DM, with or without DFU. Furthermore, higher levels of MCP-1 have been seen in patients with DFU, compared to the T2DM cases without DFU, which was consistent with the previous report (Afarideh et al., 2016). The comparison of the MCP-1 expression in the study population, according to the genotypes of the rs1024611 polymorphism shows that in both non-DFU and DFU groups, the subjects harboring GG genotype demonstrated apparently an elevated MCP-1 expression. This was consistent with the study of He et al. (2017). The study of Kasiewicz and Whitehead (2016), had shown that down-regulation of MCP-1 could stop the signal chronic inflammation within diabetic wound healing in an in vitro co-culture model of DFU (Li, 2018).
Afarideh, M., Ghanbari, P., Ghajar, A., 2016. Raised serum 25-hydroxyvitamin D levels in patients with active diabetic foot ulcers. Br. J. Nutr. 115 (11), 1938–1946. AL-Koofee, D.A., Mobarak, S.M., 2018. PRIMER1: A network service for tetra-arms PCR primer design based on well-known dbSNP. Res. J. Pharm. Technol. 11 (8), 3633–3637. Al-Tu'ma, F.J., Joda, B.J., Al-Yassiry, R.A., 2018. Assessment of vascular endothelial growth factor-a and insulin resistance in sera of ischemic heart diseases with type-II diabetic patients. Indian J. Nat. Sci. 9 (11), 16048–16055. Dekker, R.G., Qin, C., Ho, B.S., 2016. The effect of cumulative glycemic burden on the incidence ofdiabetic foot disease. J. Orthop. Surg. Res. 11 (1), 143. Frykberg, R.G., Gibbons, G.W., Walters, J.L., 2017. A prospective, multicentre, openlabel, single-arm clinical trial for treatment of chronic complex diabetic foot wounds with exposed tendon and/or bone: positive clinical outcomes of viable cryopreserved human placental membrane. Int. Wound J. 14 (3), 569–577. Hashemi, M., Hoseini, H., Yaghmaei, P., 2011. Association of polymorphisms in glutamate-cysteine ligase catalytic subunit and microsomal triglyceride. DNA Cell Biol. 30 (1), 569–575. He, J., Chen, Y., Zhang, W., 2017. Association study of MCP-1 promoter polymorphisms with the susceptibility and progression of sepsis. PLoS One 12 (5), e0176781. Hussian, S.K., Al-Karawi, I.N., Sahab, K.S., 2013. Role of Fas/Fas ligand pathway in a sample of Iraqi diabetic foot patients. Diyala J. Med. 5 (1), 95–107. Jain, A.K., Apoorva, H.C., Kumar, H., 2018. Analyzing diabetic foot ulcer through Amit Jain's classification: a descriptive study analyzing diabetic foot ulcer through Amit Jain's classification: a descriptive study. Int. J. Surg. Sci. 2 (4), 25_32. Kasiewicz, L.N., Whitehead, K.A., 2016. Silencing TNFα with lipidoid nanoparticles downregulates both TNFα and MCP-1 in an in vitro co-culture model of diabetic foot ulcers. Acta Biomater. 32 (1), 120–128. Khodaeian, M., Enayati, S., Amoli, M.M., 2015. Association between genetic variants and diabetes mellitus in Iranian populations: a systematic review of observational studies. J. Diabetes Res. 2015 (5), 21. Lee, Y.H., Bae, S.C., 2016. Monocyte chemoattractant protein-1 promoter -2518 polymorphism and susceptibility to vasculitis, rheumatoid arthritis, and multiple sclerosis A meta-analysis. Cell. Mol. Biol. 62 (5), 65–71. Li, X., 2018. The association between MCP-1, VEGF polymorphisms and their serum levels in patients with diabetic foot ulcer. Medicine 97 (24), e10959. Madmoli, M., Madmoli, Y., Taqvaeinasab, H., 2019. Some influential factors on severity of diabetic foot ulcers and predisposing of limb amputation: a 7-year study on diabetic patients. Int. J. Ayurvedic Med. 10 (1), 75–81. Mishra, S.C., Chhatbar, K.C., Mehndiratta, A., 2017. Diabetic foot. Bmj 357 (1), 1–7. Parial, C.R., Islam, M., Kasru, A., 2013. Association of HbA1c, creatinine and lipid profile in patients with diabetic foot ulcer. Middle-East J. Sci. Res. 16 (11), 1508–1511. Pilla, E.D., Desai, R., Kokiwar, P.R., 2019. A multi centric study of diabetic foot ulcer: causes and complications. Int. Surg. J. 6 (4), 1327–1331. Piwernetz, K., Home, P.D., Snorgaard, O., Antsiferov, M., Staehr-Johansen, K., Krans, M., May 1993. Monitoring the targets of the St Vincent declaration and the implementation of quality management in diabetes care: the DIABCARE initiative. The DIABCARE monitoring Group of the St Vincent Declaration Steering Committee. Diabet. Med. 10 (4), 371–377. Raina, p., Matharoo, K., Bhanw, A.J.S., 2015. Monocyte chemoattractant protein-1 (MCP1) g.-2518A > G polymorphism and susceptibility to type 2 diabetes (T2D) and end stage renal disease (ESRD) in the North-West Indian population of Punjab. Ann. Hum. Biol. 42 (3), 278–284. Raisifar, Z., Nia, A.A., Madmoli, M., 2018. The relationship between using insulin and suffering Alzheimer's disease in patients with diabetes: a two-year study. Int. J. Ecosyst. Ecol. Sci. 8 (3), 623–628. Solan, Y.M., Kheir, H.M., Mahfouz, M.S., 2017. Diabetic foot care: knowledge and practice. Endocrinol. Metab. 6 (6), 172–177. Su, N., Zhao, N., Wang, G., 2018. Association of MCP-1 rs1024611 polymorphism with diabetic foot ulcers. Medicine 97 (28), e11232. Tan, J.H., Hong, C.C., Shen, L., 2015. Costs of patients admitted for diabetic foot problems. Ann. Acad. Med. Singap. 44 (12), 567–570. Uçkay, I., Gariani, K., Dubois-Ferrière, V., 2016. Diabetic foot infections: recent literature and cornerstones of managemen. Curr. Opin. Infect. Dis. 29 (2), 145–152. Van Asten, S.A., Nichols, A., Fontaine, J.L., 2014. The value of inflammatory markers to diagnose and monitor diabetic foot osteomyelitis. Int. Wound J. 14 (1), 40–45. Wang, Y., Cao, H.J., Wang, L.Q., 2019. The effects of Chinese herbal medicines for treating diabetic foot ulcers: a systematic review of 49 randomized controlled trials. Complement. Ther. Med. 44 (1), 32–43. Yang, Y., Zhai, C., Zhou, L., 2016. High expression of chemokine CCL2 is associated with recurrence after surgery in clear-cell renal cell carcinoma. Urol. Oncol. 34 (5), 219–238. Yasir, I.O., Mahdi, M.M., 2019. Risk factors for major amputation in diabetic foot patients. Thi-Qar Med. J. 17 (1), 40–53.
5. Conclusion The rs1024611 polymorphism of MCP-1 gene was slightly associated with elevated susceptibility to DFU in the Iraqi subjects with DM. Subjects carrying the GG genotype have more than three folds' chance of developing DFU, while the risk in heterozygous AG carriers was only two and half folds. Declaration of competing interest The Authors declare no conflict of interest work. Acknowledgments I would like to thank Dr. Fadhil Jawad Al-Tu'ma and Haider Ali Mohammed/Department of Microbiology, University of Kerbala for his assistance and insight. In addition, I would like to thank all the patients who willingly participated in the study.
4
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Role of polymorphism (rs1024611) in monocyte chemoattractant protein-1 gene in diabetic foot ulcer of Iraqi patients
T
Mariam Riyadh Obieda, , Fadhil Jawad Al-Tu'maa, Hammed Hussein Al-jameelb ⁎
a b
Department of Biochemistry, College of Medicine, University of Kerbala, Iraq Department of Internal Medicine, College of Medicine, University of Kerbala, Iraq
ARTICLE INFO
ABSTRACT
Keywords: DFU T2DM A/G alleles MCP-1
Background: Diabetic foot ulcer (DFU) is one of the frequent diabetic problems, and the main reason for hospitalization and lower extremity amputation, among patients with type 2 diabetes mellitus (T2DM). The factors correlated with vascular functions and angiogenesis may be involved in the development and prevalence of DFU. Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory cytokine, playing a principal role in the inflammatory process. The influence of MCP-1 –2518A/G polymorphism and its association with the MCP-1 expression level had hardly been reported among patients with DFU. Objective: Our objective was to study the risk of genetic variation of MCP-1 -2518A/G in DFU and its association with the MCP-1 expression level and some biomarkers in serum of DFU patients. Subjects and methods: A cross-sectional study was performed on 235 subjects, including 135 individuals with DFU and another 100 with T2DM but without foot ulcer. Phenotypic data, including serum MCP-1 level, lipid profile, blood glucose, and HOMA-IR was investigated. DNA was extracted from the whole blood and the genotyping of the rs1024611 was determined, using tetra amplification refractory mutational system-polymerase chain reaction (tetra ARMS-PCR), followed by electrophoresis on 1.5% agarose gel. Results: The amplification of the MCP-1 gene gives three genotypes as indicated by (255 bp 379 bp) bands for those with homozygous wild type (AA), homozygous mutant (GG) genotypes and heterozygous (255 bp, and 379 bp) respectively, in addition to control with 594 bp band. Genotype frequencies of the rs1024611 polymorphism were consistent with Hardy–Weinberg equilibrium (HWE). Allele frequencies were 17%, 60% and 22% for AA, AG and GG, respectively, in DFU group. While the frequencies in the non-DFU group were 36%, 52% and 12% for AA, AG and GG, respectively. The homozygous genotype (GG) significantly increased the risk of DFU with respect to the AA wild type (OR = 3.35, CI 95% = 1.3–8.62, P = 0.01). The AG genotype significantly raised the risk of DFU (OR = 2.8, CI 95% = 1.42–5.7, P = 0.002). Co-dominant genotypes of the rs1024611 polymorphism exhibited significant association with high serum MCP-1 level, insulin resistance and lipid profile, in DFU group. Conclusion: Accordingly, we conclude that the rs1024611 polymorphism was significantly correlated with increased DFU susceptibility in the Iraqi Patients, and the frequency of AG and GG genotypes was higher in DFU, compared to T2DM without DFU.
1. Introduction Diabetes mellitus is a chronic, metabolic disease and known to
affect every vital organ in the human body (Madmoli et al., 2019). The global prevalence of this condition continues to rise and it is projected to affect 439 million people by the year 2030 (Jain et al., 2018). Type 2
Abbreviation: T-ARMS-PCR, Tetra amplification refractory mutational system-polymerase chain reaction; bp, Base pair; BMI, Body Mass Index; CCL2, C-C Motif Chemokine Ligand 2; DFU, Diabetes foot ulcer; DM, Diabetes mellitus; DNA, Deoxyribonucleic acid; EDTA, Ethylene diamine tetra acetic acid; ELISA, Enzyme-linked immunosorbent assay; HbA1c, Glycated hemoglobin A1c; HDL, High density lipoprotein; HOMA-IR, Homeostatic Model Assessment of Insulin Resistance; HWE, Hardy–Weinberg equilibrium; LDL-C, Low density lipoprotein cholesterol; MCP-1, Monocyte chemoattractant protein; SNPs, Single nucleotide polymorphisms; T2DM, Type 2 diabetes mellitus; TC, Total cholesterol; TG, Triglyceride; VLDL, Very low density lipoprotein; χ2, Chi-square test; FO, Forward outer primer; RO, Reverse outer primer; FI, Forward inner primer; RI, Reverse inner primer ⁎ Corresponding author. E-mail address: [email protected] (M.R. Obied). https://doi.org/10.1016/j.genrep.2019.100502 Received 22 August 2019; Accepted 5 September 2019 Available online 10 September 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
Gene Reports 17 (2019) 100502
M.R. Obied, et al.
Table 1 Primer sequences for alleles of MCP-1 gene. Alleles of MCP-1 gene External primers Inner primers
Primer sequences Forward outer primer (FO) Reverse outer primer (RO) Forward inner primer (FI) (A allele) Reverse inner primer (RI) (G allele)
diabetes mellitus (T2DM) is one of the most common metabolic disorders and its occurrence shows an upward trend in the past years (Piwernetz et al., 1993; Al-Tu'ma et al., 2018; Khodaeian et al., 2015) Diabetic foot ulcer (DFU) is one of the typical diabetic problems, and primary reason for amputation and hospitalization among patients affected by T2DM (Uçkay et al., 2016). DFUs are the major reason for lower extremity amputation; 15% of diabetic foot patients develop foot problems during their life and a significant number of individuals with diabetic foot required a lower extremity amputation (Yasir and Mahdi, 2019). DFUs normally developed by nervous and vascular lesion, and infection in the lower limbs of patients affected by DM. DFU could affect the blood vessel, nerve, tendon, skin, and surprisingly bone. DM occurrence is increasing, and the morbidity of DFUs demonstrates an upward trend (Su et al., 2018). In terms of cost, the DFU represents 12–15% of the overall cost of health care and up to 40% in developing countries, including Iraq (Solan et al., 2017). Recent studies have shown multiple risk factors are correlated with the progress of DFU, such as lifespan of diabetes > 10 years, gender, old age of patients and high Body Mass Index (BMI), and other co-morbidities, including diabetic peripheral neuropathy, glycated hemoglobin level, retinopathy, foot deformity, peripheral vascular disease, inappropriate foot self-care habits, high plantar pressure, and infections (Wang et al., 2019; Pilla et al., 2019; Mishra et al., 2017). Monocyte chemoattractant protein-1 (MCP-1), also called chemokine (C-C motif) ligand 2 (CCL2), is a chemokine, which could activate lymphocytes, macrophages, and monocytes (Su et al., 2018; Kasiewicz and Whitehead, 2016; Li, 2018). The gene encoding the protein is situated on chromosome 17, q11.2; human MCP-1 is 13 kDa in size and made of 76 amino acids (He et al., 2017). Atypical expression of MCP-1 has been found in numerous diseases, including coronary artery disease, cerebral ischemic stroke and clear-cell renal cell carcinoma (Yang et al., 2016). Hyperglycemia can induce the expression of MCP-1 in vascular endothelial cells, and its atypical expression may cause problems related to vascular functions and angiogenesis in T2DM patients. Lately, developing studies have shown that the polymorphisms of MCP1 (–2518A/G) may affect the expression of MCP-1 (Afarideh et al., 2016). Van Asten et al. recommended that the MCP-1 expression level was elevated in patients with diabetic foot osteomyelitis (Van Asten et al., 2014). Serum MCP-1 level was significantly associated with DFUs (He et al., 2017). However, the effects of MCP-1 –2518A/G polymorphism and its relationship with MCP-1 level had been hardly described in DFU patient. The aim of the current study was to examine the association of the rs1024611 polymorphism with DFU susceptibility, and the serum protein level of MCP-1 and some biomarkers in the Iraqi patients.
5′-TAACTGAGGATTCTGGACAG-3′ 5′-TTATCTGATAAAGCCACAATC-3′ 5′-GTGGGAGGCAGACAGATA-3′ 5′-AGAAAGTCTTCTGGAAAGTTAC-3′
similar age range, and including 56% males and 44% females. Blood sample (5 ml) was collected from each DFU and non-DFU patient, 2 ml added to an EDTA tube for molecular studies, the remaining sample, 3 ml was used for serum separation at 3000 ×g and utilized for various biochemical investigations, including lipid profile, blood glucose and MCP-1 level. The MCP-1 protein level was measured by ELISA, the other biomarkers were assessed, using the Roche COBAS c311, and HOMA-IR by equation. The DNA was extracted from frozen whole blood of both DFU and non-DFU groups, using the Geneaid kit and stored at −20 °C. The amplification refractory mutation system polymerase chain reaction (ARMS-PCR) is a rapid and simple technique to detect a SNP (Hashemi et al., 2011; AL-Koofee and Mobarak, 2018). A tetra primer ARMS-PCR was used for the detection of –2581 A/G polymorphism of MCP-1 (rs1024611). Sequences of primers are presented in Table 1. The PCR products were analyzed by agarose gel electrophoresis, using 1.5% agarose gel, and visualized by staining with ethidium bromide (Promega, USA). PCR cycling conditions were as follows (Table 2). The data were expressed as mean ± SD, the student t-test was used for calculating the possibility. The PAST version 3.09 used for calculating the probability value (P value), chi- square (X2), odds ratio (OR) and confidence interval 95% (CI 95%), and to express a significance between the study groups (polymorphisms and biochemical parameters). In all statistical analysis, the P = 0.05 was considered significant and P˂0.01 as highly significant. 3. Results The patient characteristics such as age, gender, and biochemical parameters such as HDL and blood glucose had no significant difference among DFU and non-DFU groups (Table 3, P > 0.05). Excepting the MCP-1 level, other characteristics were significantly different among the study groups (Table 3, P < 0.05). Genotype frequencies of the MCP-1 gene were not consistent with the Hardy–Weinberg equilibrium (HWE) in DFU group. While, with the non-DFU group were consistent with HWE (Table 4). The genetic power found to be 56.9%. Comparing the DFU with the non-DFU group, the rs1024611 GG and AG genotypes were significantly related to the increased susceptibility to DFUs (Table 4, AG vs AA, P = 0.002, OR = 2.8, 95%CI = 1.42–5.7; GG vs AA, P = 0.01, OR = 3.35, 95%CI = 1.3–8.62). The association was examined between G allele and susceptibility to DFU (P = 0.06, OR = 1.6, 95% CI = 0.9–2.9). Table 2 PCR condition for the SNP analysis of rs1024611.
2. Materials and methods This study was a cross-sectional study. The total number of DFU patients was 135 with an age range of 35–74 years, of whom 61% were males and 38% females, recruited from the Al-Hassan Center for Endocrinology at the Al-Hussein Medical City/Kerbala Health Directorates/Kerbala, Iraq, from Nov 2018 to July 2019. The exclusion criterion was T2DM patients with no DFUs, but with other clinical complications. Subsequently, 100 patients with T2DM with no other problems were randomly chosen, as the non-DFU group, having the 2
Type of cycle
Temperature (°C)
Time
No. of cycles
Initial denaturation Denaturation Annealing Extension Final extension Hold Total time: 1 h and 20 min
95 95 60 72 72 4
5 min 30 s 30 s 60 s 5 min Infinite
1 cycle 35 cycles 1 cycle
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Table 3 Biochemical parameters of study subjects.
Table 5 Biochemical parameters compared in different MCP-1 genotypes.
Characteristics
DFU (N, %) Total N = 135
Non-DFU (N, %) Total N = 100
P-value
Gender
83(61%) 52(38%) 5(3%) 35 (25%) 56(41%) 39(28%) 236.2 ± 99.94 196.98 ± 37.93 205.14 ± 96.95 136.74 ± 32.53 39.39 ± 10.64 247.8 ± 88.67 13.69 ± 6.6
56(56%) 44(44%) 1(1%) 36(36%) 35(35%) 28(28%) 193.2 ± 85.28 162.4 ± 37.15 173.1 ± 55.6 106.8 ± 39.4 37.07 ± 7.5 239.9 ± 90.2 9.23 ± 3.9
0.38
Male Female Age (Years) 35–44 45–54 55–64 65–74 MCP-1 level (pg/mL) Total cholesterol (mg/dL) Triglycerides (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) Blood glucose (mg/dL) HOMA-IR ⁎
Parameters
MCP-1 (pg/mL)
0.17
Blood glucose (mg/ dL)
0.001 0.001⁎ 0.005⁎ 0.001⁎ 0.09 0.53 0.001⁎ ⁎
Triglycerides (mg/ dL) Total-cholesterol (mg/dL) HDL-C (mg/dL)
Significant P-value˂0.05.
LDL-C (mg/dL)
Table 4 MCP-1 genotypes correlated with DFU and non-DFU groups. Genotype
DFU, (N, %) Total N = 135
Non-DFU, (N, %) Total N = 100
χ2
OR
95% CI
P-value
AA AG GG X2 P-HWE A G
24(17%) 81(60%) 30(22%) 5.53 0.01 0.49 0.51
36 (36%) 52 (52%) 12 (12%) 1.07 0.3 0.62 0.38
10.3 4.05 1.41 – – – –
– 2.8 3.35 – – – 1.6
– 1.42–5.7 1.3–8.62 – – – 0.9–2.9
– 0.002⁎ 0.01⁎ – – – 0.06
⁎
HOMA-IR
⁎
MCP-1 gene, Total N = 235, (Mean ± SD)
AA AG GG AA AG GG AA AG GG AA AG GG AA AG GG AA AG GG AA AG GG
DFU
Non-DFU
267.8 ± 106.7 221.41 ± 94.5 262.3 ± 101.12 219.1 ± 115.8 247.2 ± 79.24 275.6 ± 84.82 154.06 ± 77.76 216.6 ± 106.9 208.6 ± 41.29 155.2 ± 38.74 209.1 ± 20.87 189.7 ± 54.89 35.61 ± 6.99 38.48 ± 10.4 46.11 ± 11.36 99.13 ± 33.69 147.8 ± 18.22 129.7 ± 44.19 10 ± 5.09 14.71 ± 6.26 13.22 ± 7.78
192.3 ± 76.05 202.6 ± 94.13 155.5 ± 51.67 228 ± 84.78 258.4 ± 94.82 195.9 ± 54.83 213.1 ± 51.36 140.2 ± 39.02 208.7 ± 26.37 152.9 ± 31.84 165.4 ± 41.59 174.8 ± 18.1 86.09 ± 35.57 124.8 ± 36.9 90.5 ± 11.87 37.36 ± 9.02 38.55 ± 5.89 29.8 ± 2.82 9.38 ± 4.49 8.96 ± 3.14 10.01 ± 4.94
P-value
0.02⁎ 0.28 ˂0.001⁎ 0.79 0.49 0.004⁎ 0.018⁎ ˂0.001⁎ 0.99 0.7 ˂0.001⁎ 0.29 0.46 0.96 ˂0.001⁎ 0.22 ˂0.001⁎ 0.001⁎ 0.53 ˂0.001⁎ 0.17
Significant P-value˂0.05.
population. As a result, the individuals harboring GG and AG genotypes had a higher chance of having DFU. Moreover, DFU and non-DFU groups had high levels of MCP-1 protein. Our data demonstrated the important role of MCP-1 polymorphism in the etiology of DFU. Whereas, Li (2018) has reported that patients carrying GG genotype demonstrated a higher MCP-1 expression level than those with the AA genotype. No significant difference was found between AA and AG genotype. The population of diabetic women in many studies is greater than men, suggesting that this may be attributed to their gender characteristics, but men develop more diabetes complications than women (Madmoli et al., 2019). In the study of Li, 71 men and 50 women were studied (Li, 2018), while in the study of Frykberg et al., 90.3% of the population were male (Frykberg et al., 2017), and in the study of Tan and colleagues, the number of men and women was equal (Tan et al., 2015) The relationship between TG, cholesterol and LDL and DFU group was significant (P-value < 0.05). However, there was no significant difference in HDL in DFU group (P-value > 0.05), which is in the line with the results of studies (Madmoli et al., 2019; Parial et al., 2013; Hussian et al., 2013).
Significant P-value˂0.05.
The amplification of MCP-1–2581A/G allele resulted in 379 bp for G allele, 255 bp for A allele, and 594 bp for two outer primers (control band) (Fig. 1). Data analysis demonstrated that patients, harboring GG genotype showed a higher significant MCP-1 expression level, blood glucose, LDL-C and HDL-C, compared to those with the AA genotype (P˂0.05). No significant difference of parameters was found in AG genotype (p˃0.05). Expect TG, HOMA-IR and total cholesterol, there was a significant difference in other biological parameters in AG genotype among patients (Table 5, P = 0.05). 4. Discussion In the current study, the genetic association of the MCP-1 rs1024611 polymorphism with DFU susceptibility was investigated in the Iraqi
Fig. 1. Electrophoresis pattern of T-ARMS-PCR for detection of the SNP (–2581 A/G) in MCP-1. M = DNA marker (100 bp), line 1–4 patient 1/heterozygote and line 5–8 patient 2/homozygote genotype. Line 1 (G allele 379 bp) (FO + RI), line 2 and 5 (A allele 255 bp) (FI + RO) line 3 and 7 (control bands 594 bp)(FO + RO), line 4 and 8 (Tetra ARMS) four primers (FO + RO + FO + RI).
3
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References
It was a significant association between the blood glucose and DFU susceptibility in study groups, meaning that people who had higher blood sugar, had more severe leg ulcers (Raisifar et al., 2018; Dekker et al., 2016). MCP-1 gene is situated on chromosome 17q12, harboring three exons. The evidences have shown that the polymorphisms in MCP-1 gene may affect the genetic predisposition to the problems associated with DM (Wang et al., 2019). The rs1024611 is a commonly investigated polymorphism in the promoter region of MCP-1 gene, which has been involved in numerous inflammatory and immune diseases (Lee and Bae, 2016). He et al. found that the G allele of the rs1024611 SNP was associated with the high MCP-1 expression (He et al., 2017). Therefore, we speculated that the rs1024611 SNP might be contributed to the progression of DFUs. The homozygous wild type genotype AA was found to increase the risk of DFU two folds, with respect to those of the heterozygous genotype AG. On the other hand, the homozygous variation in GG genotype statistically and significantly increased the risk of DFU by 3.35 folds, compared to the wild type AA genotype. Our data show that a high significant association was found between the rs1024611 G allele and high susceptibility to DFUs, as about 1.6 times in total patients. The rs1024611 SNP was positively related to the increased susceptibility of diabetic retinopathy in patients with T2DM, based on a recent meta-analysis study (Wang et al., 2019). Furthermore, in patients with T2DM, the rs1024611 SNP was markedly related to the end stage renal disease (Raina et al., 2015). Increased levels of MCP-1 have been observed in the patients with DM, with or without DFU. Furthermore, higher levels of MCP-1 have been seen in patients with DFU, compared to the T2DM cases without DFU, which was consistent with the previous report (Afarideh et al., 2016). The comparison of the MCP-1 expression in the study population, according to the genotypes of the rs1024611 polymorphism shows that in both non-DFU and DFU groups, the subjects harboring GG genotype demonstrated apparently an elevated MCP-1 expression. This was consistent with the study of He et al. (2017). The study of Kasiewicz and Whitehead (2016), had shown that down-regulation of MCP-1 could stop the signal chronic inflammation within diabetic wound healing in an in vitro co-culture model of DFU (Li, 2018).
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5. Conclusion The rs1024611 polymorphism of MCP-1 gene was slightly associated with elevated susceptibility to DFU in the Iraqi subjects with DM. Subjects carrying the GG genotype have more than three folds' chance of developing DFU, while the risk in heterozygous AG carriers was only two and half folds. Declaration of competing interest The Authors declare no conflict of interest work. Acknowledgments I would like to thank Dr. Fadhil Jawad Al-Tu'ma and Haider Ali Mohammed/Department of Microbiology, University of Kerbala for his assistance and insight. In addition, I would like to thank all the patients who willingly participated in the study.
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