Journal of The Egyptian Society of Nephrology and Transplantation

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 20  |  Issue : 2  |  Page : 98--102

Role of engulfment and cell motility 1 gene polymorphism in type 2 diabetic nephropathy and its association with renal biomarkers: case–control stody


Hawraa J Mohammed1, Riyadh M Al-Saegh2, Narjis H Al-Saadi3,  
1 Department of Biochemistry, College of Medicine, University of Kerbala, Kerbala Hole, Iraq
2 Department of Medicine, College of Medicine, University of Kerbala, Kerbala Hole, Iraq
3 Department of Biochemistry, College of Sciences, University of Kerbala, Kerbala Hole, Iraq

Correspondence Address:
Dr. Riyadh M Al-Saegh
Department of Biochemistry, College of Medicine, Al-Kafeel Center for Organ Transplantation, University of Kerbala, Al-Husain Medical City, Kerbala Hole, Iraq. +964 P.O.box 1125
Iraq

Abstract

Background Diabetic nephropathy (DN) is a serious kidney-related complication of diabetes. It is also called diabetic kidney disease. Up to 40% of people with diabetes eventually develop kidney disease. Several genome-wide association studies have introduced engulfment and cell motility 1 (ELMO1) as a candidate gene that is associated with DN. This study assessed the association of ELMO1 gene polymorphisms with DN to investigate the effects of ELMO1 gene on susceptibility to DN in Kerbala/Iraqi province. Aim The current study aims to identify the role of ELMO1 gene polymorphism, single nucleotide polymorphism (SNP) rs741301, as a candidate gene for susceptibility to DN among patients with type 2 diabetes mellitus and its association with the development and progression of this disease and to verify the relationship between the investigated SNPs of ELMO1 gene with the phenotype changes in particular kidney function tests and other kidney biomarkers that may be seen in patient. Patients and methods A case–control study was conducted for a period of 14 months, starting from January 2018 to March 2019, in Al-Husain Medical City and Al-Kafeel Super Specialty Hospital in Kerbala. A total of 72 participants were divided into two groups: 36 patients with type 2 diabetes with nephropathy and 36 nonnephropathic patients with type 2 diabetes. DNA was extracted from the blood, and then genotyping of the SNP rs741301 was carried out by Tetra ARMS-PCR by using special primers. Results The genotype and allele frequencies were examined under the codominant, dominant, and recessive models with the use of multinomial logistic regression analysis. The patient with DN with the heterozygous genotype GG+AG (odds ratio=5.28, confidence interval=1.35–20.73, P=0.017) were higher than diabetic patients with GG+AG (odds ratio=4.231, confidence interval=1.06–16.97, P=0.042) under with dominant model. Conclusion SNP rs741301 of ELMO1 gene was associated with DN due type 2 diabetes complication in Kerbala/Iraqi province.



How to cite this article:
Mohammed HJ, Al-Saegh RM, Al-Saadi NH. Role of engulfment and cell motility 1 gene polymorphism in type 2 diabetic nephropathy and its association with renal biomarkers: case–control stody.J Egypt Soc Nephrol Transplant 2020;20:98-102


How to cite this URL:
Mohammed HJ, Al-Saegh RM, Al-Saadi NH. Role of engulfment and cell motility 1 gene polymorphism in type 2 diabetic nephropathy and its association with renal biomarkers: case–control stody. J Egypt Soc Nephrol Transplant [serial online] 2020 [cited 2023 Jun 10 ];20:98-102
Available from: http://www.jesnt.eg.net/text.asp?2020/20/2/98/283242


Full Text



 Introduction



Diabetes mellitus (DM) is a common metabolic disorder, with a worldwide prevalence of 8.3%, and is the leading cause of visual loss, end-stage renal disease (ESRD), and amputation. DM poses a severe threat to global public health. Diabetic nephropathy (DN) is one of the most common complications of diabetes and the leading cause of ESRD [1].

DN is clinically defined as an increase in urinary albumin excretion and a decrease in kidney function. Classification of DN is based on estimated glomerular filtration rate and the degree of proteinuria [2].

DN is a multifactorial progressive disorder caused by the interaction between environmental factors and genetic factors [3].

Previous studies have shown that ∼30–40% of patients with DM progress to ESRD [4],[5], suggesting that genetic variations may have an effect on the initiation and development of DN and ESRD [1].

Recently, genome-wide association studies (GWASs) have identified genetic risk factors for diabetic microvascular complications of retinopathy, nephropathy, and neuropathy [1],[2], suggesting that genetic variations may affect the initiation and development of DN and ESRD. It is well known that gene susceptibility to DN plays an important role in individuals, even with the same environmental exposure [1].

Engulfment and cell motility 1 (ELMO1) is a new and strong candidate gene. It is a protein consisting of 720 amino acids. It is encoded by the ELMO1 gene on chromosome 7p14.2-p14.1 with 22. ELMO1 encodes for one of the ELMO proteins that promotes phagocytosis and cell migration by interacting with the cytokinesis protein. Polymorphisms in ELMO1 are strongly associated with susceptibility to DN [1],[6].

First validated in a Chinese population, Wu et al. [7] confirmed that both variants, rs10951509 and rs741301, were associated with DN. Moreover, the rs741301 polymorphism and duration of type 2 diabetes mellitus (T2DM) were identified as independent predictors of DN, whereas functional studies have found that a high level of ELMO1 expression aggravated the progression of DN and vice versa [1].

ELMO1 is involved in the pathogenesis of DN by multiple mechanisms. This includes ELMO1 gene, identified through replication studies and GWAS, to be a candidate gene for DN in African American, Japanese, American Indian, European American [8], Caucasian, and Chinese patients with T2DM.

Independent studies showed that the GWAS for DN could not be replicated, with a few exceptions, including those in APOL3-MYH9, ELMO1, CARS, FRMD3, and MYO16/IRS2 genes. These findings highlighted the importance of phagocytosis of apoptotic cells, insulin signaling, fibroblast migration, cytoskeleton reorganization, and epithelial clonal expansion in the pathogenesis of DN [9].

The ELMO1 gene was first found to be associated with DN in a GWAS in Japanese patients

with T2DM, two African American cohorts [1136 (ESRD) diabetes cases and T2DM 1160 controls] [10], and a Chinese population [9].

The GWAS have the benefits of rapid development of human genome research, particularly single nucleotide polymorphism (SNP) databases, and international GWAS have successfully facilitated rapid progress in genetic studies of T2DM. A number of SNPs in more than 50 genes are found to be associated with T2DM [11].

The progress of GWAS in DN has slowed down mainly owing to the difficulty of collection and characterization of participants.

In the recent 4 years, six GWAS in DN and ESRD (three each in T1DM and T2DM) have been reported. Six genes have been implicated in T2DM with DN and ESRD [12].

Several recent studies have discovered associations with human disease and genetic mutations of components of the engulfment signaling machinery. For example, several point mutations in the intronic regions of ELMO1 have been linked to DN and diabetes [13].

 Patients and methods



Study participants

This case–control study took a period of 14 months starting from January 2018 to March 2019. This study was conducted in the Department of Biochemistry, College of Medicine, the University of Kerala. A total of 72 participants were included: 36 of them with T2DM and nephropathy and the other 36 had T2DM without nephropathy. Participants with urinary albumin/creatinine ratio (PCR) more than or equal to 30 mg/g and protein excretion history caused by diabetes were considered as the DN group. DM group was indicated by albumin/creatinine ratio less than 30 mg/g and ADA criteria for diabetes. Mean±SD glycosylated hemoglobin was 8.36±2.10.

The age of DN and DM groups was matched and ranged between 35 and 75 years at disease onset, and the mean duration of diabetes without nephropathy was 65.97±58.24 months and diabetes with nephropathy was 139.37±78.84 months. Informed consent was obtained from all the study participants. All investigations were done in accordance with the Health and Human Ethical Clearance Committee guidelines for Clinical Researches. Local ethical committee approved the study protocol.

Clinical characteristics of participants

Several demographic and anthropometric measurements were taken. Blood pressure was measured after 20 min of rest. The weight and the height were measured and the BMI was calculated (kg/m2). Urine and random blood samples were obtained from the participants for biochemical analysis and DNA extraction. Glycosylated hemoglobin, random blood sugar, urea, creatinine, and also urine protein creatinine ratio were measured. Estimated glomerular filtration rate was calculated using Cockroft and Gault equation.

Genotyping

DNA was extracted from the sample of the whole blood for each patient by using a Mini Kit and applying a protocol Geneaid/Korea), according to the manufacture company. DNA concentration was quantified by using NanoDrop Spectrophotometer (Thermo Fisher Sceintific TM Nanodrop spectrophotometer US) and held in 1% gel agarose electrophoresis.

The ARMS-primers used for detection of mutation screening PIT1 are provided in [Table 1].{Table 1}

Amplification reaction mixture for ELMO1 (rs741301) containing four primers was used in a single PCR reaction. The PCR mixture is prepared by addition of 1.5 µl from each of outer primers (forward amd reverse) and 1 µl from each of inner primers (forward amd reverse) of 10 pmol/µl with 4 µl of extracted DNA to 12 µl of lyophilized PCR master mix formula and completed the volume to 25 µl by using nuclease-free water. The reaction mixture has been mixed by vortex and incubated in the PCR GTC thermal cycler using the following program to amplify the mixture:

[INLINE:1]

Statistical analysis

Data are descriptively presented as mean±SD. Significant differences were confirmed by the analysis of variance test for continuous variables among more than two groups, and to compare mean levels of continuous characteristics across genotypes, Student t test was used using PAST3.20, 2018, and SPSS, version 24 software (IBM SPSS version 26, 2016 US). Categorical data (alleles and genotypes) were expressed as frequency according to Hardy–Weinberg equilibrium. For all of the statistical analysis, P values equal or less than 0.05 were considered statistically significant. Allele frequencies and genotyping of rs741301 in ELMO1 gene were compared in groups using a χ2 test. To assess the relative risk conferred by a particular allele and genotype, odds ratio (OR) and confidence intervals (95% CI) were calculated.

 Results



The genotype and allele frequencies were examined under the codominant, dominant, and recessive models with the use of multinomial logistic regression analysis. Patients with DN with the heterozygous genotype GG+AG (odds ratio=5.28, confidence interval=1.35–20.73, P=0.017) were higher than diabetic patients with GG+AG (odds ratio=4.231, confidence interval=1.06–16.97, P=0.042) under the dominant model.

 Discussion



Most studies reported rs741301 polymorphism as the strongest SNP associated with DN. In 2005, GWAS was first done in Japanese patients with T2DM, and the results illustrated the strongest association between rs741301 polymorphism and DN. They showed that GG genotype in rs741301 and G allele are associated with risk of DN in Japanese patients, which is consistent with our results [14],[15].The risk of DN for patient with AG and GG genotypes collectively in the rs741301 locus was 10.3 times higher than that for AA genotype, with a statically significant relationship (P=0.042) ([Table 2]). This study concludes that other mutations of genotype GG and allele G in rs741301 locus like deletion, substitution, and insertion of one or more of nucliec acid can decrease their additional genotype risk of DN.{Table 2}

A meta-analysis of DN genetic associations showed rs741301 of ELMO1 was associated with DN in Asians with T2DM (OR, 1.58; 95% CI, 1.28–1.94) [16]. We recommended further cohort control studies to check the effect of G allele in loci rs741301 on the development of T2DM with or without DN [6].

There was no statically significant effect of genotyping of all allele frequencies of AA, AG, and GG type in rs741301 loci regarding difference in phenotyping when compering them in DN ([Table 3]).{Table 3}

 Conclusion



The result clearly illustrate geographical and racial differences for the genotypic frequency of allele G in the rs741301 loci and it risk factor in Iraqi population, similar to Asian population, but not in American, Indian, and Mexican population. The presence of other risk factors such as age and control of systolic blood pressure may explain this difference in our results when compared with other studies. Moreover, the results showed that dyslipidemia might be a risk factor for developing DN.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Wei L, Xiao Y, Li L, Xiong X, Han Y, Zhu X et al. The susceptibility genes in diabetic nephropathy. Kidney Dis 2018; 4:226–237.
2Kwak SH, Park KS. Genetic studies on diabetic microvascular complications: focusing on genome-wide association studies. Endocrinol Metab 2015; 30:147–158.
3Mehrabzadeh M, Pasalar P, Karimi M, Abdollahi M, Daneshpour M, Asadolahpour E et al. Association between ELMO1 gene polymorphisms and diabetic nephropathy in an Iranian population. J Diabetes Metab Disord 2016; 1:1–7.
4Marshall SM. Natural history and clinical characteristics of CKD in type 1 and type 2 diabetes mellitus. Adv Chronic Kidney Dis 2014; 21:267–272.
5Hill C, Cardwell C, Patterson C, Maxwell A, Magee G, Young R et al. Chronic kidney disease and diabetes in the National Health Service: a cross-sectional survey of the UK National Diabetes Audit. Diabetic Med 2014; 31:448–454.
6Yahya MJ, Yusoff MJ. Association of CCL2, CCR5, ELMO1, and IL8 polymorphism with diabetic nephropathy in malaysian type 2 diabetic patients. Int J Chronic Dis 2019; 2019:2053015.
7Wu H, Wang Y, Chen M, Zhang X, Wang D, Pan Y et al. Association of ELMO1 gene polymorphisms with diabetic nephropathy in Chinese population. J Endocrinol Investig 2013; 36:298–302.
8Craig D, Millis M, DiStefano J. Genome-wide SNP genotyping study using pooled DNA to identify candidate markers mediating susceptibility to end-stage renal disease attributed to type 1 diabetes. Diabetic Med 2009; 26:1090–1098.
9Chang Y-C., Chang EY-C., Chuang L-M. Recent progress in the genetics of diabetic microvascular complications. World J Diabetes 2015; 6:715.
10Leak T, Perlegas P, Smith S, Keene K, Hicks P, Langefeld C et al. Variants in intron 13 of the ELMO1 gene are associated with diabetic nephropathy in African Americans. Ann Human Genet 2009; 73:152–159.
11Billings LK, Florez JC. The genetics of type 2 diabetes: what have we learned from GWAS? Ann New York Acad Sci 2010; 1212:59–77.
12Gu HF, Brismar K. Genetic association studies in diabetic nephropathy. Curr Diabetes Rev 2012; 8:336–344.
13Elliott MR, Ravichandran KS. Clearance of apoptotic cells: implications in health and disease. J Cell Biol 2010; 189:1059–1070.
14Shimazaki A, Kawamura Y, Kanazawa A, Sekine A, Saito S, Tsunoda T et al. Genetic variations in the gene encoding ELMO1 are associated with susceptibility to diabetic nephropathy. Diabetes 2005; 54:1171–1178.
15Kim S, Abboud HE, Pahl MV, Tayek J, Snyder S, Tamkin J et al. Examination of association with candidate genes for diabetic nephropathy in a Mexican American population. Clin J Am Soc Nephrol 2010; 5:1072–1078.
16Mooyaart AL, Valk EJ, van Es LA, Bruijn JA, de Heer E, Freedman BI et al. Genetic associations in diabetic nephropathy: a meta-analysis. Diabetologia 2011; 54:544–553.