- Email: [email protected]
Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy
Accepted Manuscript Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy
Farideh Razi, Marzieh Arshadi Meshkani, Fariba Zarrabi, Maryam Sadr, Saeedeh Asgarbeik, Fatemeh Bandarian, Katayoon Forouzanfar, Mahsa Mohammad Amoli PII: DOI: Reference:
S0378-1119(18)31051-5 doi:10.1016/j.gene.2018.10.017 GENE 43273
To appear in:
Gene
Received date: Revised date: Accepted date:
25 July 2018 30 September 2018 9 October 2018
Please cite this article as: Farideh Razi, Marzieh Arshadi Meshkani, Fariba Zarrabi, Maryam Sadr, Saeedeh Asgarbeik, Fatemeh Bandarian, Katayoon Forouzanfar, Mahsa Mohammad Amoli , Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy. Gene (2018), doi:10.1016/j.gene.2018.10.017
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ACCEPTED MANUSCRIPT Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy Farideh Razi, MD 1; MarziehArshadiMeshkani2; Fariba Zarrabi3; Maryam Sadr4; Saeedeh Asgarbeik5; Fatemeh Bandarian, MD, PhD 6; Katayoon Forouzanfar, 7; Mahsa Mohammad Amoli, MD, PhD 8 1
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Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of
Medical Sciences, Tehran, Iran 2
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Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran,
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Iran 3
Thrombosis Hemostasis Research Center, Tehran University of Medical Sciences, Tehran, Iran Molecular Immunology Research Center, Children’s Medical Center, Tehran University of Medical Sciences,
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4
Tehran, Iran 5
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Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
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Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute,
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Tehran University of Medical Sciences, Tehran, Iran 7
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Elderly Health Research Center, Endocrinology and Metabolism Population Sciences Institute, Tehran University
of Medical Sciences, Tehran, Iran 8
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Metabolic Disorders Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute,
Tehran University of Medical Sciences, Tehran, Iran
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Corresponding author: Mahsa Mohammad Amoli, MD, PhD Metabolic Disorders Research Center, Endocrinology and Metabolism Research Institute, 5th floor, Shariati Hospital, North Kargar Ave., Tehran, Iran Tel: +9821 88220081 Fax: +9821 88220051 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Background: Diabetic Nephropathy (DN) is one of the microvascular complications of diabetes and its early diagnosis can improve patient’s quality of life. Genetic factors may increase the risk of DN development. This study aimed to evaluate the association of vitamin D receptor (VDR) gene polymorphisms and DN.
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Method: A total of 313 Iranian participants including 104 diabetic patients with nephropathy (DN), 100
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diabetic patients without nephropathy (D) and 109 healthy people (HC) were studied. The frequencies of
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rs7975232, rs731236 and rs4516035 variants of VDR gene were determined and compared between three groups. Estimated haplotype frequencies between polymorphisms in the cases and controls were also
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calculated.
Results: No significant differences were identified for allele /genotype frequencies in HC, D and DN
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groups. However haplotype analysis showed that haplotype encompassing CCC alleles for rs7975232, rs731236 and rs4516035 variants, respectively was more frequent in DN subjects compared to HC (p-
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value= 0.01) and also, haplotype comprising TCC alleles was more frequent in DN group compared to
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both HC and D groups (p-value= 0.004 and 0.007, respectively).
with diabetes type 2.
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Conclusion: Our study identified that CCC and TCC VDR haplotypes are risk factors for DN in patients
rs4516035 Highlights
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Keyword: Diabetes type 2, Diabetic nephropathy, Vitamin D receptor gene, rs7975232, rs731236 and
-There is no significant difference in allele /genotype frequencies between HC, D and DN groups. -CCC and TCC VDR haplotypes are risk factors for DN in patients with type 2diabetes.
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ACCEPTED MANUSCRIPT Introduction Diabetes and hyperglycemia can cause metabolic changes that lead to basement membrane thickness, mesangial expansion, and glomerular sclerosis in the kidney which lead to urinary protein secretion [1].Diabetic Nephropathy (DN) is one of the most devastating microvascular complication of diabetes and the leading cause of end stage renal diseases (ESRD). DN patients are also at high risk for development of
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cardiovascular disease [2].
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In DEMAND (Developing Education on Microalbuminuria for Awareness of renal and cardiovascular
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risk in Diabetes)global study on 32,208 type 2 diabetes patients from 33 countries, overall prevalence of microalbuminuria was reported 39% which was higher in Asian and Hispanic groups[3]. The higher
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prevalence of microalbuminuria in Asian populations has been confirmed also by the MicroAlbuminuria Prevalence (MAP) Study[4]. Studies about urinary albumin excretion in Iran have reported the prevalence
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of microalbuminuria and macroalbuminuriaas 14.2-26% and 12–23%, respectively which is higher than
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reports from neighboring countries[5].
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Vitamin D plays a key role in bone metabolism but it is involved in many other functions in the biological systems such as immunomodulatory, anti-inflammatory, antioxidant, anti-angiogenic, and antiproliferative properties. Furthermore vitamin D has an important role in numerous diseases including
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[7-11].
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type 1 and type 2 diabetes [6] as well as metabolic syndrome, insulin resistance, obesity and osteoporosis
Vitamin D influence target tissues through its receptor so called vitamin D receptor (VDR), a member of the steroid/thyroid hormone receptor family. VDR gene is located on chromosome 12q13.1[12]. Association of vitamin D and also vitamin D pathway gene polymorphisms with diabetes and its complications have been investigated by several researchers [12-18] but the results are controversial. Many studies have been carried out in Iran on diabetes complication [19] and the association between
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ACCEPTED MANUSCRIPT VDR gene variants and gestational diabetes as well as diabetic foot ulcer and osteoporosis has been studied previously [20-24]. By finding the genetic risk factors of diabetic nephropathy, patients at risk of diabetic nephropathy can be identified and special preventive measure can be applied to prevent or postpone the occurrence of diabetic
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nephropathy which is a personalized approach to diabetes complications. Different studies have also reported association of various gene variations such as ELMO1, AGTR1,
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APOE and MYH9 with DN [25-29]. Current study aimed to evaluate association between DN and
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rs7975232, rs731236 and rs4516035 polymorphisms in VDR gene in a group of patients with type 2
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diabetes.
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Materials and Methods General characteristic
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A total of 313 participants including 104 diabetic patients with nephropathy (DN), 100 diabetic patients
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without nephropathy (D) and 109 healthy people (HC) were recruited between 2015 and 2017 at Diabetes Clinic of Diabetes Research Center affiliated to Endocrinology and Metabolism Research Institute of …
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University of Medical Sciences, …, ….
Diabetic patients with a minimum of 5 years diabetes duration were included. Patients with urinary
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albumin/creatinine ratio (ACR) ≥30 mg/g were considered as the DN group and a group of diabetic patients without albuminuria who were matched with DN group for diabetes duration were selected as diabetic patients without nephropathy (D) group. Healthy control subjects were participants without history of diabetes or kidney disease. Subjects of non-Iranian origin, patients with Type 1diabetes, HbA1c> 9 %, urinary tract infection, uncontrolled hypertension, pregnancy, hematuria, smoking and any other cause of protein excretion
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ACCEPTED MANUSCRIPT except for diabetes type 2 were excluded. Eligible individuals for each study group were selected by census sampling method. The study protocol was approved by the Ethics Committee of Endocrinology and Metabolism Research Institute and all participants signed written informed consent before enrollment. After an 8-hour overnight fasting, blood and urine samples were obtained from the participants for
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biochemical analysis and DNA extraction. Glucose, urea, creatinine, uric acid and also urine albumin and creatinine were measured by commercial
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kits (Pars Azmun, Karaj, Iran). Total imprecision of assays express as coefficient of variation (CV%)
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were, 2%, 3.5%, 4%, 3% and 4%, respectively. Detection limits were 5, 2, 0.1, 0.3, 3 and 0.1 mg/dL.
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HbA1c was measured by HPLC method (TOSOH G8, Tokyo, Japan) with total imprecision of 1.5% and reportable range between 4.0 and 16.9 %.
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Estimated glomerular filtration rate (eGFR) was calculated using Cockcroft-Gault equation.
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DNA extraction
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Blood samples were collected each one in a 4 ml EDTA blood tube and DNA were extracted from nucleated cells according to the phenol–chloroform protocol using proteinase K [30] .
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In order to assess nucleic acid purity, the optical density (OD) was measured. The DNAs was then stored
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at -20°C prior to genotyping. Polymorphism genotyping
The VDR (Vitamin D Receptor) gene polymorphism genotyping (rs731236, rs4516035 and rs7975232) was performed by Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA) using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) via Allelic Discrimination method. Thermal cycling conditions and data analysis were according to the standard manufacturer’s instructions. The identified genotypes were 5
ACCEPTED MANUSCRIPT named accordingly, TT for homozygous wild type, CT for heterozygous and CC for homozygous variant of rs731236 and rs4516035 and CC for homozygous wild type, CA for heterozygous and AA for homozygous variant of rs7975232. Statistical analysis
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For analysis of the biochemical and demographic data, one-way ANOVA followed by Bonferroni’s test, were applied. SPSS (version 19.0) was used for this analysis. Chi-square distribution and odds ratios
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(ORs) were used to test Hardy-Weinberg equilibrium (HWE) of genotypes and allele frequencies of
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mutated alleles. Statistical significance was defined as p-value ≤0.05. To estimate the haplotype frequencies and linkage disequilibrium between polymorphisms in the cases and controls, the EHPLUS
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program [31] was used.
Results
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A total number of 313 participants were evaluated in this study. Female to male ratio was 71/38, 54/46
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and39/65 in HC, D and DN groups, respectively. Table 1 shows demographic characteristics and biochemical parameters of three study groups. Although the mean age of cases was significantly different
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between the groups but the duration of the disease in D and DN groups was in the same range. Highest BMI was found in DN group which was significantly higher than HC but was not different with the D
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group.
Genotype and allele frequencies of VDR polymorphisms in the HC, D and DN are presented in Table 2. No significant difference was found for genotype and allele frequencies between three groups. Allele and genotype distributions in the HC and D subjects were in Hardy-Weinberg equilibrium (pvalue>0.05). Table 3 shows haplotype frequencies in HC, D and DN groups. We observed that the frequency of carrying haplotype with CCC alleles for all three variants was significantly higher in DN subjects 6
ACCEPTED MANUSCRIPT compared to HC group and frequency of carrying TCC haplotype was significantly higher in DN group compared to both HC and D groups (Table 3).
Discussion The present case-control study was conducted to examine the relationship between VDR gene
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polymorphism (rs7975232 C>A, rs731236 T>C and rs4516035 T>C) and risk of DN in a group of type 2 diabetic patients. No significant differences were identified in allele/genotype frequencies between HC, D
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and DN groups. However, haplotype analysis showed that two haplotype (CCC, TCC) were significantly
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more frequent in DN group.
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It has been demonstrated that VDR mediates the effect of vitamin D by making a heterodimer with the retinoid X receptor molecule which is regulating the expression of many target genes involved in
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enormous disease mechanisms including inflammatory, oxidative stress and etc [32]. Previous studies proved that VDR gene polymorphisms have functional effect on VDR activity and
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protein expression. Functional studies have revealed that rs4516035 variant is located at a GATA binding
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site causing decreased promoter activity of VDR gene in individuals carrying allele C while allele T increase the promoter activity to 1.9 fold [33].
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rs7975232, rs731236 variants are located in the 3′UTR of the VDR genes and have been shown to have a role in mRNA stability and therefore regulating gene expression [34].
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There is a growing interest in potential importance of vitamin D and VDR gene in diabetes mellitus but the results are controversial. In a case-control study in a group of southeastern Iranian diabetic patients reported by Nosratabadi et al[18] a significant association was found for Taq1 polymorphism genotype frequencies in diabetes while in another study with higher sample size by Shab-Bidar et al, no evidence for the association between VDR gene polymorphisms (FokI, BsmI, ApaI, and TaqI) and the risk for type 2 diabetes and metabolic syndrome were found[35].The study by Aslani et al., in Iran, found a significant association between VDR FokI polymorphism and gestational diabetes [20]. Studies in Turkish, Tunisian, 7
ACCEPTED MANUSCRIPT Brazilian and polish patients failed to show a significant correlation between VDR gene polymorphism and diabetes [14-17]. There are few studies about association of VDR gene variation and diabetes complications. In a study on post-menopausal diabetic women no significant association was found between TaqI and FokI polymorphisms and macro and microvascular complications [16] while in another case-control study,
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Vedralováet al identified significant association between FokI variant and DN[36].In a study in Iran by Soroush et al., a significant association was observed between VDR FokI functional variant and diabetic
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foot ulcer [21].
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A research on diabetes type 1 patients from GoKinD study (Genetics of Kidneys in Diabetes) in UK,
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showed no significant association with genotype or allele frequencies of VDR gene variants but they suggested that haplotype AGT (rs1544410, rs7975232 and rs731236 markers) was protective against
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DN in type 1 diabetes.
Reno-protection of vitamin D has proposed by many authors [37], but there are few studies about VDR
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gene variants and diabetic nephropathy and many of them have failed to show a correlation between VDR
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gene variation (ApaI, BsmI, FokI and TaqI) and DN in different population[38]. Diversity between various study results can be related to heterogeneity of DN, genetic or nutrition and also climate
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difference in different countries.
Small samples size and studying only three variants of VDR gene were the main limitations of present
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study. Lack of matching for age and sex, DN onset age and medication used are other limitations of current study. This study was conducted in a group of diabetic patients and replication in a greater sample size is required to confirm the findings of present study. Future studies examining other variants in VDR gene is also recommended. In conclusion, the results of current study demonstrated that some VDR genetic variants as haplotype combination might be protective against DN development while CCC and TCC haplotypes encode risk for DN development. 8
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ACCEPTED MANUSCRIPT Table 1: Demographic characteristics and biochemical parameters in three study groups
HC group
D group
DN group
p-Value
p-Value
p-Value
(n=109)
(n=100)
(n=104)
HC-D
HC-DN
D-DN
Mean (SD)
Mean (SD)
Mean (SD)
Age (year)
50.5(10.7)
57.5(8.2)
62.4(9.2)
SBP (mmHg)
115.9(12.7)
122.8(12.4)
123.7(13.7) 0.001
DBP (mmHg)
79.2(5.9)
78.9(6.3)
75.5(9.7)
BMI (kg/m2 )
27.4(4.0)
28.5(4.2)
29.7(4.9)
-
10.8(6.2)
GFR (mL/min/1.73m2)
75.5(30.0)
FBS (mg/dL)
Variables
0.001
0.001
1
1
0.001
0.004
0.270
0.002
0.196
12.5(7.0)
-
-
0.065
76.7(20.0)
64.4(29.7)
1
0.011
0.004
91.4(7.7)
140.2(34.6)
133.8(45.9) 0.001
0.001
0.525
HbA1c (%)
5.5(0.46)
7.1(0.74)
7.4(0.74)
0.001
0.001
0.006
Cr (mg/dL)
1.0(0.16)
1.11(0.26)
1.28(0.48)
0.036
0.001
0.001
Urea (mg/dL)
29.5 (7.4)
36.7(13.0)
42.6(17.0)
0.001
0.001
0.005
UA (mg/dL)
4.9(2.8)
5.2(1.4)
5.4(1.8)
0.958
0.453
1
59.3 (31.1)
73.0(48.9)
59.1(30.3)
0.379
1
0.142
24.4(6.0)
246(666)
1
0.001
0.001
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D
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ACR (mg/g)
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(year)
25(OH)D (nmol/L)
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0.001
Diabetes Duration
23.5(8.0)
0.001
Data are presented as mean (SD) Abbreviation: HC: healthy controls, D: diabetes without nephropathy, DN: diabetic nephropathy, SBP: systolic blood pressure, DBP: diastolic blood pressure, BMI: body mass index, GFR: glomerular filtration rate, FBS: fasting blood sugar, Cr: creatinine, UA: uric acid, 25(OH) D : 25 hydroxyvitamin D, ACR: urine albumin to creatinine ratio
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ACCEPTED MANUSCRIPT
Genotype/
HC=109
D=100
DN=104
N(%)
N(%)
N(%)
16(16.0)
23(22.1)
Allele
OR
p-Value HC-D
p-Value
HC-D
HC-DN
OR HC-DN
p-Value D-DN
OR D-DN
rs731236 Taq1
TT
20 (18.3)
1.00
1.00
1.00
(reference)
(reference)
(reference)
1.51 45(41.3)
51(51.0)
45(44.6)
0.18
(0.82-2.76)
0.37
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0.71
CT
0.61 0.20
1.07 CC
44(40.4)
33(33.0)
36(34.6)
0.71
85(39.0)
83(41.5)
91(43.8)
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(reference)
0.90
133(61.0)
117(58.5)
117(56.3)
0.60
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C
T
C
16(14.6)
131(60.1)
45(45)
16(16)
87(39.9)
30(28.8)
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CC
55(50.5)
39(39)
54(51.9)
20(19.2)
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CT
38(34.9)
123(61.5)
AC
TT
77(38.5)
D
rs4516035
0.76 0.50
(0.42-1.80)
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1.00
(0.29-1.30)
0.87
0.87 (0.48-2.37)
T
RI
(0.34-1.49)
(0.34-1.68) 1.0
1.00 (reference) (reference)
0.82
0.65
0.91
0.32
(0.6-1.33)
(0.56-1.21)
(0.61-1.35)
1.00
1.00
1.00
(reference)
(reference)
(reference)
0.8
0.46
1.58 0.27
(0.43-1.44)
(0.70-3.57)
0.97
0.95
1.62 0.24 (0.72-3.66)
1.24 0.48
1.56 0.16
(0.43-2.22)
(0.68-2.28)
(0.84-2.90)
1.00
1.00
1.00
(reference)
(reference)
(reference)
114(54.8)
0.94 94(45.2)
0.77
1.24 0.27
1.31 0.17
(0.64-1.39)
(0.85-1.82)
(0.89-1.95)
1.00
1.00
1.00
(reference)
(reference)
(reference)
rs7975232ApaI
CC
16 (14.7)
21 (21.0)
22 (21.2)
0.54 AC
52 (47.7)
50 (50.0)
50 (48.1)
0.13
0.57 0.16
(0.24-1.2)
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1.05 0.89
(0.26-1.25)
(0.51-2.14)
ACCEPTED MANUSCRIPT 0.73 AA
C
41 (37.6)
84 (38.5)
29 (29.0)
92 (46.0)
32 (30.8)
0.70
0.421
0.35 (0.34-1.56)
(0.33-1.48)
(0.44-2.07)
1.00
1.00
1.00
(reference)
(reference)
(reference)
94 (45.2)
0.74 A
134 (61.5)
108 (54.0)
0.95 0.89
114 (54.8)
0.76
0.12
0.16 (0.49-1.07)
(0.52-1.11)
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Table 2: Genotype and allele frequencies in three study groups
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1.03 0.87 (0.70-1.52)
ACCEPTED MANUSCRIPT Table 3: Comparison of haplotypes frequencies of VDR gene polymorphisms between three study groups
Haplotypes
D (2N=200)
DN (2N=208)
HC (2N=218)
(0.109173)
(0.084751)
(0.119266)
(0.025827)
(0.040249)*
(0.000000)*
(0.244357)
(0.225791)
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rs731236/ rs4516035/
A
CCC C
T
A
(0.218734)
C
T
C
(0.261266)
T
C
A
T
C
T T
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C
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C
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rs7975232
(0.255861)
(0.025000)
(0.049864)
(0.064220)
C
(0.005000)&
(0.046290)&#
(0.000000)#
T
A
(0.187092)
(0.169104)
(0.205402)
T
C
(0.167908)
(0.186665)
(0.129460)
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D
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(0.178720)
*P- value: 0.01, OR: 8.7 (1.1-388.5) # P-value: 0.004, OR: 10.0 (1.4-438.2) & P-value: 0.007, OR: 10.9 (1.5-477.6)
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ACCEPTED MANUSCRIPT Abbreviation list ESRD: end stage renal diseases ACR: albumin/creatinine ratio VDR: vitamin D receptor
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DN: Diabetic Nephropathy
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D
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MAP: Micro Albuminuria Prevalence
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Farideh Razi, Marzieh Arshadi Meshkani, Fariba Zarrabi, Maryam Sadr, Saeedeh Asgarbeik, Fatemeh Bandarian, Katayoon Forouzanfar, Mahsa Mohammad Amoli PII: DOI: Reference:
S0378-1119(18)31051-5 doi:10.1016/j.gene.2018.10.017 GENE 43273
To appear in:
Gene
Received date: Revised date: Accepted date:
25 July 2018 30 September 2018 9 October 2018
Please cite this article as: Farideh Razi, Marzieh Arshadi Meshkani, Fariba Zarrabi, Maryam Sadr, Saeedeh Asgarbeik, Fatemeh Bandarian, Katayoon Forouzanfar, Mahsa Mohammad Amoli , Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy. Gene (2018), doi:10.1016/j.gene.2018.10.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Haplotypes in vitamin D receptor gene encode risk in diabetic nephropathy Farideh Razi, MD 1; MarziehArshadiMeshkani2; Fariba Zarrabi3; Maryam Sadr4; Saeedeh Asgarbeik5; Fatemeh Bandarian, MD, PhD 6; Katayoon Forouzanfar, 7; Mahsa Mohammad Amoli, MD, PhD 8 1
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Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of
Medical Sciences, Tehran, Iran 2
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Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran,
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Iran 3
Thrombosis Hemostasis Research Center, Tehran University of Medical Sciences, Tehran, Iran Molecular Immunology Research Center, Children’s Medical Center, Tehran University of Medical Sciences,
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4
Tehran, Iran 5
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Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
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Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute,
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Tehran University of Medical Sciences, Tehran, Iran 7
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Elderly Health Research Center, Endocrinology and Metabolism Population Sciences Institute, Tehran University
of Medical Sciences, Tehran, Iran 8
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Metabolic Disorders Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute,
Tehran University of Medical Sciences, Tehran, Iran
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Corresponding author: Mahsa Mohammad Amoli, MD, PhD Metabolic Disorders Research Center, Endocrinology and Metabolism Research Institute, 5th floor, Shariati Hospital, North Kargar Ave., Tehran, Iran Tel: +9821 88220081 Fax: +9821 88220051 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Background: Diabetic Nephropathy (DN) is one of the microvascular complications of diabetes and its early diagnosis can improve patient’s quality of life. Genetic factors may increase the risk of DN development. This study aimed to evaluate the association of vitamin D receptor (VDR) gene polymorphisms and DN.
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Method: A total of 313 Iranian participants including 104 diabetic patients with nephropathy (DN), 100
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diabetic patients without nephropathy (D) and 109 healthy people (HC) were studied. The frequencies of
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rs7975232, rs731236 and rs4516035 variants of VDR gene were determined and compared between three groups. Estimated haplotype frequencies between polymorphisms in the cases and controls were also
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calculated.
Results: No significant differences were identified for allele /genotype frequencies in HC, D and DN
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groups. However haplotype analysis showed that haplotype encompassing CCC alleles for rs7975232, rs731236 and rs4516035 variants, respectively was more frequent in DN subjects compared to HC (p-
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value= 0.01) and also, haplotype comprising TCC alleles was more frequent in DN group compared to
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both HC and D groups (p-value= 0.004 and 0.007, respectively).
with diabetes type 2.
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Conclusion: Our study identified that CCC and TCC VDR haplotypes are risk factors for DN in patients
rs4516035 Highlights
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Keyword: Diabetes type 2, Diabetic nephropathy, Vitamin D receptor gene, rs7975232, rs731236 and
-There is no significant difference in allele /genotype frequencies between HC, D and DN groups. -CCC and TCC VDR haplotypes are risk factors for DN in patients with type 2diabetes.
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ACCEPTED MANUSCRIPT Introduction Diabetes and hyperglycemia can cause metabolic changes that lead to basement membrane thickness, mesangial expansion, and glomerular sclerosis in the kidney which lead to urinary protein secretion [1].Diabetic Nephropathy (DN) is one of the most devastating microvascular complication of diabetes and the leading cause of end stage renal diseases (ESRD). DN patients are also at high risk for development of
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cardiovascular disease [2].
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In DEMAND (Developing Education on Microalbuminuria for Awareness of renal and cardiovascular
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risk in Diabetes)global study on 32,208 type 2 diabetes patients from 33 countries, overall prevalence of microalbuminuria was reported 39% which was higher in Asian and Hispanic groups[3]. The higher
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prevalence of microalbuminuria in Asian populations has been confirmed also by the MicroAlbuminuria Prevalence (MAP) Study[4]. Studies about urinary albumin excretion in Iran have reported the prevalence
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of microalbuminuria and macroalbuminuriaas 14.2-26% and 12–23%, respectively which is higher than
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reports from neighboring countries[5].
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Vitamin D plays a key role in bone metabolism but it is involved in many other functions in the biological systems such as immunomodulatory, anti-inflammatory, antioxidant, anti-angiogenic, and antiproliferative properties. Furthermore vitamin D has an important role in numerous diseases including
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[7-11].
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type 1 and type 2 diabetes [6] as well as metabolic syndrome, insulin resistance, obesity and osteoporosis
Vitamin D influence target tissues through its receptor so called vitamin D receptor (VDR), a member of the steroid/thyroid hormone receptor family. VDR gene is located on chromosome 12q13.1[12]. Association of vitamin D and also vitamin D pathway gene polymorphisms with diabetes and its complications have been investigated by several researchers [12-18] but the results are controversial. Many studies have been carried out in Iran on diabetes complication [19] and the association between
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ACCEPTED MANUSCRIPT VDR gene variants and gestational diabetes as well as diabetic foot ulcer and osteoporosis has been studied previously [20-24]. By finding the genetic risk factors of diabetic nephropathy, patients at risk of diabetic nephropathy can be identified and special preventive measure can be applied to prevent or postpone the occurrence of diabetic
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nephropathy which is a personalized approach to diabetes complications. Different studies have also reported association of various gene variations such as ELMO1, AGTR1,
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APOE and MYH9 with DN [25-29]. Current study aimed to evaluate association between DN and
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rs7975232, rs731236 and rs4516035 polymorphisms in VDR gene in a group of patients with type 2
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diabetes.
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Materials and Methods General characteristic
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A total of 313 participants including 104 diabetic patients with nephropathy (DN), 100 diabetic patients
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without nephropathy (D) and 109 healthy people (HC) were recruited between 2015 and 2017 at Diabetes Clinic of Diabetes Research Center affiliated to Endocrinology and Metabolism Research Institute of …
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University of Medical Sciences, …, ….
Diabetic patients with a minimum of 5 years diabetes duration were included. Patients with urinary
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albumin/creatinine ratio (ACR) ≥30 mg/g were considered as the DN group and a group of diabetic patients without albuminuria who were matched with DN group for diabetes duration were selected as diabetic patients without nephropathy (D) group. Healthy control subjects were participants without history of diabetes or kidney disease. Subjects of non-Iranian origin, patients with Type 1diabetes, HbA1c> 9 %, urinary tract infection, uncontrolled hypertension, pregnancy, hematuria, smoking and any other cause of protein excretion
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ACCEPTED MANUSCRIPT except for diabetes type 2 were excluded. Eligible individuals for each study group were selected by census sampling method. The study protocol was approved by the Ethics Committee of Endocrinology and Metabolism Research Institute and all participants signed written informed consent before enrollment. After an 8-hour overnight fasting, blood and urine samples were obtained from the participants for
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biochemical analysis and DNA extraction. Glucose, urea, creatinine, uric acid and also urine albumin and creatinine were measured by commercial
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kits (Pars Azmun, Karaj, Iran). Total imprecision of assays express as coefficient of variation (CV%)
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were, 2%, 3.5%, 4%, 3% and 4%, respectively. Detection limits were 5, 2, 0.1, 0.3, 3 and 0.1 mg/dL.
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HbA1c was measured by HPLC method (TOSOH G8, Tokyo, Japan) with total imprecision of 1.5% and reportable range between 4.0 and 16.9 %.
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Estimated glomerular filtration rate (eGFR) was calculated using Cockcroft-Gault equation.
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DNA extraction
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Blood samples were collected each one in a 4 ml EDTA blood tube and DNA were extracted from nucleated cells according to the phenol–chloroform protocol using proteinase K [30] .
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In order to assess nucleic acid purity, the optical density (OD) was measured. The DNAs was then stored
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at -20°C prior to genotyping. Polymorphism genotyping
The VDR (Vitamin D Receptor) gene polymorphism genotyping (rs731236, rs4516035 and rs7975232) was performed by Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA) using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) via Allelic Discrimination method. Thermal cycling conditions and data analysis were according to the standard manufacturer’s instructions. The identified genotypes were 5
ACCEPTED MANUSCRIPT named accordingly, TT for homozygous wild type, CT for heterozygous and CC for homozygous variant of rs731236 and rs4516035 and CC for homozygous wild type, CA for heterozygous and AA for homozygous variant of rs7975232. Statistical analysis
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For analysis of the biochemical and demographic data, one-way ANOVA followed by Bonferroni’s test, were applied. SPSS (version 19.0) was used for this analysis. Chi-square distribution and odds ratios
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(ORs) were used to test Hardy-Weinberg equilibrium (HWE) of genotypes and allele frequencies of
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mutated alleles. Statistical significance was defined as p-value ≤0.05. To estimate the haplotype frequencies and linkage disequilibrium between polymorphisms in the cases and controls, the EHPLUS
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program [31] was used.
Results
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A total number of 313 participants were evaluated in this study. Female to male ratio was 71/38, 54/46
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and39/65 in HC, D and DN groups, respectively. Table 1 shows demographic characteristics and biochemical parameters of three study groups. Although the mean age of cases was significantly different
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between the groups but the duration of the disease in D and DN groups was in the same range. Highest BMI was found in DN group which was significantly higher than HC but was not different with the D
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group.
Genotype and allele frequencies of VDR polymorphisms in the HC, D and DN are presented in Table 2. No significant difference was found for genotype and allele frequencies between three groups. Allele and genotype distributions in the HC and D subjects were in Hardy-Weinberg equilibrium (pvalue>0.05). Table 3 shows haplotype frequencies in HC, D and DN groups. We observed that the frequency of carrying haplotype with CCC alleles for all three variants was significantly higher in DN subjects 6
ACCEPTED MANUSCRIPT compared to HC group and frequency of carrying TCC haplotype was significantly higher in DN group compared to both HC and D groups (Table 3).
Discussion The present case-control study was conducted to examine the relationship between VDR gene
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polymorphism (rs7975232 C>A, rs731236 T>C and rs4516035 T>C) and risk of DN in a group of type 2 diabetic patients. No significant differences were identified in allele/genotype frequencies between HC, D
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and DN groups. However, haplotype analysis showed that two haplotype (CCC, TCC) were significantly
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more frequent in DN group.
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It has been demonstrated that VDR mediates the effect of vitamin D by making a heterodimer with the retinoid X receptor molecule which is regulating the expression of many target genes involved in
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enormous disease mechanisms including inflammatory, oxidative stress and etc [32]. Previous studies proved that VDR gene polymorphisms have functional effect on VDR activity and
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protein expression. Functional studies have revealed that rs4516035 variant is located at a GATA binding
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site causing decreased promoter activity of VDR gene in individuals carrying allele C while allele T increase the promoter activity to 1.9 fold [33].
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rs7975232, rs731236 variants are located in the 3′UTR of the VDR genes and have been shown to have a role in mRNA stability and therefore regulating gene expression [34].
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There is a growing interest in potential importance of vitamin D and VDR gene in diabetes mellitus but the results are controversial. In a case-control study in a group of southeastern Iranian diabetic patients reported by Nosratabadi et al[18] a significant association was found for Taq1 polymorphism genotype frequencies in diabetes while in another study with higher sample size by Shab-Bidar et al, no evidence for the association between VDR gene polymorphisms (FokI, BsmI, ApaI, and TaqI) and the risk for type 2 diabetes and metabolic syndrome were found[35].The study by Aslani et al., in Iran, found a significant association between VDR FokI polymorphism and gestational diabetes [20]. Studies in Turkish, Tunisian, 7
ACCEPTED MANUSCRIPT Brazilian and polish patients failed to show a significant correlation between VDR gene polymorphism and diabetes [14-17]. There are few studies about association of VDR gene variation and diabetes complications. In a study on post-menopausal diabetic women no significant association was found between TaqI and FokI polymorphisms and macro and microvascular complications [16] while in another case-control study,
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Vedralováet al identified significant association between FokI variant and DN[36].In a study in Iran by Soroush et al., a significant association was observed between VDR FokI functional variant and diabetic
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foot ulcer [21].
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A research on diabetes type 1 patients from GoKinD study (Genetics of Kidneys in Diabetes) in UK,
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showed no significant association with genotype or allele frequencies of VDR gene variants but they suggested that haplotype AGT (rs1544410, rs7975232 and rs731236 markers) was protective against
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DN in type 1 diabetes.
Reno-protection of vitamin D has proposed by many authors [37], but there are few studies about VDR
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gene variants and diabetic nephropathy and many of them have failed to show a correlation between VDR
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gene variation (ApaI, BsmI, FokI and TaqI) and DN in different population[38]. Diversity between various study results can be related to heterogeneity of DN, genetic or nutrition and also climate
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difference in different countries.
Small samples size and studying only three variants of VDR gene were the main limitations of present
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study. Lack of matching for age and sex, DN onset age and medication used are other limitations of current study. This study was conducted in a group of diabetic patients and replication in a greater sample size is required to confirm the findings of present study. Future studies examining other variants in VDR gene is also recommended. In conclusion, the results of current study demonstrated that some VDR genetic variants as haplotype combination might be protective against DN development while CCC and TCC haplotypes encode risk for DN development. 8
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ACCEPTED MANUSCRIPT Table 1: Demographic characteristics and biochemical parameters in three study groups
HC group
D group
DN group
p-Value
p-Value
p-Value
(n=109)
(n=100)
(n=104)
HC-D
HC-DN
D-DN
Mean (SD)
Mean (SD)
Mean (SD)
Age (year)
50.5(10.7)
57.5(8.2)
62.4(9.2)
SBP (mmHg)
115.9(12.7)
122.8(12.4)
123.7(13.7) 0.001
DBP (mmHg)
79.2(5.9)
78.9(6.3)
75.5(9.7)
BMI (kg/m2 )
27.4(4.0)
28.5(4.2)
29.7(4.9)
-
10.8(6.2)
GFR (mL/min/1.73m2)
75.5(30.0)
FBS (mg/dL)
Variables
0.001
0.001
1
1
0.001
0.004
0.270
0.002
0.196
12.5(7.0)
-
-
0.065
76.7(20.0)
64.4(29.7)
1
0.011
0.004
91.4(7.7)
140.2(34.6)
133.8(45.9) 0.001
0.001
0.525
HbA1c (%)
5.5(0.46)
7.1(0.74)
7.4(0.74)
0.001
0.001
0.006
Cr (mg/dL)
1.0(0.16)
1.11(0.26)
1.28(0.48)
0.036
0.001
0.001
Urea (mg/dL)
29.5 (7.4)
36.7(13.0)
42.6(17.0)
0.001
0.001
0.005
UA (mg/dL)
4.9(2.8)
5.2(1.4)
5.4(1.8)
0.958
0.453
1
59.3 (31.1)
73.0(48.9)
59.1(30.3)
0.379
1
0.142
24.4(6.0)
246(666)
1
0.001
0.001
RI
SC
NU
D
PT E
CE
AC
ACR (mg/g)
MA
(year)
25(OH)D (nmol/L)
PT
0.001
Diabetes Duration
23.5(8.0)
0.001
Data are presented as mean (SD) Abbreviation: HC: healthy controls, D: diabetes without nephropathy, DN: diabetic nephropathy, SBP: systolic blood pressure, DBP: diastolic blood pressure, BMI: body mass index, GFR: glomerular filtration rate, FBS: fasting blood sugar, Cr: creatinine, UA: uric acid, 25(OH) D : 25 hydroxyvitamin D, ACR: urine albumin to creatinine ratio
14
ACCEPTED MANUSCRIPT
Genotype/
HC=109
D=100
DN=104
N(%)
N(%)
N(%)
16(16.0)
23(22.1)
Allele
OR
p-Value HC-D
p-Value
HC-D
HC-DN
OR HC-DN
p-Value D-DN
OR D-DN
rs731236 Taq1
TT
20 (18.3)
1.00
1.00
1.00
(reference)
(reference)
(reference)
1.51 45(41.3)
51(51.0)
45(44.6)
0.18
(0.82-2.76)
0.37
PT
0.71
CT
0.61 0.20
1.07 CC
44(40.4)
33(33.0)
36(34.6)
0.71
85(39.0)
83(41.5)
91(43.8)
NU
(reference)
0.90
133(61.0)
117(58.5)
117(56.3)
0.60
MA
C
T
C
16(14.6)
131(60.1)
45(45)
16(16)
87(39.9)
30(28.8)
PT E
CC
55(50.5)
39(39)
54(51.9)
20(19.2)
CE
CT
38(34.9)
123(61.5)
AC
TT
77(38.5)
D
rs4516035
0.76 0.50
(0.42-1.80)
SC
1.00
(0.29-1.30)
0.87
0.87 (0.48-2.37)
T
RI
(0.34-1.49)
(0.34-1.68) 1.0
1.00 (reference) (reference)
0.82
0.65
0.91
0.32
(0.6-1.33)
(0.56-1.21)
(0.61-1.35)
1.00
1.00
1.00
(reference)
(reference)
(reference)
0.8
0.46
1.58 0.27
(0.43-1.44)
(0.70-3.57)
0.97
0.95
1.62 0.24 (0.72-3.66)
1.24 0.48
1.56 0.16
(0.43-2.22)
(0.68-2.28)
(0.84-2.90)
1.00
1.00
1.00
(reference)
(reference)
(reference)
114(54.8)
0.94 94(45.2)
0.77
1.24 0.27
1.31 0.17
(0.64-1.39)
(0.85-1.82)
(0.89-1.95)
1.00
1.00
1.00
(reference)
(reference)
(reference)
rs7975232ApaI
CC
16 (14.7)
21 (21.0)
22 (21.2)
0.54 AC
52 (47.7)
50 (50.0)
50 (48.1)
0.13
0.57 0.16
(0.24-1.2)
15
1.05 0.89
(0.26-1.25)
(0.51-2.14)
ACCEPTED MANUSCRIPT 0.73 AA
C
41 (37.6)
84 (38.5)
29 (29.0)
92 (46.0)
32 (30.8)
0.70
0.421
0.35 (0.34-1.56)
(0.33-1.48)
(0.44-2.07)
1.00
1.00
1.00
(reference)
(reference)
(reference)
94 (45.2)
0.74 A
134 (61.5)
108 (54.0)
0.95 0.89
114 (54.8)
0.76
0.12
0.16 (0.49-1.07)
(0.52-1.11)
AC
CE
PT E
D
MA
NU
SC
RI
PT
Table 2: Genotype and allele frequencies in three study groups
16
1.03 0.87 (0.70-1.52)
ACCEPTED MANUSCRIPT Table 3: Comparison of haplotypes frequencies of VDR gene polymorphisms between three study groups
Haplotypes
D (2N=200)
DN (2N=208)
HC (2N=218)
(0.109173)
(0.084751)
(0.119266)
(0.025827)
(0.040249)*
(0.000000)*
(0.244357)
(0.225791)
PT
rs731236/ rs4516035/
A
CCC C
T
A
(0.218734)
C
T
C
(0.261266)
T
C
A
T
C
T T
SC
C
NU
C
RI
rs7975232
(0.255861)
(0.025000)
(0.049864)
(0.064220)
C
(0.005000)&
(0.046290)&#
(0.000000)#
T
A
(0.187092)
(0.169104)
(0.205402)
T
C
(0.167908)
(0.186665)
(0.129460)
AC
CE
PT E
D
MA
(0.178720)
*P- value: 0.01, OR: 8.7 (1.1-388.5) # P-value: 0.004, OR: 10.0 (1.4-438.2) & P-value: 0.007, OR: 10.9 (1.5-477.6)
17
ACCEPTED MANUSCRIPT Abbreviation list ESRD: end stage renal diseases ACR: albumin/creatinine ratio VDR: vitamin D receptor
PT
DN: Diabetic Nephropathy
AC
CE
PT E
D
MA
NU
SC
RI
MAP: Micro Albuminuria Prevalence
18