Association of VEGF -2549 I/D and VEGF +936 C/T Polymorphisms with Chronic Kidney Disease in North-West Indian Patients

Selection of subjects and collection of genetic material

The present case-control study was carried out after receiving approval from the Institutional Ethics Committee. The physician identified 166 unrelated adult CKD stage V patients (112 males and 54 females) on or starting hemodialysis therapy who were contacted from local hospitals of Amritsar, Punjab. Related, minors, and individuals who were seropositive for hepatitis C or B or HIV or had bacterial infections or any cancer, were not included in this study. Unrelated, healthy, age and gender-matched 166 individuals (107 males and 59 females) from the general population belonging to same geographical area formed the control group. The eGFR values based on the creatinine levels were used to establish the healthy kidney status of the controls. The demographic characteristics, clinical profile and disease history of all the subjects were recorded on a pre-designed structured proforma. After informed written consent, 5 ml venous blood was collected from each subject in EDTA vial.

Analysis of VEGF-2549 I/D and VEGF +936 C/T polymorphisms

Genomic DNA was extracted from the peripheral blood using standard phenol chloroform method[21] with few modifications. The DNA samples were quantified on 1% agarose gel and screened for VEGF-2549I/D and VEGF +936 C/T polymorphisms. The VEGF-2549I/D promoter polymorphism was screened by direct PCR whereas 3'UTR polymorphism VEGF +936 C/T was screened usingpolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The region of VEGF promoter harboring -2549I/D polymorphism was amplified using Forward: 5′-GCTGAGGATGGGGCTGACTAGGTA-3′ and Reverse: 5′GTTTCTGACCTGGC TATTTCCAGG-3′primers. The PCR reaction with a total volume of 10μl, contained 50ng of genomic DNA, 1X Taq buffer with 1.5mM MgCl₂ (Merck, India), 4 picomoles of each primer (Sigma), 0.4μl of dNTPs mix (Merck, India), and one unit of Taq polymerase (Merck, India). For VEGF -2549I/D analysis, PCR conditions were the following: initial denaturation at 95°C for 5 minutes followed by 35 cycles with denaturation at 95°C for 45 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 45 seconds, and final extension at 72°C for 10 minutes. The amplified products were analyzed on 2.4% ethidium bromide-stained agarose gel. A band of 229 bp represents I allele whereas band of 211bp represents D allele[Figure 1].

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Figure 1

A photograph of 2.4% ethidium bromide –stained agarose gel demonstrating VEGF -2549I/D polymorphism. Lane 1, 4, 5 represents ID genotype, Lane 2, 6, +ve control represents DD genotype and Lane 3, 7 represents II genotype

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The specific region of VEGF containing +936 C/T polymorphism was amplified using Forward: 5′-AGGAAGAGGAGACTCTGCGCAGAGC-3′ and Reverse: 5′-TAAATGTATGTATGTGGG TGGGTGTGTCTACAGG-3′ primers. PCR reaction with a total volume of 15μl, contained 50ng of genomic DNA, 1X Taq buffer, 1mM MgCl₂ (Merck, India), 6 picomoles of each primer (Sigma), 0.2μl dNTPs mix (Merck, India) and one unit of Taq polymerase (Merck, India). The amplification conditions used were: initial denaturation at 95°C for 5 minutes, followed with a denaturation at 95°C for 45 seconds, annealing at 59°C for 30 seconds and an extension at 72°C for 45 seconds for 35 cycles with final extension at 72°C for 10 minutes. The amplified PCR products of 217bp were digested with NlaIII restriction enzyme (New England Biolabs, Beverly, MA) at 37°C overnight. Restriction digestion reaction products were analyzed on 2.4% ethidium bromide–stained agarose gel. Two fragments of 122bp and 85bp indicates +936T allele, whereas the undigested fragment of 207bp represents +936C allele [Figure 2]. The details of reaction composition and conditions used have been presented in our published study.[22] To ensure genotyping accuracy, positive and negative controls were used in every batch of reaction. The PCR assay-based results were validated by reanalyzing the 10% of randomly selected samples.

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Figure 2

A photograph of 2.4% ethidium bromide–stained agarose gel demonstrating restriction digestion pattern of VEGF +936C/T polymorphism. Lane 1, 3, 5 represents CC genotype, Lane 2, 4 represents CT genotype and +ve control represents TT genotype

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Statistical analyses

The data on variables are expressed as number or as percentage, and as mean ± standard deviation. Differences between the patient and control groups were analyzed by independent Student's t test while multiple comparisons were carried out using one-way ANOVA. The allele frequencies were tested for the Hardy–Weinberg equilibrium (HWE) for both patients and controls using the Chi-square test. This test was also used to evaluate the differences in the VEGF genotype and allele frequencies between the patient and control groups, and also as a function of various general, demographic, and clinical parameters. Odds ratio (OR) and its 95% confidence interval (CI) were used to assess the association between genotypes and alleles with the disease risk. Haplotypes were constructed using SNPStats.[23] Statistical significance level was set at P < 0.05.

General demographic and clinical profile of subjects

The patient group (n = 166) comprised of unrelated patients in CKD stage V in the age range of 18–80 years (mean = 50.87 ± 13.36 years) with high frequency of males (67.47%). Mean age of unrelated healthy individuals was 48.24 ± 12.23 years [Table 1]. About 74.69% of the patients were on hemodialysis therapy for 8 months to 6 years. Ongoing hemodialysis therapy comprised of groups on once-a-week (23.49%), twice-a-week (25.30%), thrice-a-week (12.05%), and fortnightly (15.66%) regimens with 25.30% yet to initiate hemodialysis. Serum creatinine (7.37 ± 3.37 mg/dl) and urea (125.46 ± 34.90 mg/dl) levels were elevated in the patients. Patients were on prescribed medications from past 9.24 ± 0.70 years.

VEGF-2549 I/D and VEGF +936 C/T polymorphisms and disease risk

Genotype and allele frequencies of VEGF-2549 I/D and VEGF +936 C/T polymorphisms in patient and control groups are detailed in Table 2. The genotype distributions of VEGF -2549 I/D and VEGF +936 C/T polymorphisms were in HWE (P > 0.05; except VEGF -2549 I/D in controls). The frequency of DD (33.73 vs 22.29%), ID (46.99 vs 60.84%) and II (19.28 vs 16.87%) genotypes of VEGF -2549 I/D was significantly different between patients and controls (P < 0.05). ID genotype of VEGF -2549 I/D polymorphism was significantly associated with decreased risk of CKD (P = 0.009). The frequency of CC (82.53% vs 83.13%) and CT (16.27% vs 16.87%) genotypes of VEGF +936 C/T polymorphism was not significantly different between the patient and control groups. The variant genotype TT was only observed in the patient group (1.20%). The frequency of T allele was slightly higher in patients (9.34%) compared to controls (8.43%).

The genetic model analysis of VEGF -2549 I/D polymorphism revealed a significant decreased risk of CKD in co-dominant (OR: 0.510, 95% CI: 0.307–0.849; P = 0.009), dominant (OR: 0.563, 95% CI: 0.346-0.917; P = 0.021) and over-dominant (OR: 0.570, 95% CI: 0.369–0.882; P = 0.012) models [Table 3]. However, none of the genetic models of VEGF +936 C/T polymorphism showed any significant disease association.

The frequency of distribution of VEGF -2549 I/D and VEGF +936 C/T polymorphisms as a function of general demographic (age, gender) and disease-specific variables showed no association with the disease (data not shown). Genotype combination analysis [Table 4] showed that ID genotype of VEGF -2549 I/D and CT genotype of VEGF +936 C/T polymorphisms was associated with decreased CKD risk (OR: 0.450, P = 0.047). Haplotypes constructed for VEGF-2549 I/D and VEGF +936 C/T polymorphisms did not reveal any significant disease-risk association [Table 4]. Analysis of association of VEGF -2549 I/D and VEGF +936 C/T polymorphism with creatinine level showed non-significant results (data not shown).

Financial support and sponsorship

This study is partially supported by the MHRD grant under RUSA scheme sanctioned to Kamlesh Guleria and Vasudha Sambyal. Postdoctoral fellowship to Gurleen Kaur Tung under RUSA scheme is dully acknowledged. The clinical classification of patients by late Dr. PS Mokha, Mokha Hospital and Kidney Care Centre Amritsar is gratefully acknowledged.

Conflicts of interest

There are no conflicts of interest.