Abstract
Back ground and objectives: Vitamin D receptor (VDR) gene polymorphism is reported to be associated with end-stage renal disease (ESRD). We have investigated the potential role of VDR gene polymorphisms among ESRD. Design and methods: The influence of VDR gene polymorphism in 258 ESRD patients comprising of 226 (87.5%) male and 32 (12.5%) females was investigated in this study. We compared ESRD patients with 569 healthy controls. The distribution of male and female among controls was 485 (85.3%) males and 84 (14.7%) females. This polymorphism was studied by using polymerase chain reaction (PCR). The product was digested by using restriction enzymes Apa1, Taq1, Fok1, and Bsm1. Results: We observed a significant difference in the genotype frequencies of the Apa1-aa (p = 0.0001, OR = 2.1, 95% CI = 1.45–3.08), Fok1-ff (p = 0.001, OR = 3.44, 95% CI = 1.76–6.76), and Bsm1-BB (p = 0.0004, OR = 6.8, 95% CI = 2.2–21.58). At allelic level B allele of Bsm1 was significantly different among ESRD patients as compared to controls (p = 0.0001). The combined analysis revealed that ESRD patients with Fok1 and Bsm1 polymorphism were at increased risk of 4.33-fold. The haplotype analysis revealed individuals with a/t/F/b haplotype were at greater risk of 11.0-fold (95% CI = 1.38–87.69). The serum calcium levels were significantly higher (p = 0.001) in Bsm1 “BB” genotype. Interpretations and conclusions: Bsm1 and Fok1 gene polymorphism of VDR gene were associated with ESRD among north Indians.
INTRODUCTION
Several common polymorphisms in the vitamin D [1,25(OH)2D3] receptor (VDR) gene have been reported. It is reported to be involved in calcium absorption, excretion, and modulation of cellular proliferation and differentiation. The functional significance and potential effect on disease susceptibility have been investigated in different diseases like prostate cancer, urolithiasis, and osteoporosis. However, there are very few reports related to Apa1, Taq1, Fok1, and Bsm1 polymorphisms of VDR gene among end-stage renal disease (ESRD) patients.
At the 3′-end of the VDR gene, a Bsm1 restriction fragment length polymorphism (RFLP) is found, which is strongly associated with other polymorphisms, including Apa1 and Taq1.Citation1,Citation2 These polymorphisms produce no coding region differences and thus do not change the structure of the protein. However, a translation initiation codon polymorphism, Fok1, has been identifiedCitation3 that is not in linkage disequilibrium (LD) with other VDR polymorphisms.Citation4 There are some studies which showCitation5–7 that the shorter truncated version “F” allele is responsive to 1,25(OH)2D3.Citation7 This F allele may have greater transcriptional activity.Citation6 The F allele is found to be associated with greater lumbar bone mineral density in several European-descent populations.Citation3
It has been reported that ESRD patients show abnormal metabolism of calcium,Citation7 which may lead to secondary hyperparathyroidism. Excess of parathyroid hormone (PTH) in secondary hyperparathyroidism accelerates bone turnover, causing the high turnover bone disease known as osteitis fibrosa. Activation of bone resorption results into the elevated level of calcium that inhibits intact PTH (iPTH) and 1,25(OH)2D3 synthesis and calcium absorption. This causes calcium deficiency and secondarily stimulates iPTH synthesis to restore calcium balance. Vitamin D3 regulates calcium and phosphate homeostasis; hence, genetic variant of VDR may be helpful in assessing bone metabolism.Citation8 In this study we have investigated whether allelic differences in the VDR gene polymorphisms (Apa1, Taq1, Fok1, and Bsm1) are associated with ESRD, and also whether allelic differences in the VDR gene could determine differences in parathyroid cell function which may be partially responsible for the wide variation in the degree of secondary hyperparathyroidism observed in ESRD patients.
MATERIALS AND METHODS
A total number of 258 ESRD patients [male = 226 (87.5%), female = 32 (12.5%)] were included in the study. They were on regular follow-up in the Department of Nephrology, a superspecialty center at Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow. The inclusion criteria for patient selection were constantly elevated serum creatinine level above normal range (ranging from 3.4 mg/dL to 15.8 mg/dL) and creatinine clearance <15 mL/min/1.73 m2 and were recommended for renal transplantation. All the patients were on hemodialysis. The type of chronic kidney disease was established by doing ultrasound and/or computed tomography (CT) scan of the kidneys followed by histopathological evaluation of the renal biopsy specimen. Patients were categorized on the basis of the histopathological subtypes chronic glomerulonephritis (CGN = 168), chronic interstitial nephritis (CIN = 68), hypertensive nephrosclerosis (HN = 15), and polycystic kidney disease (PKD = 7). Patients with a history of diabetes mellitus, or receiving corticosteroids, vitamin D, or vitamin D derivatives, were excluded from this study. Serum iPTH concentration was measured by a two-site chemiluminometric assay only in 136 ESRD patients and 85 controls. Serum levels of calcium, phosphorus, creatinine, and CCr were measured simultaneously in each subject.
Five hundred and sixty-nine [male = 485 (85.3%), female = 84 (14.7%)] healthy, age- and sex-matched north Indians from the same geographic areas were selected as controls. Controls with risk factors like family history of hypertension, diabetes mellitus, and hyperlipidemia were excluded from the study. Controls were free from any renal impairment based upon the proteinuria, blood urea nitrogen levels, and creatinine clearance. Both patients and controls were from the state of Uttar Pradesh. An informed consent was obtained from both patients and controls. The study was approved by the ethical committee of SGPGIMS.
BLOOD COLLECTION, DNA EXTRACTION, AND GENOTYPING
Blood samples for measuring the serum biochemical parameters were obtained in the morning after 8 h of fasting. Genomic DNA was obtained using genomic DNA extraction kit from Quiagen (Brand GMbH and Co KG, Cat #51104).
The genotyping was performed by using polymerase chain reaction (PCR) RFLP technique. The genotyping for the detection of ApaI (db SNP ID rs7975232), TaqI (db SNP ID rs731236), FokI (db SNP ID rs10735810), and BsmI (db SNP ID rs1544410) was according to Vupputuri et al.Citation9
To improve the genotyping quality, ∼15% of the samples (both in the patient and control groups) were confirmed by DNA sequencing (Applied Biosystems, Foster City, California, USA).
STATISTICAL ANALYSIS
Allele frequencies were calculated as the number of occurrences of the test allele in the population divided by the total number of alleles. The carriage rate was calculated as the number of individuals carrying at least one copy of the test allele divided by the total number of individuals. Synergistic effect of VDR gene polymorphisms was evaluated by using logistic regression analysis through SPSS software (version 11.5). p-Values less than 0.05 were considered statistically significant. Yates correction was applied wherever required. Allele frequencies of both patients and controls were tested for Hardy–Weinberg equilibrium. Haplotypes were constructed using Arlequin software (version 3.1).
LD was calculated using Haploview v 3.11 program (http://www.broad.mit.edu/mpg/haploview/index.php).
RESULTS
Demographic profile and clinical characteristics of patients and controls
The demographic profile and clinical characteristics of ESRD patients and controls is shown in . The male-to-female ratio in each group was comparable (87.5:12.5 in patients, as compared to 85.3:14.7 in controls). The controls were age- and sex-matched with patients; the mean age of the patients was 34.2 years as compared to the mean age of controls (35.8 years). Two most important renal function parameters, that is, serum creatinine level and proteinuria, showed highly significant differences (p < 0.0001) between patient and controls except serum triglyceride and serum very-low-density lipoprotein (VLDL) values.
TABLE 1. Demographic profile and clinical characteristics of ESRD patients and controls
Distribution of VDR genotypes
Different genotypic, allele, and allele carriage frequencies for VDR are shown in . Allele frequencies in patients and controls were in Hardy–Weinberg equilibrium. The aa and AA genotype of Apa1 in patient's group was 24.1% and 31.0%. Among controls it was 13.0% and 30.0%, respectively, and both the groups differed significantly (p = 0.0001; OR = 2.1, 95% CI = 1.45–3.08). In case of Taq1, the TT genotype was present in 40.7% and tt was observed only in 14.7%, which was comparable to controls and was not significantly associated with ESRD (p = 0.128). We observed that the ff genotype of Fok1 was present in 8.5% of the ESRD patients while in control it was present only in 2.6% of the individuals and both the groups differed significantly (p = 0.0003; OR = 3.44, 95% CI = 1.76–6.76) (). We observed that the mutant BB genotype of Bsm1 polymorphism was higher (4.6%) in ESRD patients while in control it was only 0.7%. Our results demonstrated that ESRD patients revealed strong association with the BB genotypes and also with the B allele (p = 0.0004; OR = 6.89, 95% CI = 2.2–21.58 and p = <0.0001; OR = 2.25, 95% CI = 2.2–21.58) (). The allele carriage frequency of Bsm1 in the ESRD group was statistically different as compared to control ().
TABLE 2. Distribution of VDR among patients and healthy control group
We compared all the studied polymorphisms in ESRD patients with different types of primary kidney diseases, that is, CGN, CIN, HN, and PKD. None of the genotypes or allele frequencies was statistically different among different groups of ESRD patients (). ( describes the difference of frequency of genotype/allele between the subgroups CGN, CIN, HN, PKD, and the * are denoted for comparison between subgroup (CGN and CIN) and control. We have not done any comparison with the subgroup HN = 15, PKD = 7 with controls because of the small number of the sample size and to avoid false significant p-values.) Further, on comparing various kidney diseases with 569 healthy controls, we observed significant association at allelic and genotypic level of Apa1 and Bsm1 gene polymorphisms.
TABLE 3. VDR genotype and allele frequency distribution among primary kidney disease patients and normal controls
Combined analysis, haplotype distribution, and linkage disequilibrium
To evaluate the synergistic effect, the genotypes of VDR genes were combined and compared in both the groups. The heterozygous and mutant types were combined together. The combined genotypes showed significant difference in the frequency distribution of patients and controls (). In double gene combination, ESRD patients with a combination of Fok1 and Bsm1 showed the highest risk, 4.33-fold (p = <0.0001, 95% CI = 2.88–6.53); however, it was reduced to ∼2.3-fold (p = <0.0001, 95% CI = 1.58–3.27) in triple gene combination. Further, the significance was reduced when all the polymorphisms were taken together.
TABLE 4. Combined analysis of VDR genotypes among ESRD patients and controls
The haplotype analysis () revealed that a/t/F/b haplotype is comparatively higher in patients (9.8%) as compared to the controls (1.3%) and the differences were statistically significant (p = 0.0131; OR = 11.0; 95% CI = 1.38–87.69). We have avoided any calculations for haplotypes which are less frequent than 5%. Strong LD was observed for three polymorphisms, Bsm1, Apa1, and Taq1, localized at 3′-end of the VDR gene. The strongest LD (D′ = 1.0) was observed for Bsm1 and Apa1 and weaker for Apa1 and Taq1 (D′ = 0.87).
TABLE 5. Haplotype distribution of Apa1, Taq1, Fok1, and Bsm1 gene polymorphism among ESRD patients and control groups
The plasma levels (mean ± SD) of iPTH, serum calcium, and serum phosphorous in ESRD patients according to VDR genotypes are shown in . We observed no correlation of serum phosphorous in ESRD patients according to VDR genotypes. The mean iPTH level in ESRD was higher than that in control subjects (p > 0.05). The carrier of BB and ff genotypes of Bsm1 and Fok1 had lower iPTH level than those with bb and FF genotypes; however, the differences were not significant. Upon performing the R-statistics we observed that two-tailed p-value was non-significant between iPTH level and Apa1, Taq1, Fok1, and Bsm1 polymorphism of VDR gene among ESRD patients. The serum calcium levels were increased in patients with Bsm1 non-bb (Bb + BB) variants, but were significantly decreased in the bb variants (9.6 ± 1.17 vs. 9.08 ± 0.87 mg/dL, p = 0.001) (). We correlated the serum calcium levels with VDR gene polymorphism by using Pearson's correlation coefficient and observed that Bsm1 was inversely correlated with calcium levels (r = −0.1347, p = 0.348).
TABLE 6. Biochemical parameter of patients (n = 136) with VDR gene polymorphism
DISCUSSION
This study deals with the potential role of VDR gene polymorphism for susceptibility to ESRD and their influence on parathyroid response in hemodialysis patients with ESRD. There are some reports where this gene polymorphism has been investigated in different malignancies among the Indian populationCitation10–12; however, there is no study on ESRD patients from India. Our results showed that Apa1, Fok1, and Bsm1 gene polymorphism may be one of the genetic risk factors for ESRD among north Indian population.
We have observed that the genotype frequency distribution of Apa1 (a), Fok1 (f), and Bsm1 (B) was significantly different among patients and control groups. No significant difference was observed in case of t at Taq1 restriction site. When two high-risk genotype f of Fok1 and B of Bsm1 were combined, we found that the risk increased to 4.33-fold.
Differentiation of VDR genotypes may trigger breakdown of the cytokine relationship directly or indirectly and may be associated with the pathogenesis of the ESRD, or it may cause alteration of the VDR structure, ultimately leading to altered receptor function, which may increase or decrease the expression of VDR protein thereby causing the disease. The VDR gene contains two potential translation initiation (ATG) sites.Citation13 The Fok1 polymorphism, which occurs at the first start codon in exon 2, changes the nucleotide sequence to ACG.Citation14 Alleles with this polymorphism initiate translation three codons downstream, resulting in a protein (424 amino acids, the F allele) which is three amino acids shorter than wild type and is considered to be more active.Citation15 This is the only known protein polymorphism in the VDR gene.Citation15 In our study we observed that the ff genotype of Fok1 was present in 8.5% of the ESRD patients, while in controls it was found in 2.6% of the individuals and both the groups differed significantly (p = 0.0003). The carriers of ff genotypes were at a higher risk of developing ESRD; this may be due to truncated protein levels and hence a less efficient vitamin D receptor. In several cancers it has been shown that exposure to higher than median vitamin D was able to completely overcome the risks.Citation12 The same mechanisms may also hold true for ESRD.
Though the Bsm1 and Apa1 polymorphism lie in the intronic region, these polymorphisms may influence the expression of VDR which includes the disruption of a splice site for VDR mRNA transcription, hence resulting into the truncated or alternatively spliced protein productCitation16 or these may alter the mRNA product.Citation17 We have observed BB genotypes of Bsm1 were strongly associated with ESRD, indicating production of truncated protein and hence less binding with the vitamin D receptor, which may lead to ESRD. The Taq1 polymorphism may cause a silent mutation in exon 9, with ATT and ATC both coding for isoleucine,Citation18 thus not affecting the mRNA transcripts. Such types of associations have been reported in type 2 diabetes from north India.Citation19
The combined analysis revealed that ESRD patients with Fok1 and Bsm1 polymorphism were at increased risk of 4.33-fold. The haplotype analysis revealed individuals with a/t/F/b haplotype were having a higher risk of 11.0-fold.
VDR polymorphism has been shown to modify parathyroid cell differentiation and function. In ESRD patients, there are wide variations in the degree of secondary hyperparathyroidism. Whereas some patients develop severe and uncontrollable hyperparathyroidism, others develop only modestly elevated iPTH levels that fail to promote adequate bone turnover and finally result in a dynamic bone disease.Citation20 The reason for this heterogeneity in the clinical behavior is not well defined. However, it has been reported that Bsm1 is associated with low bone mineral density (BMD), risk of osteoporotic fractures, and tendency for hyperparathyroidism in general and among patients undergoing hemodialysis.Citation21 The carrier of BB and ff genotypes of Bsm1 and Fok1 had a lower iPTH level than those with bb and FF genotypes in the limited number of patients that we have studied. The serum calcium levels were significantly higher (p = 0.001) in the BB genotype of Bsm1, showing inverse correlation, which may act as a secondary check to the severity of secondary hyperparathyroidism.
Conclusively, VDR gene polymorphisms appear to be important genetic determinants found to be associated with ESRD. We observed that aa of Apa1, ff of Fok1, and BB of Bsm1 were strongly associated with ESRD among north Indians. No association was found at Taq1 polymorphism among ESRD patients. However, further studies are required on VDR polymorphisms as there are no data available from the Indian subcontinent.
Acknowledgments
The authors are thankful to P.B. Vinod for clinical details and Dr. Minal Borkar for haploview analysis. G. Tripathi is thankful to Jawaharlal Nehru Memorial Fund, New Delhi, for awarding junior research fellowship. There are no competing interests of the authors.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Related Research Data
REFERENCES
- Whitfield GK, Remus LS, Jurutka PW, Functionally relevant polymorphisms in human nuclear vitamin D receptor gene. Mol Cell Endocrinol. 2001;177:145–159.
- Ingles SA, Haile RW, Henderson BE, Strength of linkage disequilibrium between two vitamin D receptor markers in five ethnic groups: Implications for association studies. Cancer Epidemiol Biomarkers Prev. 1997;6:93–98.
- Gross C, Eccleshall TR, Malloy PJ, The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women. J Bone Miner Res. 1996;11:1850–1855.
- Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene. 2004;338:143–156.
- Whitfield GK, Remus LS, Jurutka PW, Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. Mol Cell Endocrinol. 2001;177:145–159.
- Colin EM, Weel AE, Uitterlinden AG, Consequences of vitamin D receptor gene polymorphisms for growth inhibition of cultured human peripheral blood mononuclear cells by 1,25-dihydroxyvitamin D3. Clin Endocrinol (Oxford). 2000; 52:211–216.
- Lomonte C, Vernaglione L, Chimienti D, Does vitamin D receptor and calcium receptor activation therapy play a role in the histopathologic alterations of parathyroid glands in refractory uremic hyperparathyroidism? Clin J Am Soc Nephrol. 2008;3:794–799.
- Kisakol G, Kaya A, Gonen S. Bone and calcium metabolism in subclinical autoimmune hyper and hypothyroidism. Endocr J. 2003;50:657–661.
- Vupputuri MR, Goswami R, Gupta N, Prevalence and functional significance of 25-hydroxyvitamin D deficiency and vitamin D receptor gene polymorphisms in Asian Indians. Am J Clin Nutr. 2006;83:1411–1419.
- Vidyarani M, Selvaraj P, Raghavan S, Narayanan PR. Regulatory role of 1,25-dihydroxyvitamin D3 and vitamin D receptor gene variants on intracellular granzyme A expression in pulmonary tuberculosis. Exp Mol Pathol. 2009;86:69–73.
- Alagarasu K, Selvaraj P, Swaminathan S, Narendran G, Narayanan PR. 5′ Regulatory and 3′ untranslated region polymorphisms of vitamin D receptor gene in south Indian HIV and HIV-TB patients. J Clin Immunol. 2009; 29:196–204.
- Onsory K, Sobti RC, Al-Badran AI, Hormone receptor-related gene polymorphisms and prostate cancer risk in north Indian population. Mol Cell Biochem. 2008;314:25–35.
- Baker AR, McDonnell DP, Hughes M, Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA. 1988;85:3294–3298.
- Saijo T, Ito M, Takeda E, A unique mutation in the vitamin D receptor gene in three Japanese patients with vitamin D dependent rickets type II: Utility of single-strand conformation polymorphism analysis for heterozygous carrier detection. Am J Hum Genet. 1991;49:668–673.
- Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene. 2004;338:143–156.
- Nesic D, Cheng J, Maquat LE. Sequences within the last intron function in RNA 3′-end formation in cultured cells. Mol Cell Biol 1993;13:3359–3369.
- Holick MF. Vitamin D. Importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am J Clin Nutr. 2004;79:362–371.
- Marco MP, Martinez I, Amoedo ML, Vitamin D receptor genotype influences parathyroid hormone and calcitriol levels in predialysis patients. Kidney Int. 1999;56:1349–1353.
- Bid HK, Konwar R, Aggarwal CG, Vitamin D receptor (FokI, BsmI and TaqI) gene polymorphisms and type 2 diabetes mellitus: A north Indian study. Indian J Med Sci. 2009;63:187–194.
- Ott SM. Bone density in patients with chronic kidney disease stages 4–5. Nephrology. 2009;14:395–403.
- Karkoszka H, Chudek J, Strzelczyk P, Does the vitamin D receptor genotype predict bone mineral loss in hemodialysed patients? Nephrol Dial Transplant. 1998;13:2077–2080.