Indian J Pediatr DOI 10.1007/s12098-015-1783-6
ORIGINAL ARTICLE
Influence of Vitamin D Receptor Gene Fok1 Polymorphism on Bone Mass Accrual Post Calcium and Vitamin D Supplementation Neha Sanwalka 1 & Anuradha Khadilkar 2 & Shashi Chiplonkar 2 & Kavita Khatod 3 & Nikhil Phadke 3 & Vaman Khadilkar 2
Received: 15 December 2014 / Accepted: 27 April 2015 # Dr. K C Chaudhuri Foundation 2015
Abstract Objective To examine the association of vitamin D receptor (VDR) gene polymorphisms of the Fok1 locus on bone mass accrual in Indian girls used to a low calcium intake. Methods An intervention trial was undertaken in 102 girls aged 8–16 y, attending a state run school in Pune city, India. All girls received 500 mg calcium daily and 30,000 IU of vitamin D3 quarterly for one year. Dietary calcium intake was evaluated. Bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) were measured at total body using Dual Energy X-ray Absorptiometry (Lunar DPXPRO). Polymorphisms of the Fok1 locus of the vitamin D Receptor (VDR) gene were detected using SYBR Green quantitative polymerase chain reaction. Results The prevalence of Fok1 polymorphism was 43.1 %(Ff), 9.8 %(ff) and 47.1 %(FF). At baseline, FF genotype had significantly lower BMD as compared to ff and Ff genotype (p0.1)
Blood samples were drawn early in the morning between 8 am to 10 am after a fasting period of 8 h. Serum concentrations of ionized calcium (iCa) using an ion selective electrode (AVL-9180, Roche Company, USA) and, intact serum parathyroid hormone (PTH) using the enzyme immunoassay (Center, Siemens, USA) were measured for all girls at baseline and follow-up. Serum concentrations of 25-hydroxy vitamin D [25(OH)D] were assessed for all girls at baseline and on a subset of 32 girls at follow-up using radioimmunoassay (DiaSorin, Stillwater, Minnesota, USA). 2–3 ml venous blood was collected in a BD Vaccutainer K2 EDTA tube. Genomic DNA was extracted from 200 μL of the peripheral leukocytes in the venous blood using AccuPrep® spin-column based Genomic DNA Extraction Kit (Bioneer Corporation, South Korea). Allelic variants of the VDR gene at the Fok1 locus were detected using a real-time ARMS (amplification refractory mutation system) based SYBR Green quantitative Polymerase Chain Reaction (qPCR) as described previously by modifications to the method of Quesada et al. [24, 25]. Primers complimentary to each of the allelic variants ‘F’ or ‘f’ were used to differentially amplify the variants in parallel real-time polymerase chain reactions. Table 2
Baseline dietary intake of the study group
Energy (kcal/d) RDA energy (%) Protein (g/d) RDA Protein (%) Calcium (mg/d) RDA Calcium (%) Phosphorus (mg/d) Oxalate (mg/d) Phytate (g/d) Fibre (g/d)
Ff (n=44)
ff (n=10)
FF (n=48)
1357±44 68.6±2.8 32±2 79.8±4.1 280±23 37.7±2.7 652±34 78±13 164±13 12±1
1641±132 86.7±6.8 33±3 91.7±7.3 263±40 36.9±4.4 639±66 105±39 143±12 15±2
1307±52 67.3±2.8 31±2 79.4±4.0 252±20 35.2±2.4 621±38 54±9 151±15 11±1
Analyses were performed using SPSS software for Windows (version 16.0, 2007, SPSS Inc, Chicago, IL). Data are presented as Mean±SE. The Chi-square test was used to compare observed genotype frequencies with those expected under Hardy-Weinberg equilibrium. Paired sample T-test was used to analyse the difference in anthropometric, biochemical and bone parameters before and after supplementation. The differences in the mean values of the anthropometric, biochemical parameters and percentage increase in bone mass over the period of intervention in subjects with 3 different genotype groups of Fok1 loci of VDR gene polymorphisms were analysed using One-Way ANOVA with post hoc Tukey’s test for pairwise comparisons. General linear model procedure was used to test the difference in means of bone parameters according to either Fok1 genotype after adjusting for effects of age, height and weight. For the result to be statistically significant, a level of significance, p0.1). Table 1 shows baseline as well as post supplementation anthropometric characteristics of the study group.There was a significant increase in all the anthropometric characteristics at the end of the supplementation trial for all 3 groups of Fok1 locus (p0.1). Table 2 describes the baseline dietary intake of the study subjects. There was no significant difference in the nutrient
Indian J Pediatr Table 3 Biochemical characteristics of the study group before and after supplementation
Before Ff (n=44)
After ff (n=10)
FF (n=48)
Ff (n=44)
ff (n=10)
FF (n=48)
Ionized Calcium (mmol/L)
1.03±0.02
1.03±0.02
0.99±0.01
1.13±0.01
1.15±0.02
1.13±0.01
PTH (pmol/L) Vitamin Da (nmol/L)
6.79±0.72 27.77±3.05
4.51±0.58 28.70±2.00
5.78±0.52 16.91±2.08
5.08±0.42 61.72±6.24
3.55±0.44 47.30±10.3
4.70±0.48 46.74±7.55
a
Vitamin D on a sub-sample of 32 girls is presented. Data presented as Mean±SE. There were no significant difference in biochemical parameters of the girls at baseline or end-line when divided according to Fok1 genotype (p>0.1)
intake or percentage RDA intake of the 3 Fok1 groups (p>0.05). All the girls had dietary calcium intake below RDA. Table 3 describes the biochemical characteristics of study subjects at baseline and follow-up. At baseline, 82.4 % were hypocalcaemic (iCa < 1.12 mmol/L), 98 % girls had hypovitaminosis D (25[OH]D 6.4 pmol/L). There was a significant increase in serum iCa, and 25[OH]D levels (in subset studied) and significant decrease in PTH levels of the study groups post supplementation (p0.05). Post supplementation, hypocalcemia was observed in 44.6 % girls whereas 20.8 % girls had PTH above the reference range. However, there were no significant differences in the biochemical characteristics of the girls when classified according to Fok1 polymorphism (Table 2) (p>0.05). The percentage increase in iCa [Ff(11.9±1.3 %), ff(12.0±0.9 %), FF(14.0±1.2 %)] and 25[OH]D [Ff(166.5±46.4 %), ff(68.1±47.6 %), FF(261.1± 107.5 %)] or decrease of PTH levels [Ff(19.8±3.2 %), ff(11.7 ±10.9 %), FF(19.2±2.2 %)] was not significantly different for the three groups (p>0.1) post supplementation. Age, height and weight have been shown to significantly influence bone mass accrual in children [26]. Therefore, bone parameters were adjusted for age, height and weight (Table 4) to examine their difference across Fok1 genotype. At baseline, Table 4 Bone parameters of the study group adjusted for age, height and weight before and after supplementation
FF genotype had significantly lower BMD as compared to ff and Ff genotype. FF genotype also had significantly lower BA as compared to ff genotype. A significant increase in BMC, BA, BMD and LBM were seen post supplementation in all the 3 genotype groups (p value0.05) over the period of intervention. Even after adjusting for age, height and weight, no
Before
BMC (g) BA (g/cm2) BMD (g/cm2) Lean Body Mass (g)
Ff (n=44)
ff (n=10)
FF (n=48)
Ff (n=44)
ff (n=10)
FF (n=48)
1069±19 1267±11** 0.826±0.008$$ 20,690±243
1123±41* 1321±26$ 0.832±0.019$ 21,523±533*
1042±18 1253±11 0.812±0.008 20,470±232
1240±19 1401±14 0.870±0.008** 22,266±338
1314±41$ 1451±31$ 0.889±0.017$ 22,955±742
1198±18 1378±13 0.851±0.008 22,123±323
Data presented as Mean±SE *
ff>FF (pFF (pFF (pFF (p0.1)
significant differences were noted in percentage change in bone parameters among Fok1 genotypes post intervention (Fig. 1).
Discussion In the current study, girls with FF genotype had significantly lower bone mass as compared to Ff and ff genotype both before and after supplementation. There was a significant increase in bone mass in all the three Fok1 genotype groups post supplementation. However, there was no significant difference in the percentage increase in bone mass when classified according to Fok1 genotype groups. VDR gene codes for the VDR protein which is widely expressed in the parathyroid glands and also affects the cutaneous production of vitamin D and prevalence of vitamin D deficiency [27]. Studies in adolescent girls have shown no significant difference in 25[OH]D or PTH levels when classified according of Fok1 polymorphisms [8, 11, 28]. Similar results were seen in the current study. Studies on VDR gene polymorphisms of Fok1 locus and its associations with bone mass in children have shown varied results. Laaksonen et al. found no significant association of Fok1 polymorphisms with BMD at radius and ulna or
Fig. 1 Percentage change in bone parameters post supplementation adjusted for age, weight and height. Data presented as Mean±SE
calcaneus in Finnish girls [9]. Similarly, Fok1 polymorphism gene failed to show an association with total body BMD in Danish girls or Lebanese girls [10, 11]. As against this, in a cross sectional study in Japanese girls aged 13.4±0.9 y ff genotype of Fok1 locus polymorphism was associated with significant risk of acquiring low BMD [7]. Similarly, studies in Caucasian children have also demonstrated a significant association of Fok1 polymorphism and bone mass in children with FF genotype having significantly higher bone mass as compared to ff genotype [8, 29]. In the current study, authors found a significant association of Fok1 polymorphism with bone mass in adolescent girls. However, as opposed to the Caucasian girls, Indian girls in the current study with FF genotype had significantly lower bone mass as compared to the other two genotypes, possibly due to differences in ethnicity [8, 29]. Abrams et al. demonstrated a significant association of Fok1 polymorphism on bone mass accrual in children having calcium intake >800 mg/d whereas in children with calcium intake ≤800 mg/d the Fok1 genotype did not have a significant effect on bone mass accrual [29]. In line with these results, in the current study, Fok1 locus polymorphism failed to show any added effect on bone mass accrual which may be due to the habitual low dietary intakes of calcium in the index study subjects. Although the associations of Fok1 gene polymorphism needs to be confirmed on a larger sample size, the index study suggests a significant association of Fok1 polymorphism with bone mass; however, no added effect on bone mass accrual was observed over the period of supplementation in Indian girls habituated to a low calcium intake. A limitation of the index study was that it did not have a control group to compare the effect of supplementation when classified according to Fok1 polymorphism. Further, for logistic reasons, vitamin D was repeated only on a random subset of girls at the end of the study, the authors could thus not study the change in vitamin D post supplementation. In conclusion, the present study demonstrates that girls with Fok1 locus polymorphism of VDR gene as defined by FF genotype had significantly lower bone mass as compared to other genotypes of Fok1 locus. However, Fok1 genotype
Indian J Pediatr
had no added influence on bone mass accrual in Indian girls habituated to a low calcium intake. Acknowledgments The authors thank the participating girls and their parents for their participation in this study. They are also grateful to Dr. Uma Divate, Director, Hirabai Cowasji Jehangir Medical Research Institute for her support in the study. They would like to thank Dr. M Zulf Mughal, Royal Manchester Children’s Hospital, Manchester, UK for his valuable guidance during the study and manuscript preparation. They are grateful to Ms Deepa Pillay, Mrs. Shamim Momin and Mr. Jagdish Sanwalka for their help with the study. They are also grateful to Dr. Ravinder Goswami, Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, India for providing them with controls for genotype for this study. Contributions NS: Manuscript preparation and data collection; AK and VK: Conceptualization of the study and manuscript preparation; SC: Data analysis and conceptualization of the study; NP and KK: Genetics. AK will act as guarantor for this paper. Conflict of Interest None. Source of Funding Government of India.
Department of Science and Technology,
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ORIGINAL ARTICLE
Influence of Vitamin D Receptor Gene Fok1 Polymorphism on Bone Mass Accrual Post Calcium and Vitamin D Supplementation Neha Sanwalka 1 & Anuradha Khadilkar 2 & Shashi Chiplonkar 2 & Kavita Khatod 3 & Nikhil Phadke 3 & Vaman Khadilkar 2
Received: 15 December 2014 / Accepted: 27 April 2015 # Dr. K C Chaudhuri Foundation 2015
Abstract Objective To examine the association of vitamin D receptor (VDR) gene polymorphisms of the Fok1 locus on bone mass accrual in Indian girls used to a low calcium intake. Methods An intervention trial was undertaken in 102 girls aged 8–16 y, attending a state run school in Pune city, India. All girls received 500 mg calcium daily and 30,000 IU of vitamin D3 quarterly for one year. Dietary calcium intake was evaluated. Bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) were measured at total body using Dual Energy X-ray Absorptiometry (Lunar DPXPRO). Polymorphisms of the Fok1 locus of the vitamin D Receptor (VDR) gene were detected using SYBR Green quantitative polymerase chain reaction. Results The prevalence of Fok1 polymorphism was 43.1 %(Ff), 9.8 %(ff) and 47.1 %(FF). At baseline, FF genotype had significantly lower BMD as compared to ff and Ff genotype (p0.1)
Blood samples were drawn early in the morning between 8 am to 10 am after a fasting period of 8 h. Serum concentrations of ionized calcium (iCa) using an ion selective electrode (AVL-9180, Roche Company, USA) and, intact serum parathyroid hormone (PTH) using the enzyme immunoassay (Center, Siemens, USA) were measured for all girls at baseline and follow-up. Serum concentrations of 25-hydroxy vitamin D [25(OH)D] were assessed for all girls at baseline and on a subset of 32 girls at follow-up using radioimmunoassay (DiaSorin, Stillwater, Minnesota, USA). 2–3 ml venous blood was collected in a BD Vaccutainer K2 EDTA tube. Genomic DNA was extracted from 200 μL of the peripheral leukocytes in the venous blood using AccuPrep® spin-column based Genomic DNA Extraction Kit (Bioneer Corporation, South Korea). Allelic variants of the VDR gene at the Fok1 locus were detected using a real-time ARMS (amplification refractory mutation system) based SYBR Green quantitative Polymerase Chain Reaction (qPCR) as described previously by modifications to the method of Quesada et al. [24, 25]. Primers complimentary to each of the allelic variants ‘F’ or ‘f’ were used to differentially amplify the variants in parallel real-time polymerase chain reactions. Table 2
Baseline dietary intake of the study group
Energy (kcal/d) RDA energy (%) Protein (g/d) RDA Protein (%) Calcium (mg/d) RDA Calcium (%) Phosphorus (mg/d) Oxalate (mg/d) Phytate (g/d) Fibre (g/d)
Ff (n=44)
ff (n=10)
FF (n=48)
1357±44 68.6±2.8 32±2 79.8±4.1 280±23 37.7±2.7 652±34 78±13 164±13 12±1
1641±132 86.7±6.8 33±3 91.7±7.3 263±40 36.9±4.4 639±66 105±39 143±12 15±2
1307±52 67.3±2.8 31±2 79.4±4.0 252±20 35.2±2.4 621±38 54±9 151±15 11±1
Analyses were performed using SPSS software for Windows (version 16.0, 2007, SPSS Inc, Chicago, IL). Data are presented as Mean±SE. The Chi-square test was used to compare observed genotype frequencies with those expected under Hardy-Weinberg equilibrium. Paired sample T-test was used to analyse the difference in anthropometric, biochemical and bone parameters before and after supplementation. The differences in the mean values of the anthropometric, biochemical parameters and percentage increase in bone mass over the period of intervention in subjects with 3 different genotype groups of Fok1 loci of VDR gene polymorphisms were analysed using One-Way ANOVA with post hoc Tukey’s test for pairwise comparisons. General linear model procedure was used to test the difference in means of bone parameters according to either Fok1 genotype after adjusting for effects of age, height and weight. For the result to be statistically significant, a level of significance, p0.1). Table 1 shows baseline as well as post supplementation anthropometric characteristics of the study group.There was a significant increase in all the anthropometric characteristics at the end of the supplementation trial for all 3 groups of Fok1 locus (p0.1). Table 2 describes the baseline dietary intake of the study subjects. There was no significant difference in the nutrient
Indian J Pediatr Table 3 Biochemical characteristics of the study group before and after supplementation
Before Ff (n=44)
After ff (n=10)
FF (n=48)
Ff (n=44)
ff (n=10)
FF (n=48)
Ionized Calcium (mmol/L)
1.03±0.02
1.03±0.02
0.99±0.01
1.13±0.01
1.15±0.02
1.13±0.01
PTH (pmol/L) Vitamin Da (nmol/L)
6.79±0.72 27.77±3.05
4.51±0.58 28.70±2.00
5.78±0.52 16.91±2.08
5.08±0.42 61.72±6.24
3.55±0.44 47.30±10.3
4.70±0.48 46.74±7.55
a
Vitamin D on a sub-sample of 32 girls is presented. Data presented as Mean±SE. There were no significant difference in biochemical parameters of the girls at baseline or end-line when divided according to Fok1 genotype (p>0.1)
intake or percentage RDA intake of the 3 Fok1 groups (p>0.05). All the girls had dietary calcium intake below RDA. Table 3 describes the biochemical characteristics of study subjects at baseline and follow-up. At baseline, 82.4 % were hypocalcaemic (iCa < 1.12 mmol/L), 98 % girls had hypovitaminosis D (25[OH]D 6.4 pmol/L). There was a significant increase in serum iCa, and 25[OH]D levels (in subset studied) and significant decrease in PTH levels of the study groups post supplementation (p0.05). Post supplementation, hypocalcemia was observed in 44.6 % girls whereas 20.8 % girls had PTH above the reference range. However, there were no significant differences in the biochemical characteristics of the girls when classified according to Fok1 polymorphism (Table 2) (p>0.05). The percentage increase in iCa [Ff(11.9±1.3 %), ff(12.0±0.9 %), FF(14.0±1.2 %)] and 25[OH]D [Ff(166.5±46.4 %), ff(68.1±47.6 %), FF(261.1± 107.5 %)] or decrease of PTH levels [Ff(19.8±3.2 %), ff(11.7 ±10.9 %), FF(19.2±2.2 %)] was not significantly different for the three groups (p>0.1) post supplementation. Age, height and weight have been shown to significantly influence bone mass accrual in children [26]. Therefore, bone parameters were adjusted for age, height and weight (Table 4) to examine their difference across Fok1 genotype. At baseline, Table 4 Bone parameters of the study group adjusted for age, height and weight before and after supplementation
FF genotype had significantly lower BMD as compared to ff and Ff genotype. FF genotype also had significantly lower BA as compared to ff genotype. A significant increase in BMC, BA, BMD and LBM were seen post supplementation in all the 3 genotype groups (p value0.05) over the period of intervention. Even after adjusting for age, height and weight, no
Before
BMC (g) BA (g/cm2) BMD (g/cm2) Lean Body Mass (g)
Ff (n=44)
ff (n=10)
FF (n=48)
Ff (n=44)
ff (n=10)
FF (n=48)
1069±19 1267±11** 0.826±0.008$$ 20,690±243
1123±41* 1321±26$ 0.832±0.019$ 21,523±533*
1042±18 1253±11 0.812±0.008 20,470±232
1240±19 1401±14 0.870±0.008** 22,266±338
1314±41$ 1451±31$ 0.889±0.017$ 22,955±742
1198±18 1378±13 0.851±0.008 22,123±323
Data presented as Mean±SE *
ff>FF (pFF (pFF (pFF (p0.1)
significant differences were noted in percentage change in bone parameters among Fok1 genotypes post intervention (Fig. 1).
Discussion In the current study, girls with FF genotype had significantly lower bone mass as compared to Ff and ff genotype both before and after supplementation. There was a significant increase in bone mass in all the three Fok1 genotype groups post supplementation. However, there was no significant difference in the percentage increase in bone mass when classified according to Fok1 genotype groups. VDR gene codes for the VDR protein which is widely expressed in the parathyroid glands and also affects the cutaneous production of vitamin D and prevalence of vitamin D deficiency [27]. Studies in adolescent girls have shown no significant difference in 25[OH]D or PTH levels when classified according of Fok1 polymorphisms [8, 11, 28]. Similar results were seen in the current study. Studies on VDR gene polymorphisms of Fok1 locus and its associations with bone mass in children have shown varied results. Laaksonen et al. found no significant association of Fok1 polymorphisms with BMD at radius and ulna or
Fig. 1 Percentage change in bone parameters post supplementation adjusted for age, weight and height. Data presented as Mean±SE
calcaneus in Finnish girls [9]. Similarly, Fok1 polymorphism gene failed to show an association with total body BMD in Danish girls or Lebanese girls [10, 11]. As against this, in a cross sectional study in Japanese girls aged 13.4±0.9 y ff genotype of Fok1 locus polymorphism was associated with significant risk of acquiring low BMD [7]. Similarly, studies in Caucasian children have also demonstrated a significant association of Fok1 polymorphism and bone mass in children with FF genotype having significantly higher bone mass as compared to ff genotype [8, 29]. In the current study, authors found a significant association of Fok1 polymorphism with bone mass in adolescent girls. However, as opposed to the Caucasian girls, Indian girls in the current study with FF genotype had significantly lower bone mass as compared to the other two genotypes, possibly due to differences in ethnicity [8, 29]. Abrams et al. demonstrated a significant association of Fok1 polymorphism on bone mass accrual in children having calcium intake >800 mg/d whereas in children with calcium intake ≤800 mg/d the Fok1 genotype did not have a significant effect on bone mass accrual [29]. In line with these results, in the current study, Fok1 locus polymorphism failed to show any added effect on bone mass accrual which may be due to the habitual low dietary intakes of calcium in the index study subjects. Although the associations of Fok1 gene polymorphism needs to be confirmed on a larger sample size, the index study suggests a significant association of Fok1 polymorphism with bone mass; however, no added effect on bone mass accrual was observed over the period of supplementation in Indian girls habituated to a low calcium intake. A limitation of the index study was that it did not have a control group to compare the effect of supplementation when classified according to Fok1 polymorphism. Further, for logistic reasons, vitamin D was repeated only on a random subset of girls at the end of the study, the authors could thus not study the change in vitamin D post supplementation. In conclusion, the present study demonstrates that girls with Fok1 locus polymorphism of VDR gene as defined by FF genotype had significantly lower bone mass as compared to other genotypes of Fok1 locus. However, Fok1 genotype
Indian J Pediatr
had no added influence on bone mass accrual in Indian girls habituated to a low calcium intake. Acknowledgments The authors thank the participating girls and their parents for their participation in this study. They are also grateful to Dr. Uma Divate, Director, Hirabai Cowasji Jehangir Medical Research Institute for her support in the study. They would like to thank Dr. M Zulf Mughal, Royal Manchester Children’s Hospital, Manchester, UK for his valuable guidance during the study and manuscript preparation. They are grateful to Ms Deepa Pillay, Mrs. Shamim Momin and Mr. Jagdish Sanwalka for their help with the study. They are also grateful to Dr. Ravinder Goswami, Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, India for providing them with controls for genotype for this study. Contributions NS: Manuscript preparation and data collection; AK and VK: Conceptualization of the study and manuscript preparation; SC: Data analysis and conceptualization of the study; NP and KK: Genetics. AK will act as guarantor for this paper. Conflict of Interest None. Source of Funding Government of India.
Department of Science and Technology,
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