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Keywords:

  • vitamin D receptor;
  • calcium absorption;
  • stable isotopes;
  • calcium kinetics;
  • bone mineralization

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Few studies of the VDR polymorphisms have looked at calcium metabolism or long-term effects. We measured bone mineralization and calcium metabolic parameters longitudinally in a group of 99 adolescents. We found a significant relationship between calcium absorption and skeletal calcium accretion and the Fok1, but not other VDR or related, genetic polymorphisms. It seems that the Fok1 polymorphism directly affects bone mineralization during pubertal growth through an effect on calcium absorption.

Introduction: There are few data regarding the relationship between genetic markers for low bone mass and changes in calcium metabolism in childhood or adolescence. We sought to identify the effects of polymorphisms of the vitamin D receptor (VDR) on calcium and bone mineral metabolism in a longitudinal study of pubertal adolescents.

Materials and Methods: Adolescents (n = 99) received comprehensive stable isotope studies of calcium absorption, bone calcium kinetics, and bone mineralization. Studies were repeated 12 months later. Polymorphisms of putative genetic markers were determined and related to bone mineralization and calcium metabolic finding. Results were analyzed by ANOVA in which changes over time were determined using the initial value as a covariate.

Results: Polymorphisms of the Fok1 gene of the VDR were significantly related to calcium absorption (p = 0.008) and whole body BMC (p = 0.03) and BMD (p = 0.006). The Fok1 effect on whole body BMD was significant for those with Ca intake >800 mg/day (p < 0.001), whereas for those with Ca intake ≤800 mg/day, the Fok1 genotype did not have a significant effect on whole body BMD (p = 0.40). The Fok1 genotype was significantly related to the changes during the year in whole body calcium accretion, with the ff genotype having a 63 ± 20 mg/day deficit compared with the FF genotype (p = 0.008).

Conclusions: The Fok1 polymorphism of the VDR receptor seems to directly affect bone mineral accretion during pubertal growth through an effect on calcium absorption. The relationship between different genetic polymorphisms and bone mineral metabolism may vary by life stage as well as diet.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Since the initial reports of a link between vitamin D receptor (VDR) gene polymorphisms and bone mineralization, there has been considerable interest in further understanding this association.(1,2) Previous studies have generally found evidence of a relationship between several VDR gene polymorphisms and bone mineralization, but the relationships identified have been inconsistent across population groups and genetic markers.(3,4) One of the difficulties in interpreting most reports is that the primary endpoint has been whole body or regional BMD, and the populations studied have usually been adults or postpubertal adolescents. This has allowed relatively minimal data regarding the specific effect on bone metabolism that can be affected by the VDR genes and little longitudinal information, especially relative to pubertal growth, when the majority of bone is formed and genetic effects may be most important.(5,6)

In a cross-sectional retrospective study of 72 children (65 girls and 7 boys), 7-12 years of age of varying pubertal status, we found a significant association between the Fok1 polymorphism of the VDR translation initiation site and both total calcium absorption (Va) and whole body BMD.(7) We did not find any association with bone calcium deposition rates (Vo+) determined using stable isotopes and did not evaluate longitudinal changes during puberty or regional bone mineralization. We did not find a significant relationship between calcium absorption or bone mineralization and other VDR polymorphisms. Since that report in 1999, we are unaware of further published studies looking at VDR polymorphisms and calcium absorption or kinetics in children or adolescents, probably because of the few centers capable of performing these metabolic studies. There are virtually no data in adults either.(8) However, several recent studies have found a link between Fok1 polymorphisms and bone mineral status(9,10) in children and adolescents.

It is possible that variations in VDR function directly affect calcium absorption and kinetics, and this results in the effects described on bone mineralization outcomes.(11–13) Therefore, we sought to characterize the relationship between VDR and other genetic polymorphisms related to bone metabolism and multiple measures of bone mineral metabolism including calcium absorption, bone formation, and whole body and spinal bone mineralization in a prospective longitudinal study. Our goal was to characterize the consequences of these polymorphisms, both at the time of study entry during early puberty and longitudinally during pubertal growth, a period of extremely rapid bone growth and calcium accretion.(14)

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Study design: overview

We conducted a study of 100 early pubertal adolescents to evaluate factors related to calcium absorption and bone mineralization during a year of pubertal growth. We measured calcium absorption and bone mineralization at the start of the study and 12 months later. At baseline, we also obtained biochemical and kinetic measures related to bone turnover. Results from these studies are related in this report to putative genotypic markers related to calcium and bone mineral metabolism.

Subjects

By public advertising, we identified 50 girls and 50 boys for this study. All subjects were between 9.0 and 13.0 years of age and had a body mass index between the 5th and 95th percentile for age and sex. Subjects were selected to approximately match the ethnic distribution of the greater Houston area. All subjects received a screening physical examination including Tanner staging before inclusion in the study. To be enrolled, subjects had to be healthy and Tanner stage 2 or 3 (breast stage for females or penile stage for males). Girls had to be premenarcheal. Subjects could not have any chronic illnesses requiring them to take medications on a regular basis.

Written informed consent was obtained from a parent or legal guardian for each subject; written assent was obtained from all of the study subjects. The Institutional Review Board of Baylor College of Medicine and Affiliated Hospitals approved this protocol.

Initial study visit

Within 8 weeks of the screening visit described above, subjects were admitted for 24 h to the General Clinical Research Center of Texas Children's Hospital (Houston, TX, USA). During this stay, comprehensive measurements of bone calcium metabolism and bone mineralization were carried out as described below under protocol methods.

Subjects were discharged at the end of this time and returned 12 months later for a comprehensive follow-up visit in which measurements of Va, BMC, and BMD were repeated. At the time of the initial study, subjects were randomized by sex (and matched by ethnicity) to receive supplementation with either a prebiotic inulin-type fructan(15) or placebo (maltodextrin) to be mixed with 180 ml of calcium fortified orange juice and taken with breakfast daily for 12 months. This aspect of the study had no significant effect on the genetic findings of this report except as noted in the Results section for changes in whole body BMC and BMD.

Calcium absorption and metabolism measurements

Stable isotope studies were performed as previously described.(16,17) Each subject was given a breakfast containing one-third to one-half of their usual daily intake of calcium. Toward the end of breakfast, the subjects were given 20 μg of46Ca that had been mixed with 240 ml of calcium-fortified orange juice. After breakfast,42Ca (1.2 mg) was infused over 2 minutes through a heparin lock catheter. Beginning with breakfast, a complete 24-h urine collection was obtained. Subsequently, subjects collected a second 24-h urine collection at home, and spot urine samples were collected for 4 additional days for kinetic measurements. Serum samples were also obtained intermittently during the first 8 h after dosing for kinetic analysis.(16)

Dietary methods

At the screening visit, the study dietitian asked subjects what they usually ate on a normal day, and food preferences were obtained. Inpatient menus for the overnight study visit were based on normal calcium intake.(18) All foods and beverages during the inpatient and outpatient visits were pre- and postweighed to accurately determine intake. Subjects were instructed to keep weighed food records for a total of 6 days during the study: a 2-day period after the first overnight visit, a 2-day period 2 months later, and a 2-day period after the 1-year visit. Subjects were called at home during the 1-year period to obtain a 24-h recall of the previous day's intake and to ensure that the subject maintained a relatively consistent calcium intake. To reflect the marketplace changes in dietary food contents during the study, dietary intake data were collected using Nutrition Data System for Research software versions 4.03 and 4.05, developed by the Nutrition Coordinating Center (NCC; University of Minnesota, Minneapolis, MN, USA).

Bone turnover makers

Osteocalcin was determined using a radioimmunoassay method previously described.(19) Samples were immediately spun and frozen at −60°C and assayed for osteocalcin in the laboratory of Dr Caren Gundberg at Yale University by an immunoassay that recognizes both intact and the major n-mid proteolytic fragment. All samples were run in the same batch. Interassay variability was 3%. Serum N-telopeptide was measured using a commercially available assay (Osteomark NTx; Wampole Laboratories, Princeton, NJ, USA).

Isotopic analytical methods

Urine samples were prepared for thermal ionization mass spectrometric analysis as previously described using an oxalate precipitation technique.(20) Samples were analyzed for isotopic enrichment using one of two techniques. For serum samples, measurement of enrichment was performed using a magnetic sector ICP-MS (Element 2; ThermoFinnigan, Bremen, Germany). This is a high-speed instrument capable of analysis of the42Ca/43Ca ratio with precision and accuracy of 0.3%.(21) Urine samples obtained were analyzed to determine their calcium isotope ratios using a Finnigan MAT 261 (Bremen, Germany) magnetic sector thermal ionization mass spectrometer. Accuracy and precision of this technique for natural-abundance samples compared with standard data are 0.15%.(20)

Bone mineral measurement methods

Whole body scans were performed using a Hologic QDR-4500A DXA (Hologic, Waltham, MA, USA) that operates in the fan-beam mode. Whole body BMC and areal BMD values were determined. A region-specific scan of the spine was used to assess spinal BMD. Relative bone status of the subjects was obtained using a z score model for BMC and BMD based on a reference population.(22) Whole body BMD and spine BMD precisions were <1%, whereas whole body BMC precision was <1.5%.(23)

Genetic methods

Genomic DNA was isolated from 3 ml of whole blood collected in EDTA-coated tubes using the Wizard TM Genomic DNA Purification Kit (Promega, Madison, WI, USA). Analysis of DNA for the genetic polymorphisms was carried out by the Gene Expression Core of the Texas Coast Digestive Disease Center. For these analyses, 80 ng of genomic DNA was amplified, and the Xba1 and PvuII estrogen receptor gene phenotypes were analyzed as described by Lorentzon et al.(24) VDR BsmI was analyzed according to Fleet et al.(25) The VDR phenotypes (ApaI, Taq1, and FokI sites) were analyzed as previously described.(7) Primer sequences for VDR BsmI, ApaI, TaqI, and FokI were obtained from Dr Mark Johnson (Creighton University School of Medicine, Omaha, NE, USA). Calcitonin receptor genotyping was analyzed according to Nakamura et al.(26) All PCR reactions except FokI were purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) before restriction digests. Calcitonin receptor primers were designed by the Baylor College of Medicine Microarray Core Laboratory personnel using BioRad's Beacon Designer 2.0. Restricted products were electrophoresed on a 12% PAGE gel and stained with ethidium bromide for visualization.

Calculations and modeling

The compartmental model used for calcium kinetics was similar to that described by Neer et al.(27) The model is based on three sequential pools before calcium deposition in the “deep” bone Ca pool. Bone Ca deposition (Vo+) is the rate of flow of Ca to the final pool. Compartmental modeling of the data were performed with the aid of the SAAM (Simulation, Analysis, and Modeling) program as described previously.(20) Va was calculated as the product of calcium intake and fractional absorption.

Statistical methods

Sample size determination was made to ensure that adequate power was present for determining an effect of the inulin-type fructan. The sample size determined, of 50 enrolled of each sex, with 40 of each sex completing the study, also was of adequate power to determine a genotype effect. In our preliminary study,(7) we found a mean decrease in Va of 20% in ff genotype subjects compared with Ff genotype subjects (Fok1 polymorphism). We determined that, based on expected genotype distributions in the population, a sample size of 40 each (male/female) would have a power >0.9 to identify a similar 20% difference in Va between ff and FF groups (p < 0.05). Comparisons of genotype groups were made using a generalized linear model (univariate ANOVA, ANOVA) with covariate adjustment based on the specific analysis conducted. Sex, race, and Tanner stage at enrollment were included as covariates in all models. Normality of distribution and equality of variances (Levene's test of equality of error variances) were assured using the analytical software. Differences in anthropometric and calcium metabolic parameters were evaluated for the complete datasets using SPSS 13.0 for Windows (SPSS, Chicago, IL, USA). All data are presented as the mean ± SE.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Subjects and anthropometry

Of the 100 subjects enrolled after screening in the study, 1 male subject was withdrawn for failure to complete the baseline study properly. Of the 99 subjects who completed the baseline calcium metabolism and bone mass measurements at the first study, there were 52 whites, 14 blacks (one of these was biracial black and white), 23 Hispanic (all Mexican Americans), and 10 Asians. Tanner staging (breast/penile) at study initiation showed that 73 were Tanner stage 2, and 26 were Tanner stage 3. Mean age was 11.6 ± 0.1 years, mean weight was 42.0 ± 0.9 kg, and mean height was 148.6 ± 0.9 cm. Mean weight z score was 0.23 ± 0.09, and mean height z score was 0.12 ± 0.11. Mean body mass index was 18.8 ± 0.3 kg/m2, and no subject had a BMI >25 kg/m2. Of the study subjects, 96 completed bone mass and 92 completed calcium absorption measurements at 1 year.

Results for anthropometry based on Fok1 genotype are shown in Table 1. Adjusting for sex and race, a marginally statistically significant effect of genotype on height was found at the start of the study (p = 0.06). However, when Va was included in the model, Va was identified as a significant predictor of height (p = 0.005), as were Tanner stage (p = 0.002), sex (p = 0.001), and race (p = 0.046), but genotype was no longer significantly related to height (p = 0.29).

Table Table 1.. Anthropometric Characteristics at Start of Study
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Effects of genotype on calcium absorption and vitamin D status

The relationship between calcium absorption and genotype at the start of the study is shown in Table 2. Both fractional absorption and Va at the start of the study were closely related to genotype, with a 91 mg lower Va in subjects with the ff compared with the FF genotype. The relationship between genotype and both fractional and total calcium absorption remained significant at the end of the study (Table 3). Kinetic values were not obtained at that time.

Table Table 2.. Calcium Metabolism and Kinetics at Start of Study
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Table Table 3.. Calcium Absorption and Urinary Excretion at End of Study
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Effects of genotype on bone formationand resorption

Although the Fok1 genotype was marginally significantly (p = 0.06) associated with bone calcium deposition rate, Vo+ (Table 2), when fractional absorption and calcium intake were included in this model, the effect of the Fok1 genotype was not significantly related to Vo+ (p = 0.54), although Vo+ was significantly related to fractional calcium absorption (p < 0.001), sex (p < 0.001), Tanner stage (p = 0.04), and calcium intake (p = 0.01). Similarly, inclusion of fractional absorption and calcium intake in the prediction model for Vo+ decreased the significance of the relationship between serum N-telopeptide, a measure of bone resorption, and the Fok1 genotype from p = 0.08 to p = 0.20. In this case, serum N-telopeptide was not significantly related (p > 0.3 for each) to sex, Tanner stage, calcium intake, or fractional absorption of calcium.

Effects of genotype on BMC and BMD

Data for whole body BMC, whole body areal BMD, and spinal BMD at the beginning of the study are shown in Table 4. Results for whole body measures are also given as z scores using our internal database based on age and sex.(23) Each of these bone mineral mass measures was significantly related to genotype.

Table Table 4.. BMC and BMD at Start of Study
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Effects of dietary calcium on genotype effects on bone mineralization

After adjusting for race, Tanner stage, and sex, there was a significant interaction between Fok1 genotype and dietary calcium intake (p = 0.008) on whole body BMD but not whole body BMC (p = 0.14) at the start of the study. To further evaluate a potential relationship between dietary calcium intake and genotype effects, we separated the analysis to those whose diets had >800 mg/day of calcium (n = 56) and those whose diets had ≤800 mg/day of calcium (n = 43). After correcting for race, Tanner stage, and sex, the Fok1 effect on whole body BMD was significant for those with Ca intake >800 mg/day (p < 0.001), whereas for those with Ca intake ≤800 mg/day, the Fok1 genotype did not have a significant effect on whole body BMD (p = 0.40).

At the 1-year follow-up visit, for both whole body BMC and whole body BMD, a statistical effect of the Fok1 genotype was present for those with mean calcium intake (average of intake at the start and end of the study) >800 mg/day but not for those with intakes ≤800 mg/day. Similarly, the calcium accretion over the study year was related to the mean calcium intake. For those with an average calcium intake ≤800 mg/day, the Fok1 genotype was not significantly related to calcium accretion (p = 0.27). In contrast, for those with an average intake >800 mg/day, the Fok1 genotype was significantly related to calcium accretion (p = 0.02).

Longitudinal evaluation of changes in bone mineralization during the study period

During the year after initial measurements, subjects had been randomized to receive either an inulin-type fructan or placebo (maltodextrin) daily with a 180-ml daily serving of calcium-fortified orange juice. This intervention significantly affected whole body BMD and its z score (p = 0.02 for an effect of use of the inulin type-fructan on whole body BMD), and therefore, the analyses (Table 5) are adjusted for this factor. After adjustment for this and race, Tanner stage, and sex, results showed a significant effect of the Fok1 genotype on whole body BMC but not a significant effect on whole body BMD, whether these are viewed as the actual value or as the age- and sex-adjusted z score values. These analyses were performed using the initial value for bone mineralization as a covariate in the model. If, instead, actual numerical changes of the whole body BMC or whole body BMD are used as the outcome variable, virtually identical results are obtained.

Table Table 5.. BMC and BMD at End of Study Adjusted for Initial Values
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Longitudinal evaluation of changes in calcium accretion to skeleton

To further evaluate the effect of genotype on daily changes in calcium accreted to the skeleton during the phase of rapid pubertal growth, we looked at two models for calculating daily calcium accretion during the study period. The first, based on the longitudinal measurements of whole body BMC, calculated the daily difference in whole body BMC between initial and final 1-year study and used a factor of 0.322 for the fraction of calcium in whole body BMC.(28) Correcting for sex, race, Tanner stage at enrollment, and randomization to inulin-type fructan, we found (Fig. 1) a difference of 63 ± 20 mg/day between the ff and FF genotypes (p = 0.008 for model and p = 0.02 for ff versus FF difference posthoc using Fisher's LSD). Heterozygotes (Ff) had a 21 ± 16 mg difference versus FF (p = 0.18) and a 42 ± 18 mg difference versus ff (p = 0.01). Of note is that neither race nor sex was a statistically significant (p > 0.10 for each) covariate in this model, although Tanner stage at enrollment was a significant covariate (p < 0.01).

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Figure FIG. 1.. Effect of the Fok1 genotype on calcium accretion to skeleton. Data are corrected for race, genotype, Tanner stage at enrollment, and for blinded randomization of one-half of subjects to 8 g/d inulin-type fructan. *Overall effect, p = 0.06, Fisher's LSD posthoc, ff < FF, p = 0.03 and Ff < FF, p = 0.06. **Overall effect, p = 0.008. Fisher's LSD posthoc, ff < FF, p = 0.002 and ff < Ff, p = 0.02.

Download figure to PowerPoint

A second method used the calcium balance data at the initial and final studies. In this case, because endogenous fecal excretion was not measured, there is some added uncertainty in the exact values for calcium balance, but it is unlikely that this value changed substantially between studies(18) or was related to genotype. Using the total absorbed and urinary calcium at each measurement and after corrections for race, sex, Tanner stage at enrollment, and randomization to inulin-type fructan, we found a mean average calcium balance difference of 71 ± 32 mg/day between the ff and FF genotypes (p = 0.06 for overall model, p = 0.03 for ff versus FF difference posthoc using Fisher's LSD). Heterozygotes (Ff) had a 23 ± 30 mg difference versus ff (p = 0.44) and a 48 ± 25 mg difference versus FF (p = 0.06). Race, sex, and Tanner stage were not significant covariates.

Effects of other genotypes

The effects of the ApaI, BsmI, and Taq1 genotypes on calcium absorption, bone calcium deposition rate, and whole body and spinal BMC and BMD were evaluated. Results for fractional absorption, whole body BMC and BMD are shown in Table 6. None of these approached significance (p < 0.1). There was no significant interaction between these polymorphisms and the Fok1 polymorphism. In addition, we found no effects of the estrogen receptor genotypes or the calcitonin receptor genotype on BMC, BMD, or calcium absorption (Table 6).

Table Table 6.. Relationship Between Other Genetic Polymorphisms and Calcium Absorption/Bone Mineralization at the Start of the Study
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We have shown that a specific polymorphism of the VDRFok1 gene is associated with variations in calcium absorption in early puberty and with total bone mass and bone mass accretion during puberty. The effect of the homozygote ff genotype is substantial because about 20% less calcium absorbed and accreted to bone during pubertal growth than in the favorable FF genotype. Effects of the heterozygote genotype seem to be intermediate between the two homozygote genotypes. The balance method has a greater variability than the bone mineralization method, leading to less significant differences among genotypes despite comparable numerical difference between genotypes (Fig. 1).

Our results suggest a greater effect of the Fok1 genotype on bone mineralization at higher calcium intakes. It is not obvious why this intake effect may be present nor is a calcium intake of 800 mg/day, at which we performed the analysis, necessarily the level of intake at which the Fok1 genotype may affect calcium absorption. However, in a preliminary fashion, our data support an interaction of diet and genotype such that the genotype effect is more limited in situations of substantial dietary calcium deficiency. In general, at very low intakes, in the absence of overt vitamin D deficiency, calcium absorption increases substantially because of increases in active 1,25-dihydroxyvitamin D concentration.(18) Our findings suggest that the favorable Fok1 polymorphism may in part be enhancing calcium absorption through a transcellular effect at more usual intakes where the stimulus to form 1,25-dihydroxyvitamin D is less. However, we cannot exclude an effect on paracellular absorption as well. Our results in this regard should be interpreted cautiously because this study was not designed to look at calcium absorption or bone mineralization over a wide range of calcium intakes.

We found marginally significant effects of the Fok1 polymorphism on height, bone formation, and bone resorption. These effects seem to be related primarily to its effect on calcium absorption. In particular, we found calcium absorption itself to be significantly related to height. During growth, bone formation and resorption are tightly linked to each other and to the availability of calcium for bone accretion. Our data indicate the importance of intervention strategies designed to enhance both dietary and total absorbed calcium, such as through increased intake of calcium and vitamin D and decreased intake of absorption inhibitors such as phytate. These may be especially beneficial to those at-risk from an unfavorable genotype, although this too requires a direct intervention trial to evaluate.(29,30)

Twin studies suggest that 50% or more of the variance in BMD is genetically determined.(31,32) Efforts to identify specific genes that are involved have focused primarily on the VDR receptor, especially the Bsm1, Apa1, Taq1, and Fok1 cleavage sites.(33) More recently, evidence has been presented for a relationship between BMD and other genetic sites.(24–26) In our study, we did not find any significant effect of the Bsm1, Apa1, or Taq1 genes or two genes associated with the estrogen receptor and one with the calcitonin receptor on any parameter of calcium absorption, bone mass, or mineral accretion. Our study was not large enough to completely exclude an interaction of these genotypes, but the data in Table 6 do not provide any strong evidence for an effect of any of these genotypes in our population. The contrast between these findings with those of some studies in adults suggests that different genotypes may be relatively more important at different ages.

Of note is that we did not find any specific sex or racial effect of the Fok1 genotype on calcium absorption or accretion. Our population represents a mixed sex and racial population and was not designed to specifically evaluate genotype effects in different racial groups. However, our findings, like those previously reported,(7) suggest that race and sex are not primary determinants of the Fok1 effect on calcium and bone mineral metabolism.

We found some variability in the genotype effects relative to areal BMD compared with BMC, with a trend toward a greater effect on BMC and the least effect on spinal BMD. It is not certain if this is a biological distinction, but separation of cortical and trabecular bone such as through quantitative CT would be ideal to further evaluate these issues.

Recently, several studies have supported a role for the Fok1 genotype in determining bone mineral status. In postmenopausal Czech women, the Fok1 polymorphism, but not the Bsm1 or Taq1 polymorphisms, was associated with a lower BMD at the hip.(34) Other previous studies(35,36) did not find this relationship in older adults. In two studies of adolescents in Scandinavia, the Fok1 genotype was associated with variation in BMD, although in one of these studies it was not associated in girls. Of note is that this study evaluated primarily cortical bone sites. Both studies looked at adolescents well past the point of peak bone mass accrual.(6,10) Katsumata et al.(9) recently reported a significant association of both lumbar spine BMD and femoral neck BMD with the Fok1 genotype of Japanese girls averaging 13 years of age.

Most studies of the VDR and mineral metabolism have been performed on postpubertal adolescents and have used some single time-point measure of bone mineral status as the phenotypic characteristic. One longitudinal study of Norwegian children found no effect of the Bsm1 gene polymorphism in children or adolescents,(37) a finding consistent with the results in this study.

In a limited cross-sectional reported in 1999, we reported(7) an association between the Fok1 genotype and both Va and whole body BMD of young adolescents. This remains the only report evaluating calcium absorption based on genetic polymorphisms of the Fok1 gene. In fact, even in adults, very few studies of calcium absorption related to VDR receptor polymorphisms have been reported.(8) There are substantial limits to cross-sectional studies or those focusing on genetic effects on bone mineralization of older adolescents and adults. The most important is the wide variability in both genetic and environmental factors leading to variations in bone mass after peak bone mass is achieved. As such, it is not surprising that results have not clearly identified the magnitude of genotypic effect on bone mineralization. More recently, careful consideration of lifestyle effects such as dietary intakes has shown that apparently “negative” studies may in fact show a genetic effect after these factors are properly included.(38) One recent study in Japan indicates that bone structure may differ based on genotype, with FF individuals producing stronger bones in response to impact loading.(39)

The molecular mechanism by which differences in the VDR protein encoded by the f versus F allele affect bone homeostasis remains somewhat uncertain.(40) Previously, it has been shown that Bsm1 polymorphisms with the less favorable BB genotype having higher 1,25-dihydroxyvitamin D levels.(41) Our data showing slightly lower 25(OH)D levels in the more favorable FF genotype would require confirmation and consideration of possible feedback mechanisms related to vitamin D metabolism that might be responsible for this finding.

The functional consequences of an ∼60-70 mg daily less calcium accretion during pubertal growth may be substantial. This translates to a substantial annual difference of 25 g of calcium accreted. In theory, the difference of 60-70 mg/day could be accounted for by increasing calcium intake by 350 mg/day, assuming a net retention of 20% of the added intake and accounting for the possible decrease in retention of other dietary calcium associated with the increased intake. Whether such calculations can be practically applied to a genetically at-risk population is unclear. The assertion from 1997 that “VDR genotyping is probably of little use for the detection of individuals who would benefit from increased calcium and physical activity to increase their peak bone densities”(37) may still be correct. However, ours and other recent data show that such an approach may ultimately be feasible.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank the nursing staff of the General Clinical Research Center of Texas Children's Hospital for caring for the study subjects; Cynthia Edwards for study recruitment; Holly Endris, Melissa Knox, Courtney Edwards, Lora Plumlee, Yana Kriseman, Rachel Wolfson, Michelle Lopez, and Anh Mai for subject assistance, Lily Liang for laboratory assistance; and E O'Brian Smith, PhD, for statistical advice. Calcium-fortified orange juice used in the study was provided by The Coca-Cola Company, Houston, TX. The inulin-type fructan was Raftilose Synergy1, provided by Orafti, N.V., Tienen, Belgium. This work is a publication of the U.S. Department of Agriculture (USDA)/Agricultural Research Service (ARS) Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. This project has been funded in part with federal funds from the USDA/ARS under Cooperative Agreement 58-6250-6-001, the NIH, NCRR General Clinical Research for Children Grant RR00188, NIH AR43708, and NIDDK P30 DK56338. Contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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