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Bone Mass in Prepubertal Boys Is Associated with a Gln223Arg Amino Acid Substitution in the Leptin Receptor

Service of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis Prevention, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, 1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Serge Ferrari, M.D., Service of Bone Diseases, Geneva University Hospital (HUG), 24, Rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. E-mail: serge.ferrari{at}medecine.unige.ch.

	Abstract

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Objective: The contribution of leptin to bone mass acquisition in humans remains unclear. We investigated the association of the Gln223Arg polymorphism in the leptin receptor gene (LEPR) with bone mineral content (BMC) and areal bone mineral density (aBMD) in prepubertal boys and LEPR interaction with vitamin D receptor (VDR) genotypes (Bsm1 and Fok1).

Design: In a cross-sectional design with a longitudinal follow-up, dual-energy x-ray absorptiometry measurements at the lumbar spine, hip, femoral diaphysis, and radius were performed at baseline (mean age 7.4 ± 0.4 yr) and 2 yr later in 222 healthy Caucasian males.

Results: LEPR genotypes were significantly associated with baseline BMC at the hip (P = 0.017), femur diaphysis (P = 0.019), and radius (P = 0.007) and with height (P = 0.041) as well as with physical activity (P = 0.016). Associations with height and BMC at femur diaphysis and radius remained significant after 2 yr. Significant differences in 2-yr bone mass gain at the spine and femur neck were also found among LEPR genotypes. In contrast, adjusting BMC for projected bone area (aBMD) and/or weight, height, and physical activity resulted in a weak association only at the femur (P = 0.014–0.054). VDR polymorphisms were not associated with BMC or aBMD, but significant interactions occurred between VDR Fok1 and LEPR genotypes.

Conclusions: The LEPR Gln223Arg polymorphism was associated with bone mass in growing boys. The association, however, was markedly dependent on bone area, body size, and physical activity, in addition to VDR genetic variation, suggesting that the leptin system may modulate bone mass in humans mostly through indirect mechanisms.

	Introduction

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LEPTIN HAS RECENTLY been identified as an important regulator of bone mass, first in mice (1) and then in sheep (2). Numerous experiments in rodents have clearly demonstrated that leptin exerts positive, anabolic effects on cortical bone and whole-body bone mineral mass (3, 4, 5). However, leptin deficiency has also been associated with increased, rather than decreased, trabecular bone volume density in mice (1, 6). Whether these apparently discordant results are due to a central inhibitory effect of leptin on bone formation and/or a stimulation of bone resorption through the ß-adrenergic system (7, 8), or represent an adaptive response of the trabecular bone compartment to the cortical bone defect (9), remains unclear at present.

In humans, the adipocyte-derived cytokine leptin is known to regulate food intake and energy balance, and thereby fat mass and weight, by activating a hypothalamic receptor (10). A number of studies have attempted to correlate circulating leptin levels with bone mineral density (BMD), yielding inconsistent results (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Nevertheless, fat mass has recently been associated with gain in bone mass over 2 yr in prepubertal boys (24), raising the possibility of a positive relationship between leptin and early bone mass acquisition. Unfortunately, the variability of circulating leptin levels and its dependency on food intake might explain the discrepancy of the reported findings.

The analysis of genetic polymorphisms in the leptin system, by contrast, may permit investigation of its association with bone mass independently from the biological variability of leptin measurements. Various polymorphisms of the leptin receptor (Ob-Rb) gene (LEPR) have been identified (25, 26, 27). Among them, the Gln223Arg polymorphism, characterized by an adenine to guanine transition at position 668 in exon 6, results in an amino acid substitution in the extracellular domain of all receptor isoforms. In a study of 220 Caucasian postmenopausal women, the Gln223-encoding allele was associated with increased body mass index (BMI), fat mass, and serum leptin (28). Chagnon et al. (29) reported similar results in middle-aged Caucasian males, suggesting that Gln223 decreases Ob-Rb activity in response to leptin.

Few publications have addressed the contribution of the Gln223Arg polymorphism to bone mass in humans. In a study population of 219 adult Korean males, carriers of the Gln223 allele had higher bone mineral density at the lumbar spine compared with subjects not carrying the allele (30). In 270 European men over 70 yr, however, no association was found between Gln223Arg and BMD changes at the hip and forearm (31). Therefore, the contribution of LEPR alleles to bone mass acquisition remains unknown.

To investigate whether genetic variation in the leptin system may influence bone mass acquisition in humans, we designed a cross-sectional study with a longitudinal follow-up to test the association of the Gln223Arg allele with bone mineral content (BMC) and areal BMD (aBMD) gain in a cohort of prepubertal Caucasian boys.

	Subjects and Methods

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Study cohort

The cohort included in this study formerly participated in an intervention study that analyzed the effects of calcium supplementation on bone mass gain, for which results have previously been reported in detail (32). Briefly, healthy prepubertal European-Caucasian boys were recruited through the Public Health Youth Service in Geneva, Switzerland, from 1999–2000. Candidates with physical signs of puberty, with a spontaneous calcium intake above the 75th percentile or with a weight to height ratio below the 3rd or above the 97th percentile according to Geneva reference data were excluded from the sample. Of 235 healthy prepubertal European-Caucasian boys initially enrolled, 222 had both complete dual-energy x-ray absorptiometry (DXA) measurements at baseline and DNA samples available for genotyping. Of those, 214 boys were available for follow-up 2 yr later.

The study was approved by the Ethics Committee of the Department of Pediatrics of the Geneva University Hospitals, and informed consent was obtained from the parents, with assent of the child. Of note, we were allowed to collect saliva for DNA extraction, but not blood samples, precluding the measurement of biological variables in serum.

Physical activity was assessed at baseline and during follow-up by means of a questionnaire based on self-reported time spent on sports, recreational activity, and usual walking and cycling (32). Subsequently, the collected data were converted into physical activity energy expenditure (PAEE, kilocalories per day), using established conversion formulas (33). Protein and calcium intakes were assessed with a food frequency questionnaire (32).

Evaluation of physical characteristics and BMC

Participants’ standing height and weight were measured at baseline and during follow-up with a Harpenden stadiometer. BMC (in grams) and aBMD (in grams per square centimeter) at the lumbar spine, femoral neck, trochanter, total hip, femoral mid-diaphysis, radius metaphysis, radius diaphysis, and total radius were measured by DXA on the nondominant side, using a Hologic QDR 4500 instrument (Hologic, Waltham, MA). The coefficient of variation in vivo ranged from 1.0–1.6% for BMD and from 0.3–3.0% for BMC in young healthy adults.

Genotyping

Genomic DNA was extracted from saliva using the QiaAmp blood kit according to the provider’s instructions (QIAGEN, Basel, Switzerland). The Gln223Arg LEPR polymorphism corresponds to an A to G transition at nucleotide 668 that creates an MspI restriction site (26). The PCR used the specific primer pairs 5'-ACCCTTTAAGCTGGGTGTCCCAAATAG-3' and 5'-AGCTAGCAAATATTTTTGTAAGCAATT-3', with the following PCR conditions: denaturation at 94 C for 40 sec, annealing at 55 C for 40 sec, and extension at 72 C for 40 sec for 40 cycles. After MspI digestion, the DNA fragments were discriminated by electrophoresis on a 1% agarose gel.

Additionally, genotyping of vitamin D receptor (VDR) polymorphisms (3'-untranslated region Bsm1 polymorphism and 5'-start codon Fok1 polymorphism) was performed according to standard procedures, as previously described (34, 35).

Statistical analysis

Hardy-Weinberg equilibrium was tested using the Hardy-Weinberg exact test as implemented in the Genepop software. Association of LEPR genotypes (three groups) with crude BMC, aBMD, and longitudinal changes in these measurements, calculated as the difference between values obtained at baseline and after 2 yr, was tested by ANOVA, with Fischer’s protected least significant difference for post hoc comparisons between genotypes. The contribution of age, body size, and environmental variables to the bone traits was evaluated by multiple linear regressions. The association of LEPR genotypes with BMC and aBMD was then adjusted for the independent variables that were both significantly correlated to the bone traits and associated with the genotypes. For this purpose, height, weight, PAEE, age, and protein intake were entered into a multiple regression analysis together with dummy variables accounting for the number of Arg alleles (0 = GlnGln, 1 = GlnArg, 2 = ArgArg).

Interactions between the LEPR polymorphism and VDR polymorphisms on bone traits were tested by ANOVA.

Descriptive characteristics of the sample are expressed as means and SE. In the primary analyses, the level of statistical significance was defined as an ANOVA P value < 0.05. For post hoc comparisons, a Bonferroni correction for partially linked traits was applied, in which case a P value 0.0125 was considered significant (36). All tests were computed by Statview 5.0 version of SAS.

	Results

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Associations of LEPR genotypes with body size and bone mass at baseline

Characteristics of the study population at baseline and after 2 yr are shown in Tables 1 and 2. Mean age of the sample at baseline was 7.4 yr (SD 0.4 yr), and all study participants were prepubertal (Tanner stage 1) and nonobese. The frequencies of the Gln223Arg leptin receptor genotypes shown in Table 2 were 0.498 (GlnGln), 0.404 (GlnArg), and 0.098 (ArgArg), with the alleles being in Hardy-Weinberg equilibrium. Mean BMC, BMD, and height values in these children were consistent with previously published data at this age and/or prepubertal stage (37, 38, 39).

Borderline associations of LEPR genotypes were also found with standing height and arm span but not with sitting height (Table 3), subjects with the ArgArg genotype being on average nearly 3.5 cm shorter than the other groups. There was no association with weight or BMI, but pairwise comparisons indicated a trend toward lower weight in ArgArg compared with heterozygotes (P = 0.046). Moreover, there was a significant difference in PAEE among groups, homozygosity for the Arg-encoding allele being associated with lower PAEE compared with the other genotypes.

Adjustment for size and PAEE reduces associations of BMC with LEPR genotypes

The observation that LEPR genotypes were associated with height and partially with weight, as well as with PAEE, raised the possibility that lower BMC in ArgArg carriers could at least partly be explained by their lower body and/or bone size. Weight and/or height and PAEE as well as age were indeed significantly correlated with BMC, and to a lesser degree with aBMD, at several skeletal sites (by multiple regression analysis, data not shown). Accordingly, we adjusted BMC for height, weight, and PAEE, which markedly reduced BMC association with LEPR genotypes. Borderline associations nevertheless persisted with adjusted BMC at the femoral neck (P = 0.054) and at the total hip (P = 0.04). Further adjustments for age or protein intake did not alter these results. In addition, adjusting BMC for projected bone area to obtain aBMD markedly decreased the association with LEPR genotypes (Table 3). Hence, aBMD was significantly associated with LEPR genotypes only at the femoral diaphysis, both before and after correcting for height, weight, and PAEE.

Two-year follow-up

To extend and confirm these findings, 214 boys were reanalyzed 2 yr later at a mean age of 9.6 (SD 0.4) yr, when all participants were still prepubertal (Table 1).

At that time, LEPR genotypes were also significantly associated with crude BMC and aBMD at the femur diaphysis and with radius and spine BMC (Table 4). A significant association with the Gln223Arg LEPR polymorphism persisted regarding height and arm span (4.5 cm shorter in ArgArg compared with GlnGln, P = 0.016), whereas sitting height (trunk and head) differed by only 1.5 cm between these groups (not significant) (Table 4).

Association of LEPR genotypes with bone mass gain

We further analyzed Gln233Arg associations with BMC and aBMD changes over 2 yr. As shown in Fig. 1A, a significant association with BMC gain was found at the lumbar spine (P = 0.021), with lesser bone mass gain in ArgArg compared with GlnGln and also compared with GlnArg subjects. Similar but nonsignificant trends (P = 0.063) were observed regarding lumbar spine aBMD. Moreover, significant differences in both BMC (P = 0.029) and aBMD gain (P = 0.036) between genotypes were found at the femur neck (Fig. 1B). At this site, BMC and aBMD gain were highest in the heterozygote group. After adjustment for weight gain, height gain, and PAEE, only the association regarding BMC gain at the femoral neck persisted (P = 0.038).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Associations of LEPR genotypes with BMC and aBMD gain over 2 yr. GlnGln, n = 107; GlnArg, n = 86; and ArgArg, n = 20. ANOVA P values for genotype associations were P = 0.021 for BMC and P = 0.063 for BMD at lumbar spine (A); P = 0.029 for BMC and P = 0.036 for BMD at femoral neck (B). *, Post hoc comparison between subgroups, P < 0.05; **, P < 0.01 (Fisher’s test and Bonferroni’s correction).

Interaction between LEPR and VDR genotypes

Leptin has been reported to regulate vitamin D metabolism (40), and vitamin D is a primary determinant of volumetric bone density in children (41). Hence, we hypothesized that interactions could occur between VDR and LEPR polymorphisms in our cohort.

Single-gene analyses showed that neither the 3'-untranslated region Bsm1 alleles (bb 36.0%, Bb 44.1%, BB 19.8%) nor the 5'-start codon Fok1 alleles (FF 46.4%, Ff 42.3%, ff 11.3%) of the VDR were significantly associated with BMC or aBMD at any site (supplemental Tables 1 and 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). In contrast, two-gene analyses revealed significant interactions (P < 0.05) between LEPR and VDR genotypes at most skeletal sites. In this case, differences in both BMC and aBMD between LEPR genotypes were highly significant among VDR FF homozygotes but not among carriers of the f allele, as illustrated for the femoral neck, radius metaphysis, and lumbar spine in Fig. 2.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Interaction between LEPR and VDR Fok1 genotypes. P values for interaction between LEPR genotypes and carriers/noncarriers of the VDR f allele (n = 65 GlnGln, n = 44 GlnArg, and n = 10 ArgArg among Ff/ff; n = 46 GlnGln, n = 45 GlnArg, and n = 12 ArgArg among FF), as calculated by two-factor ANOVA, were as follows: BMC (A) at femoral neck, P interaction = 0.011; radius metaphysis, P interaction = 0.047; and lumbar spine, P interaction = 0.072; and BMD (B) at femoral neck, P interaction = 0. 146 (not significant); radius metaphysis, P interaction = 0.123 (not significant); and lumbar spine, P interaction = 0.047. *, Post hoc comparison between subgroups, P < 0.05; **, P < 0.01 (Fisher’s test and Bonferroni’s correction).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

BMC and aBMD gain at the lumbar spine over 2 yr was highest among homozygous carriers of the Gln genotype. Our findings are in keeping with those reported by Koh et al. (30) who described an independent association of the Gln223-encoding allele with increased aBMD at the lumbar spine. Although the molecular mechanisms by which the LEPR Gln223Arg substitution may alter leptin activity remain unclear, it has been suggested that the Gln allele is associated with lower leptin binding affinity to its receptors (including the soluble receptor isoform) and with higher circulating leptin levels (28, 29). Hence, homozygous carriage of Gln could lead to either 1) decreased leptin activity in the central nervous system leading to a lower suppression of bone formation (by ß2-adrenergic effectors) (7) and/or 2) increased leptin concentrations/activity on receptors expressed in osteoblasts, leading to a direct increase in bone formation, particularly on cortical bone (4). Both mechanisms would be consistent with our observation of a higher bone mass in the GlnGln genotype. Additionally, the association of the GlnGln variant with higher bone mass gain specifically at the lumbar spine, but not at the femoral neck, in our study is consistent with the observation that leptin-deficient mice (ob/ob) have high cancellous bone mass in the vertebrae (1, 6).

In contrast, at the femoral neck, the largest 2-yr gain in BMC and aBMD was observed among heterozygotes. Previous studies on fat mass and leptin levels have reported similar observations, raising the possibility of heterosis, i.e. an alteration of receptor function and activity in the presence of two different subunits in the dimeric leptin receptor (namely Gln223 and Arg223) (28, 29). Alternatively, the absence of increased bone mass gain among GlnGln homozygotes at the femoral neck, contrarily to spine, may reflect the opposite effects of the leptin system on cortical and trabecular bone (3, 6).

Differences in bone mass among genotypes were markedly decreased after adjustment for projected bone area (aBMD), suggesting that the studied polymorphism affected bone mass mostly through bone size rather than through a direct effect on BMD. In addition, adjustment of BMC for weight, height, and PAEE, variables that were all associated to some extent with LEPR polymorphisms, markedly decreased genotype associations with BMC at most skeletal sites. Although data on PAEE should be interpreted with caution, given that its assessment was based on a self-reported questionnaire, these observations further suggest that the relationship between the leptin system and bone mass is partly mediated by the effects of loading (i.e. weight and physical activity) on the skeleton.

A recent study in young men found that, whereas leptin was positively correlated with whole-body and radius BMD, and more specifically with the cortical circumference of the tibia as measured by peripheral quantitative computed tomography, it became negatively associated with bone mass after adjustment for body composition, calcium intake, physical activity, and other variables (43). Altogether, these observations support the notion that, beyond its positive association with body weight and bone mass, leptin may exert some negative influences on the skeleton.

The significant association of the LEPR polymorphism with height might suggest a possible influence of the leptin system on longitudinal growth. These differences could result from the action of leptin on the growth plate. Indeed, it has been demonstrated in vitro that a functional leptin receptor is expressed by human chondrocytes and that leptin stimulates chondrocyte proliferation (44). Moreover, intraarticular injection of leptin in rats stimulated the cartilage growth factors TGFß and IGF-I (45). In humans, studies during growth have shown that appendicular growth is more rapid than axial growth before puberty, due to differences in growth velocity among skeletal regions (46, 47). Hence, it is not surprising that in our study, arm span, but not trunk length, differed between genotypes.

Follow-up assessment in our study was limited to 2 yr, and the associations between the LEPR polymorphism and height and BMD were investigated only during the prepubertal period. During puberty, major hormonal influences (GH-IGF-I and FSH/LH-gonadal steroids) will play a considerable role to further modulate bone mass and height. Our observation of an interaction between LEPR and VDR genetic variations on bone mass in these boys clearly suggests that interactions among the main hormonal systems may occur during growth. Hence, it would be too speculative at this stage to conclude about an influence of genetic LEPR variations on peak bone mass and final (adult) height. For this purpose, longer follow-up will be required. Nevertheless, we previously reported strong tracking for bone mass and height during growth as well as correlations for these parameters between prepubertal daughters and their mothers that were similar to those observed between daughters after puberty and their mothers (48, 49). Hence, it is possible that specific genetic influences on bone mass and height may persist during growth, despite major hormonal changes.

In conclusion, we found associations of the Gln223Arg leptin receptor polymorphism with bone mass at multiple skeletal sites in healthy, nonobese prepubertal boys. Our results support the notion of an involvement of the leptin system in the regulation of bone mass acquisition in humans, most of which seems to be indirect.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online September 4, 2007

Abbreviations: aBMD, Areal BMD; BMC, bone mineral content; BMD, bone mineral density; DXA, dual-energy x-ray absorptiometry; PAEE, physical activity energy expenditure; VDR, vitamin D receptor.

Received April 24, 2007.

Accepted August 27, 2007.

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