1 9 10 11 12 13 14 15 16 17 http://www.ncbi.nlm.nih.gov/SNP 18 19 20 21 22 22 21 22 22 18 18 18 23 18 16 18 23 In the present study, we investigated the association between the Ala1330Val and Val667Met polymorphisms and peak bone mass in men in a population-based study. Subjects and Methods 24 Genotyping DNA was isolated from whole blood samples using QIAamp DNA Blood Midi kits (Qiagen, Valencia, CA, USA). Sense: 5′-GACGGCGAGGCAGACTGT-3′ Antisense: 5′-GCAGGGCCAGGGTCTTG-3′ C probe: 5′-FAM-TCAAAGTCCGCCTCGT-NFQ-3′ T probe: 5′-VIC-CACAGTCCACCTCGTC-NFQ-3′ 25 Sense: 5-gccTTcTTggTcTTcAccAg-3′ Antisense: 5′-cTTTgAggcAggAAcAgAgg-3′ Anthropometrics and Lifestyle Body weight, body height, sitting height, and arm span were measured. Data regarding exercise habits were collected using a self-administered questionnaire. Non-sedentary lifestyle was defined as participation in any form of regular exercise. Dual-Energy X-ray Absorptiometry (DXA) BMD of the lumbar spine, hip, and whole-body was measured using a Hologic 4500 DXA-scanner (Waltham, MA, USA). The coefficient of variation (CV) for BMD is 1.5%, 1.5%, and 0.7% in the lumbar spine, total hip, and whole body, respectively. Lean body mass (LBM) and fat mass was obtained from the whole-body scans. Biochemistry Serum samples were drawn after an overnight fast between 08.00 and 10.00 hours and stored for later analyses at −80°C. Serum concentrations of osteocalcin were measured by a luminoimmunoassay (BRAHMS Diagnostica, Berlin, Germany) with an intra-assay and inter-assay CV of 5% and 8%, respectively. 26 Statistics D 27 28 t p Results Genotype Frequencies 1 Table 1 Covariates, BMD, and lifestyle (sedentary/non-sedentary) by LRP5 genotypes (Ala1330Val and Val667Met polymorphisms)  Polymorphisms  Ala1330Val Val667Met  CC CT TT p GG GA AA p n 589 170 20 – 699 76 4 – Age (years) 25.5 ± 2.8 25.3 ± 2.8 25.9 ± 2.7 NS 25.5 ± 2.8 25.3 ± 2.6 27.0 ± 1.8 NS Body height (cm) 181.7 ± 6.8 181.5 ± 6.1 182.8 ± 7.7 NS 181.7 ± 6.8 181.3 ± 5.6 190.7 ± 6.3 0.02 Arm span (cm) 183.4 ± 8.2  183.1 ± 7.0 185.4 ± 8.9 NS 183.3 ± 8.0 182.6 ± 6.9 193.5 ± 6.8 0.04 Body weight (kg) 81.9 ± 11.8 81.1 ± 12.4 85.9 ± 16.7 NS 81.7 ± 12.1 82.2 ± 12.9 90.4 ± 12.2 NS 2 24.8 ± 3.3 24.6 ± 3.7 25.6 ± 4.1 NS 24.8 ± 3.3 25.1 ± 4.3 24.8 ± 1.8 NS Total fat mass (kg) 14.9 ± 6.2 14.9 ± 7.3 17.7 ± 8.5 NS 15.3 ± 6.2 15.1 ± 6.7 17.9 ± 7.9 NS Lean body mass (kg) 64.0 ± 7.3 63.3 ± 6.8 65.3 ± 9.0 NS 64.7 ± 6.9  64.4 ± 6.0 70.4 ± 7.0 NS n 11.4 ± 10.2 10.7 ± 8.6 12.3 ± 8.7 NS 11.0 ± 9.4 10.8 ± 8.1 6.5 ± 7.3 NS n 0 [0–45] 0 [0–35] 0 [0–25] NS 0 [0–35] 0 [0–30] 0 [0–1] NS 3 65.1 ± 27.9 64.2 ± 26.9 67.9 ± 28.6 NS 65.2 ± 27.5 63.0 ± 29.1 60.0 ± 25.4 NS Serum IGF-I (μg/l) 199.9 ± 52.8 202.4 ± 50.3 191.0 ± 36.1 NS 27.6 ± 7.5 25.5 ± 6.6 27.3 ± 7.8 NS Serum osteocalcin (mmol/l) 3.0 ± 1.1 3.1 ± 1.3 3.0 ± 1.0 NS 3.1 ± 1.2 2.8 ± 1.0 2.3 ± 0.5 NS Serum 1CTP (μg/l) 5.0 ± 1.4 4.9 ± 1,5 5.1 ± 1.3 NS 5.0 ± 1.4 4.9 ± 1.4 4.9 ± 0.9 NS Bone-AP (U/l) 26. 6 ± 8.1 28.4 ± 10.0 26.7 ± 8.3 NS 26.7 ± 8.2 25.8 ± 7.0 33.4 ± 16.4 NS spine 2 1.08 ± 0.12 1.07 ± 0.17 1.05 ± 0.13 NS 1.08 ± 0.12 1.07 ± 0.22 1.07 ± 0.15 NS hip 2 0.95 ± 0.14 0.94 ± 0.14 0.94 ± 0.15 NS 0.95 ± 0.14 0.96 ± 0.15 0.84 ± 0.07 0.03 WB 2 1.22 ± 0.10* 1.21 ± 0.09 1.21 ± 0.11 NS 1.22 ± 0.10 1.21 ± 0.11 1.20 ± 0.08 NS n 150/440 37/133 4/16 NS 171/528 18/58 1/3 NS t 2 p 2 p p Similarly, the GG, GA, AA genotypes of the Val667Met polymorphism were found in 699 (89.7%), 76 (9.8%), and 4 (0.5%), respectively. This corresponds to allele frequencies of 0.95 and 0.05 for the G- and A-alleles, respectively. 2 p 2 p D R Covariates 1 3 p p BMD in Relation to Age p Genotypes in Relation to BMD p Regarding the Val 667Met polymorphism, a significant difference in hip BMD was observed, since subjects with the AA genotype had lower BMD (0.84 ± 0.07 vs. 0.95 ± 0.14 and 0.96 ± 0.15, p = 0.03). 1 2 3 p n Table 2 Multiple regression analysis determining the change in BMD Z-score for each copy of the T- or A-allele  Polymorphism Ala1330Val Val667Met Z-score Unadjusted a Unadjusted a Total population ( n  = 779) spine NS NS NS NS hip NS NS NS NS WB p p NS NS Non-sedentary ( n  = 589) spine p p p p hip NS NS NS NS WB p p NS NS Data are shown as mean effect and 95% confidence interval. Z-scores were derived from the study population itself a 3 n p 1 p 2 p p Fig. 1 (*) p p  Genotypes in Relation to Bone Markers 1 Haplotype Analysis 3 Table 3 The effect of haplotypes of Ala1330Val and Val667Met polymorphisms on BMD adjusted for BMI, lean body mass, smoking, drugs, and vitamin D  Haplotypes  Ala1330Val C T  Val667Met G A G A p n 1331 3 129 81  spine 2 0.008 ± 0.96 −0.42 ± 0.65 −0.09 ± 0.90 −0.04 ± 1.60 NS hip 2 −0.002 ± 1.01 0.97 ± 1.48 −0.0005 ± 0.95 −0.04 ± 1.0 NS WB 2 0.02 ± 1.01 0.70 ± 0.59 −0.10 ± 0.87 −0.14 ± 1.04 NS Data are shown as the mean ± SD. No significant associations between BMD and haplotypes were found when sedentary and non-sedentary men were analyzed separately (not shown) Discussion 18 19 21 22 18 22 29 30 18 25 n 16 12 14 14 31 32 7 33 34 23 21 21 19 n 3 22 35 36 11 20 18 37 10 38 39 40 41 42 18 29 n 19 21 22 30 21 2 2 18 20 24 We conclude that the Ala1330Val (exon 18) and Val667Met (exon 9) polymorphisms of the LRP5 gene are significantly associated with lumbar spine peak bone mass in physically active men, potentially accounting for 2.5% of BMD in the spine. This gene-environment interaction provides support for LRP5 as a mediator of load-induced bone formation and suggests that this gene is involved in the pathogenesis of osteoporosis in men.