osteoporosis is primarily a disease of older people and advancing age is one of the main determinants of osteoporosis and fragility fractures .
the estimated lifetime risk of hip , spine , or distal forearm fracture for caucasian man at the age of 50 years is 20% and approximately 30% of all hip fractures occur in men .
heredity is now generally accepted as playing an important part in the pathogenesis of osteoporosis in both men and women .
indeed , after excluding secondary risk factors and age then the other main causal factor for osteoporosis in men is genetic .
this view is supported by genetic epidemiological studies on bmd , an important surrogate of fracture risk , which show that peak bone density has a substantial heritable component .
variation in bmd is determined by multiple genes ; objective evidence for this is provided by candidate gene snp studies , as well as quantitative trait locus ( qtl ) studies in inbred animals and humans .
qtl studies have identified multiple chromosomal regions which influence bone mass and are linked to osteoporosis - related phenotypes .  
studies of classical and novel candidate genes have only been able to account for a small proportion of the variance in bone mass .
candidate gene contributions to bone mass also show gender , geographic , and ethnic variability .
therefore , studies aimed at identifying the contribution of specific genes towards bmd and the heritable component to osteoporosis should ideally be gender and ethnic specific .
one such candidate gene is bone morphogenetic protein 2 ( bmp2 ) , located on chromosome 20p12.3 .
it is a growth factor that possesses osteoinductive properties and promotes osteoblast differentiation and bone formation [ 49 ] .
linkage analysis in extended families with osteoporosis in iceland , using a phenotype that combines osteoporotic fractures and bmd measurements , showed linkage to chromosome 20p12.3 .
three variants in the bmp2 gene , a missense polymorphism , and two anonymous single nucleotide polymorphisms and the resultant haplotypes have been associated with an osteoporotic phenotype , including osteoporotic fractures as well as low bmd , both before and after the menopause .
a number of follow - up studies however have yielded inconsistent results [ 59 ] .
to date no investigation has been performed in an exclusively male cohort and no follow - up study has investigated the association with risk of osteoporosis in men and rate of bone loss . in view of the inconclusive nature of the earlier study and to determine possible gender and geography - related variability , we therefore investigated the association of ser37ala and arg190ser genotypes / haplotypes with bmd , rate of bone loss , and fracture risk in men from the uk .
participants comprised 342 male subjects , 224 controls , and 118 subjects with osteoporosis , who were all caucasian men from england , uk [ 1012 ] .
the men with osteoporosis comprised men who had a bmd t score below 2.5 at either the femoral neck or lumbar spine or had a low trauma fracture and a t score below 1.0 at either the femoral neck or lumbar spine . of the patients
89 also had a history of fractures ( 46 had distal forearm fracture , 33 symptomatic vertebral , 6 hip , and 4 other fractures ) .
their fractures had all occurred at least six months prior to their inclusion in the study , and all blood samples were taken before the initiation of therapy .
underlying secondary causes of osteoporosis were excluded by medical history , physical examination , and laboratory investigations .
the laboratory investigations included full blood count , esr , biochemical profile , thyroid function tests , serum testosterone , sex - hormone - binding globulin , gonadotrophins , and serum and urine electrophoresis . in a subset of 145 control subjects , there was a follow - up bmd estimation and annual change in bmd was determined , in order to test the association between bmp2 genotypes and change in bmd .
a similar analysis of the annual change in the bmd measurement was not performed in the men with osteoporosis , because of the confounding effect of antiresorptive treatment .  
serum testosterone , sex - hormone - binding globulins , follicular stimulating hormone , and luteinizing hormone were measured by commercially available radioimmunoassay ( sas laboratory , royal victoria infirmary , newcastle upon tyne ) .  
all bone density measurements , for both the osteoporotic and control subjects , were performed by dxa using a hologic qdr 2000 bone densitometer ( hologic , waltham , mass ) . in vivo precision for measurement with this system
is 1.0% at the lumbar spine ( l1l4 ) and 1.5% for the femoral neck .
bmd results were obtained as an areal density in g / cm , but were also given as t and z scores .
the t score is the number of standard deviation units above or below the mean for normal young men , whilst the z score is the number of standard deviation units above or below the age - related normal men ( calculated using the manufacturer 's standard normal reference database ) .
individual pcr standardisation reactions were set up prior to setting up multiplex pcr for the three bmp2 snps .
the three bmp2 snp polymorphisms ( snp1 , ser37ala ; snp2 , ala94ser and snp3 , arg190ser ) studied are shown in the schematic diagram ( figure 1 ) .
the multiplex reactions were transferred into 384 well plates and treated with shrimp alkaline phosphatase to remove the excess dntps in the reaction mixture .
extension primers designed to detect the three bmp2 snps were added to the samples and incubated as a pcr reaction for 2  h. salt ions were removed from the reaction mixture using a desalting resin and finally resuspended with 16  l  ddh2o .
15  nl of each sample was spotted onto the chip and analysed on the sequenom ( maldi - tof mass spectrometer ) .
the results were collected by spectroanalyser software and the snp information , represented by chromatogram peaks at g , t , a , gt , or at , was sorted by the typeranalyser software .
the snp information was then exported along with the sample details into ms - excel file(s ) for further statistical analysis .  
data were analyzed using appropriate statistical packages ( microsoft excel xl , spss ) ; the significance of differences in genotype and allele frequency between control subjects and men with vertebral fractures was determined using odds ratio and  tests .
anova and student 's unpaired t - test were used to determine significant differences in bmd and anthropometric measurements between different bmp2 genotypes and between control subjects and osteoporotic men , respectively .
genetic association analyses were carried out using an online hwe calculator ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) and snpstats programme ( http://bioinfo.iconcologia.net/snpstats ) which also worked out the hardy - weinberg equilibrium statistics .
the binary logistic regression analyses were performed ( spss , version 13.0 ) with disease or fracture outcome as the dependent variables .
the genotypes as categorical variables were used as specific genotypes ; the other variables included in the regression analysis were age , height , weight , bmi , and key relevant biochemical parameters .
the age , anthropometric , and bmd measurements for the men with osteoporosis and the control subjects are given in table 1 .
the men with osteoporosis , who had age range of 30 to 86  yr , were on average 6 years younger than controls with the age range of 41 to 79  yr .
for this reason when comparing bone density comparisons , to compensate for age - related decline in bmd , comparison with z score was performed .
the osteoporotic patients were also significantly shorter and lighter than the male control subjects , as well as having significantly lower bmd at the lumbar spine , total femoral and femoral neck ( p < 0.0001 ) . in both men with osteoporosis and control subjects
the bmp2 gene snp1 gg genotype was absent , and in the combined cohort only eight subjects were found to be heterozygotes , thus making this locus uninformative .
snp1 genotype , due to ser37ala variants that change a conserved amino acid , has been previously shown to have significant association with osteoporosis in both danish and icelandic cohorts . however , in our cohorts the frequency of snp1-related g allele was low in both the patients ( 0.004 ) and controls ( 0.015 ) .
snp2 , which gives rise to ala to ser change at 94th position , was rare , uninformative , and monomorphic , and of the three genotypes gg , gt , and tt , only gg was observed .
snp3 ( arg190ser ) was polymorphic , and distribution of genotypes and allele frequencies in both male controls and men with osteoporosis is shown in table 2 . in both cohorts
genotype aa was slightly higher in controls ( 14.3% versus 10.2% , p = 0.36 ) while susceptibility genotype tt was higher in patients ( 38.1% versus 31.7% , p = 0.28 ) .
the odds ratio for bmp2 snp3 genotypes and alleles * a and * t showed no statistically significant evidence of association with the risk of osteoporosis , although a trend towards carriers of the * t allele may be worth exploring . linkage disequilibrium ( ld )
the d prime is 0.989 ( p  value = 0.0264 ) , suggesting that there is a significant linkage disequilibrium between snp1 and snp3 , but caution is warranted in view of the relatively small numbers and low allele frequencies especially at snp1 locus which led to a low r value ( 0.02 ) .
haplotype analysis based on two snps ( snp1 and snp3 ) showed that two common haplotypes ( ta and tt ) accounted for > 99% of the alleles at these bmp2 loci ( table 3 ) .
haplotype - based association analysis indicated higher odds ratio for t - t haplotype ; however , it was nonsignificant ( or = 1.43 , ci 0.962.15 , p = 0.08 ) .  
the effect of bmp2 gene snp3 genotypes on weight , height , and bmd at lumbar spine , femoral neck , and total femoral was analysed by anova in both the male controls and men with osteoporosis , and genotype was found to lack any significant association ( table 4 ) .
the analysis of the pooled data , comprising controls and patients , also failed to demonstrate any significant association of snp3 genotype with the bmd or anthropometric indices ( data not shown ) .  
the association of the snp3 genotype was also determined by employing a binary logistic regression model , which accounted for between 44.5% and 62.6% of the variance in disease status and the overall model successfully predicted 72% of osteoporotic cases correctly ( table 5 ) .
an analysis of fracture incidence with reference to genotypes and age , age at fracture , and biochemical parameters was performed .
age at fracture , weight , biochemical parameters , and genotypes did not show any interaction or independent effect in the fracture prediction .
the addition of covariates to the model resulted in very sparse data categories and led to wide confidence intervals .
this investigation is the only one from the uk that has sought an association between bmp2 gene polymorphisms and bmd and susceptibility to osteoporosis .
as far as we know this is the first such investigation carried out exclusively in men with idiopathic osteoporosis .
however , the power of this study is low ; therefore any conclusions drawn should be treated with caution .
post hoc power analysis , calculated using a quanto programme and based on observed allele frequencies , sample size , and odds ratios , showed that the study had 67% power to detect an odds ratio of 1.50 ( moderate level ) and above . in order to achieve an odds ratio of 1.25 one would require a case - control sample size of 507 individuals in each group . in the original icelandic study a significant effect of bmp2 gene snp1 polymorphism , ser37ala ,
was demonstrated in a cohort of 201 subjects , comprising 153 controls and 58 subjects with fracture . just like the earlier observation we too found it to be a rare variant in our cohort with an allele frequency of 1.25% for ala37 . in previous reports ,
the allele frequency for ala37 in the icelandic cohort was 0.8% in controls and 3% to 4.9% in osteoporotic patients and 2.5% in the rotterdam study .
however , unlike the icelandic study , we did not see a significant association or increase in the relative risk ( rr ) with the snp1 genotype . in our cohort
we did not see any gt or tt genotypes of the rare variant of the snp2 genotype that arises from ala94ser .
the gene frequency for snp3 polymorphism , due to arg190ser variants , constituting three genotypes , tt ( arg190arg ) , at ( arg190ser ) , and aa ( ser190ser ) , was akin to earlier reports in european populations . in both male controls and men with osteoporosis
in addition to studying the association of the bmp2 to risk of fracture and bmd , in a subset of subjects we also investigated the association of the genotype with annual bone loss at the lumbar spine , total femoral , and femoral neck . both initial bmd as well as
this could be partly due to allele frequency differences in different geographical and gender cohorts . in our study
we observed a higher frequency of susceptible t ( ser ) allele ( 64% in patients and 59% in controls ) which is comparable to hapmap frequencies ( 58%73% in european populations ) , but other populations like turkish , korean , and african populations show a lower frequency [ 1315 ] .  
the desirability of having a large cohort for snp studies investigating polygenic disease association with putative candidate genes with phenotype or disease state is well established .
this is due to the fact that in a polygenic disorder like osteoporosis many genes make a small , but significant contribution towards the attainment and maintenance of bmd .
therefore , many genes may be involved in the pathogenesis of osteoporosis and risk of osteoporotic fractures . in view of this small contribution of a large number of genes ,
the contribution of the genotypic influence is likely to be more clearly demonstrated in a sufficiently large cohort with the required level of statistical power .
it is , however , becoming increasingly evident that the association of a large number of candidate gene snps with bmd and osteoporosis risk shows race- , geography- and gender - related differences [ 3 , 16 , 17 ] .
we would also contend that , if a given genotype does make a substantial contribution to the attainment and maintenance of bmd and pathophysiology of disease then it should be possible to demonstrate such effects even in smaller homogenous cohorts .
indeed , in the original study on bmp2 the genotype association with osteoporosis was established in a small cohort . we have been able to demonstrate associations of some novel genotypes in smaller cohorts and which were subsequently verified in much larger studies [ 5 , 18 , 19 ] .  
there have been only a handful of studies following the first report of bone morphogenetic protein 2 ( bmp2 ) as a susceptibility gene for osteoporotic fractures and low bmd in icelandic and danish populations .
even these relatively few studies of polymorphisms , within the bmp2 gene in relation to bone mineral density ( bmd ) and fracture , have produced inconsistent findings [ 49 ] .
neither a rotterdam study of a large population - based cohort of dutch whites nor a study in healthy american whites could find any contribution to bmd by variations in bmp2 genotypes [ 7 , 8 ] .
on the other hand , a snp- and haplotype - based us family study showed highly suggestive associations with bmp2 . in another analysis on a european cohort , variation in bmp2
genotype showed no association with bmd ; it did , however , find a role in aspects of bone quality , which may be age and site dependent .
the 3 region of the gene was significantly associated with the ultrasound parameters speed of sound and stiffness .
similarly in turkish women study bmp2 polymorphisms did not substantially contribute to lumbar spine bone mineral density .
overall , association studies have shown that arg190ser snp is not associated with bmd in different populations and the initial study may have been a false positive association .
alternatively , it is entirely possible that the association may show gender - related variation , as the initial study had shown association in females and our study was carried out in an exclusively male population .   in conclusion ,
in a bmp2 candidate gene polymorphism study in the uk , comprising osteoporotic men and male control subjects , there were no genotypic or haplotypic effects on phenotype and fracture risk identified .
 
 introduction . 
 the association of bone morphogenetic protein 2 ( bmp2 ) with bmd and risk of fracture was suggested by a recent linkage study , but subsequent studies have been contradictory . 
 we report the results of a study of the relationship between bmp2 genotypes and bmd , annual change in bmd , and risk of fracture in male subjects . 
 materials and methods . 
 we tested three single - nucleotide polymorphisms ( snps ) across the bmp2 gene , including ser37ala snp , in 342 caucasian englishmen , comprising 224 control and 118 osteoporotic subjects . 
 results . 
 bmp2 snp1 ( ser37ala ) genotypes were found to have similar low frequency in control subjects and men with osteoporosis . 
 the major informative polymorphism , bmp2 snp3 ( arg190ser ) , showed no statistically significant association with weight , height , bmd , change in bmd at hip or lumbar spine , and risk of fracture . 
 conclusion . 
 there were no genotypic or haplotypic effects of the bmp2 candidate gene on bmd , change in bmd , or fracture risk identified in this cohort . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion