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Keywords:

  • BONE MINERAL DENSITY;
  • QUANTITATIVE ULTRASOUND;
  • FEMORAL GEOMETRY;
  • MUSCLE MASS;
  • PRINCIPAL COMPONENT ANALYSIS;
  • GENOME-WIDE ASSOCIATION;
  • SINGLE NUCLEOTIDE POLYMORPHISMS;
  • PLEIOTROPY

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Multiple musculoskeletal traits assessed by various methods at different skeletal sites serve as surrogates for osteoporosis risk. However, it is a challenge to select the most relevant phenotypes for genetic study of fractures. Principal component analyses (PCA) were conducted in participants of the Framingham Osteoporosis Study on 17 measures including bond mineral density (BMD) (hip and spine), heel ultrasound, leg lean mass (LLM), and hip geometric indices, adjusting for covariates (age, height, body mass index [BMI]), in a combined sample of 1180 men and 1758 women, as well as in each sex. Four principal components (PCs) jointly explained ∼69% of the total variability of musculoskeletal traits. PC1, explaining ∼33% of the total variance, was referred to as the component of “Bone strength,” because it included the hip and spine BMD as well as several hip cross-sectional properties. PC2 (20.5% variance) was labeled as “Femoral cross-sectional geometry;” PC3 (∼8% variance) captured only ultrasound measures; PC4, explaining ∼7% variance, was correlated with LLM and hip geometry. We then evaluated ∼2.5 mil SNPs for association with PCs 1, 2, and 4. There were genome-wide significant associations (p < 5 × 10−8) between PC2 and HTR1E (that codes for one of the serotonin receptors) and PC4 with COL4A2 in women. In the sexes-combined sample, AKAP6 was associated with PC2 (p = 1.40 × 10−7). A single nucleotide polymorphism (SNP) in HTR1E was also associated with the risk of nonvertebral fractures in women (p = 0.005). Functions of top associated genes were enriched for the skeletal and muscular system development (p < 0.05). In conclusion, multivariate combination provides genetic associations not identified in the analysis of primary phenotypes. Genome-wide screening for the linear combinations of multiple osteoporosis-related phenotypes suggests that there are variants with potentially pleiotropic effects in established and novel pathways to be followed up to provide further evidence of their functions. © 2012 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Age-related osteoporotic fractures are common in the Untied States and represent a major public health threat that is likely to increase in importance as the population ages.1 Osteoporotic fracture is heritable; however, the fracture phenotype is a difficult one to study genetically: fractures typically do not occur until later in life, and are influenced by factors outside of the skeleton. Therefore, risk factors for osteoporotic fracture (proxy phenotypes), such as bone mineral density (BMD), bone quantitative ultrasound (QUS), and bone geometry are traditionally investigated, because these quantitative traits predict risk of osteoporotic fractures2–5 and may be reliably measured at any age. Studies over the last decades have documented the major contribution of genes to BMD,6, 7 QUS,8 and bone geometry.9, 10 Because fracture is a product of both bone strength and forces applied to the skeleton, muscle mass has also been considered to be a risk factor for fractures;11 moreover, it is characterized by a shared heritability with bone strength properties.12, 13

It is important to realize that none of these proxies is a perfect phenotype of osteoporosis, especially for a genetic study. There might be some unique information obtained from each risk factor, however, because these phenotypes are correlated and partially redundant, a study of the commonality of genetic associations among these traits may prove useful in obtaining a more complete understanding of the mechanisms underlying fracture susceptibility. Genetic markers that influence highly correlated traits can be detected by several methods. One approach to extract nonredundant information is to apply principal component analysis (PCA) to the data. PCA transforms the original phenotypes to an equal number of orthogonal factors (PCs), each defined as a specific linear combination of the original phenotypes.

In earlier work, we applied PCA to BMD and heel ultrasound measures; we identified two independent heritable PCs, which were linked to several chromosomal regions.14 quantitative trait loci (QTL) for these integrated phenotypes were further replicated by a large-scale meta-analysis of BMD,15 attesting to the power and merits of this approach. PCA analysis has been applied by others to multiple bone-related traits in mice.16, 17 We hypothesized that using a composite measure of several correlated phenotypes may provide new and complementary insights into the genetics of osteoporosis, by both reducing the measurement error of several related variables and improving genetic signals through an integrated phenotype definition. Correlated musculoskeletal phenotypes should therefore point towards gene networks that may contribute to these phenotypes.18, 19

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Sample

The sample used for our analyses was derived from two cohorts of the population-based Framingham Heart Study (FHS). FHS is a large, longitudinal population- and family-based study with multiple well-characterized phenotypes that began in 1948 in Framingham, MA, USA, with the enrollment of 5209 men and women ages 28 to 62 years (Original Cohort). In 1971, the FHS Offspring Study was initiated by enrolling 5124 adult children of the Original Cohort and their spouses. Details and descriptions of the Framingham Osteoporosis Study are provided elsewhere,20–23 as well as publicly available through the Database of Genotype and Phenotype (dbGaP) at http://view.ncbi.nlm.nih.gov/dbgap. In brief, the Original and the Offspring Cohorts in the Osteoporosis Study represent adult members of two-generational (mostly nuclear) families. The Osteoporosis Study was approved by the institutional review boards for Human Subjects Research of Boston University and Hebrew SeniorLife and all participants provided written informed consent.

Osteoporosis-related musculoskeletal phenotypes

The following measures were available in members of both Framingham Cohorts.

BMD

The participants underwent bone densitometry by dual-energy X-ray absorptiometry (DXA) with a Lunar DPX-L (Lunar Corp., Madison, WI, USA) between 1996 and 2001. The coefficients of variation (CV) in normal subjects for the DPX-L have been previously reported to be 0.9% for the lumbar spine (LS), 1.7% for the femoral neck (FN), and 2.5% for the trochanter BMD.20

QUS

Calcaneal broadband ultrasound attenuation (BUA) and speed of sound (SOS) were measured with a Sahara® bone sonometer (Hologic, Inc., Waltham, MA, USA), between 1996 and 2001. Based on duplicate, same-day measurements on 29 subjects, CVs for BUA and SOS were 5.3% and 0.4%, respectively.24

Hip geometry

DXA scans were measured with an interactive computer program.25 The program derived a number of proximal femoral structural variables, including (1) gross anatomic measures, such as femoral neck length (FNL), neck-shaft angle (NSA), and subperiosteal diameter (width, cm); and (2) cross-sectional indices, such as cross-sectional bone area (CSA, cm2), section modulus (Z, cm3), and buckling ratio (unitless) at the two femoral regions (narrow neck, NN, and the femoral shaft, S). CVs were previously reported to range from 3.3% (NN outer diameter) to 9.1% (FNL).25

Body composition

Whole-body DXA scans were obtained from the study participants with the same Lunar DPX-L machine used for BMD and at the same visit. The scans were collected at medium speed for all subjects regardless of weight or body thickness. Regions of interest were analyzed using the standard Lunar software for body composition. Leg lean mass (LLM, kg) was derived by subtracting regional bone mineral content from the fat-free mass of the lower extremities.

Nonvertebral fractures

Using the assessment protocol in the Framingham Osteoporosis study that has been reported previously:26 incident fractures were reported in the Offspring, by interview at each examination (conducted every 4 years) or by telephone interview for participants unable to attend examinations. Fractures included hip, wrist, pelvis, femur, humerus, rib, sternum, lower arm, ankle, and lower leg fractures, and were confirmed by review of medical records and radiographic and operative reports, when available. Whenever possible, fractures because of high trauma (eg, a fall from higher than standing height, severe traffic accidents) were excluded; multiple simultaneous fractures in different sites were counted as one fracture. The Offspring cohort's participants were followed for fractures from the date of the DXA scan through December 31, 2007.

Other measurements (covariates)

Information on age, sex, height, body mass index (BMI), and in women, menopausal status, and estrogen use, were obtained for each participant at the time of the musculoskeletal measurement. Details for these measurements are available elsewhere.20, 27

Genotyping, quality control, and imputation

Genotyping was conducted through the FHS SHARe (SNP Health Association Resource) project initiated in 2007 on all Framingham Study participants with DNA available, using the Affymetrix 500K (250K Sty and 250K Nsp) mapping array and the Affymetrix 50K supplemental gene-centric array (50K MIP). A total of 549,827 single nucleotide polymorphisms (SNPs) were genotyped in 9274 FHS subjects. We excluded 793 individuals with an average SNP call rate <0.97. We also excluded SNPs with call rate <0.95 (34,868 SNPs); Hardy-Weinberg equilibrium test p-value <10−6 (8531 SNPs); minor allele frequency (MAF) <0.01 (66,829 SNPs); or unknown genomic annotation (6,089 SNPs). Genotyping from 433,510 SNPs in 8481 individuals passed these quality control measures.

SNP imputation in the FHS was performed on all 8481 individuals, using MACH2, which outputted imputed dosages for all autosomal 2,543,887 SNPs on HapMap CEU release 22, build 36. Imputation of 57,197 SNPs for the X chromosome was performed with the program IMPUTE. SNPs with the imputed variance ratio <0.3 were excluded.

Population substructure

A principal component analysis was performed using EIGENSTRAT28 to model differences of individuals' ancestral genetic background (to infer axes of variation) using a subset of 425,173 SNPs with MAF ≥0.01, HWE p ≥ 10−6, and call rate ≥0.95. SNP weights for 10 principal components (EIGENSTRAT-PCs) were calculated using a maximal set of independent individuals (n = 882); the PCs for the remaining individuals were computed using the SNP weights obtained from this subset of unrelated individuals. EIGENSTRAT-PCs that were significantly associated with musculoskeletal PC phenotypes (p < 0.05) were adjusted for in our GWAS analyses to minimize spurious associations because of population substructure.

Statistical analysis

PCA of the musculoskeletal traits

Measures including BMD (hip and spine), heel ultrasound, leg lean mass, and hip geometric indices were adjusted for covariates: age, height, BMI (and sex in combined sample). PCA was conducted in a combined sample of 1180 men and 1758 women who had all the studied traits measured (nonmissing), using the information from the entire correlation coefficient matrix of these phenotypes (provided in Supplementary Table 3). Our analyses were also performed in men and women separately, because musculoskeletal traits, especially BMD, bone geometry and muscle mass, are sexually dimorphic. PCA was conducted in order to (1) examine the interrelationship among the variables and (2) produce a substantially smaller number of independent (uncorrelated) hypothetical underlying factors.29 This analysis produces linear combinations of the original measures that capture most of the information (variance) of these measures. The first principle component (PC1) accounts for as much of the variability in the data as possible. The analysis was performed in two stages: (1) factor extraction and (2) rotation of the principal components using the Varimax option. Those factors with eigenvalues >1.0 were retained for further investigation. We calculated PCs using weights from PCA of unrelated subjects in our data. To obtain representative estimates of the weights for the rest of the sample, we used averages from 10,000 PCAs of resampled unrelated subjects.

Genome-wide association study (GWAS)

We performed GWAS analyses of musculoskeletal PCs using population-based additive linear mixed effects (LME) models30 with ∼2.5 mil SNPs, in sexes-combined sample, as well as in the women-only subsample. In addition to adjusting for covariates, LME regression models account for correlations because of family relationships in pedigrees of arbitrary sizes and varying degrees of relationship. Because all the SNPs were imputed, we used the expected dosages (number ranging between 0 and 2) in our regression models. Further, to minimize missing genotypes in our data, we used only well-imputed SNPs in our analyses. SNP associations at p < 5 × 10−8 were considered to be genome-wide significant (GWS); SNPs with p < 5 × 10−5 were considered “suggestive” associations.31

SNP association with the nonvertebral fractures

Using logistic regression, each GWS SNP was tested for association with the nonvertebral fractures outcome using an additive genetic model. The mean dosage of one of the alleles (a value between 0 and 2) was the predictor variable, in the sex-combined sample (adjusting for sex) and in women-only sample. We used generalized estimating equations (GEE) to account for familial correlations. Because we assessed associations between top SNPs identified for PCs with fracture risk, significance was set with Bonferroni adjustment for the number of tests.

Bioinformatic analysis

Annotation

For the SNPs associated with PCs at p < 5 × 10−5, gene annotation was based on the UCSC table browser for all RefSeq (hg18) genes. If a SNP was located outside known genic regions, the nearest RefSeq gene was assigned as the gene annotation. The distance between nongenic SNPs to the nearest gene ranged from 99 bp to 1514 kbp.

Identification of enriched physiological function in identified genes

To better understand potential functional roles and the biological validity of the top associated genes on a gene-level as well as a gene network level, we inferred novel gene networks for each PC using Ingenuity Pathways Analysis (IPA) Software (Ingenuity Systems, Redwood City, CA, USA). In IPA, there are 43 high-level physiological functional categories, and “skeletal and muscular development and function” is one of these categories. Each high-level physiological function contains a number of lower level (or more specific) physiological functions. The functional analysis identified the biological functions that were significantly associated with the data set. Annotated genes associated with biological functions and/or diseases in Ingenuity's Knowledge Base were considered for the analysis. Right-tailed Fisher's exact test was used to calculate a p-value determining the probability that the “skeletal and muscular development and function” assigned to that data set was because of chance alone. A p-value of <0.05 was considered significant.

Gene network inference via knowledge-based data mining

We next analyzed biological interactions among identified genes using the IPA tool. The gene annotations from the GWAS SNPs were entered into the IPA analysis tool to construct the biological networks of the top associated genes. Networks are generated from the gene set by maximizing the specific biological relationship of the input genes, which represents their interconnectedness with each other relative to other molecules they are connected to in Ingenuity's Knowledge Database. All biological relationships are supported by at least one reference from the literature or from canonical information stored in the Ingenuity Pathways Knowledge Base. Networks were limited to 35 molecules each to keep them to a functional size. The p-value of probability for the genes forming a network was calculated with the right-tailed Fisher's Exact Test based on the hypergeometric distribution. To gain biological insights on whether this novel gene network was associated with any known canonical pathways, we further overlaid the gene network with canonical pathways using IPA.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Characteristics of the sample are provided in Table 1, by cohort and gender. In each cohort, men and women were of similar age. As expected, male participants were heavier, taller and in general had greater average BMI, leg lean mass, BMD, QUS, and geometric measures than females (but lower for shaft buckling ratio). Because of missing values for some musculoskeletal traits, 1180 men and 1758 women were included in the final analysis. Of the total 1758 women, there were 641 postmenopausal women, mostly from the Offspring Cohort, who were not on estrogen.

Table 1. Characteristics of the Studied Sample, by Cohort and Gender
VariableOriginal cohortOffspring cohort
MalesFemalesMalesFemales
Mean ± SDMean ± SDMean ± SDMean ± SD
  • a

    Number of subjects for all available participants with both genotypes and phenotypes.

Na1442619621362
Age (years)77.3 ± 3.677.7 ± 4.061.3 ± 9.260.3 ± 9.3
Height (meter)1.70 ± 0.071.56 ± 0.061.74 ± 0.071.61 ± 0.06
Weight (kg)79.2 ± 12.465.8 ± 12.186.6 ± 13.070.8 ± 14.2
BMI (kg/m2)27.4 ± 3.827.0 ± 4.928.4  ± 3.827.3  ± 5.3
Premenopausal or currently on estrogen, N (%)19 (7.28%)625 (45.88%)
BMD (g/cm2)
 Femoral neck0.866 ± 0.1250.723 ± 0.1190.966 ± 0.1320.868 ± 0.140
 Total femoral0.937 ± 0.1410.760 ± 0.1291.036 ± 0.1400.911 ± 0.147
 Trochanter0.826 ± 0.1330.624 ± 0.1210.877 ± 0.1360.714 ± 0.133
 Lumbar spine1.313 ± 0.2131.053 ± 0.2111.311 ± 0.1971.153 ± 0.197
Quantitative ultrasound
 BUA (dB/MHz)77.78 ± 22.4353.37 ± 16.5383.14 ± 19.0371.40 ± 18.44
 SOS (m/s)1546.25 ± 38.191513.49 ± 30.121559.73 ± 34.271549.60 ± 35.14
Bone geometry
 NSA (degrees)131.7 ± 6.2128.0 ± 5.5129.4 ± 4.9127.5 ± 5.2
 FNL (cm)5.4 ± 0.84.6 ± 0.66.0 ± 0.85.2 ± 0.6
 Narrow neck outer diameter (cm)3.4 ± 0.32.9 ± 0.33.8 ± 0.53.3 ± 0.4
 Narrow neck CSA (cm2)2.4 ± 0.41.8 ± 0.42.8 ± 0.42.3 ± 0.4
 Narrow neck section modulus (cm3)1.4 ± 0.30.9 ± 0.21.8 ± 0.41.3  ± 0.3
 Narrow neck buckling ratio12.9 ± 3.213.6 ± 4.711.4 ± 6.210.4 ± 6.0
 Shaft Outer diameter (cm)3.2 ± 0.23.0 ± 0.23.5 ± 0.43.2 ± 0.4
 Shaft CSA (cm2)4.2 ± 0.62.8 ± 0.54.5 ± 0.63.3 ± 0.5
 Shaft Section modulus (cm3)2.6 ± 0.41.6 ± 0.32.8 ± 0.51.9 ± 0.4
 Shaft buckling ratio3.6 ± 0.94.7 ± 1.33.9 ± 1.14.7 ± 1.5
 Muscle mass    
 Leg Lean mass (kg)17.04 ± 2.2311.53 ± 1.5417.21 ± 2.2111.57 ± 1.57

PCA of osteoporosis-related phenotypes

We conducted PCA in sexes-combined as well as in 1180 men and 1758 women, separately. In sexes-combined analysis as well as in women, the PCA found four components above the 1.0-eigenvalue threshold, which jointly explained 69.3% of the total variability of musculoskeletal characteristics, in each sample (Table 2a). In men, the PCA revealed an additional principal component with an eigenvalue >1.0 (cumulatively explaining up to 71.8% variance; Table 2b). Based on the loadings of the musculoskeletal phenotypes, principal component 1 (PC1), explaining 33.9% of the total variance in the combined sample (33.5% in women and 33.2% in men), may be referred to as representing “Bone strength,” because it correlates with BMD and several hip cross-sectional properties, such as narrow neck and shaft CSA and section modulus (also, inversely correlated with buckling ratio). PC1 weights were very similar in all three analyses for most variables except for shaft section modulus, shaft buckling ratio, and LLM in men.

Table 2. Component Loading Matrix After Varimax Rotation
a) Sexes combined
 PC1PC2PC3PC4
Bone strengthFemoral cross-sectional geometryHeel ultrasoundFemoral shaft stability
Total body BMD 88−1124−15
Femoral neck BMD 84−519−20
Trochanter BMD 82−1124−7
LS BMD 63−229−1
Heel ultrasound/BUA302 894
Heel ultrasound/SOS302 900
Femoral neck Length8 62−2−7
Femoral neck shaft Angle−10−159 50
Narrow neck outer diameter−10 8945
Narrow neck CSA 813410−11
Narrow neck Section modulus49 727−6
Narrow neck Buckling ratio−3510948
Shaft outer diameter−13 91−1−6
Shaft CSA 849623
Shaft section modulus 59 61120
Shaft buckling ratio55 67−5−16
Leg lean mass22−1−17 69
Eigenvalue5.753.491.381.15
Explained variance (%)33.920.58.156.75
b) Men
 PC 1PC 2PC 3PC 4PC 5
Bone strengthFemoral cross-sectional geometryHeel ultrasoundFemoral shaft stabilityFemoral neck geometry
Total body BMD 88−12279−10
Femoral neck BMD 88−6192−4
Trochanter BMD 82−122613−7
LS BMD 65327118
Heel ultrasound/BUA260 916−3
Heel ultrasound/SOS282 913−1
Femoral neck length149612 −57
Femoral neck shaft angle−8−507 89
Narrow neck outer diameter−12 8953−4
Narrow neck CSA 843012131
Narrow neck section modulus 52 7488−2
Narrow neck buckling ratio5011142210
Shaft outer diameter−10 91−4−7−13
Shaft CSA 68019 54−11
Shaft section modulus45 61940−13
Shaft buckling ratio−42 73−14−32−4
Leg lean mass1−41 834
Eigenvalue5.983.631.841.301.09
Explained variance (%)33.220.210.27.26.0
c) Women
 PC 1PC 2PC 3PC 4
Bone strengthFemoral cross-sectional geometryHeel ultrasoundFemoral shaft stability
  1. Values are multiplied by 100 and rounded to the nearest integer.

  2. The highest weights (≥0.5) are marked in bold.

Total body BMD 87−1324−16
Femoral neck BMD 83−520−21
Trochanter BMD 80−1225−6
LS BMD 64−3291
Heel ultrasound/BUA301 893
Heel ultrasound/SOS301 89−1
Femoral neck length7 63−2−1
Femoral neck shaft angle−5−811 56
Narrow neck outer diameter−7 9023
Narrow neck CSA 77388−18
Narrow neck section modulus49 705−11
Narrow neck buckling ratio−2896 54
Shaft outer diameter−13 910−3
Shaft CSA 8712215
Shaft section modulus 63 58−316
Shaft buckling ratio59 65−3−10
Leg lean mass20−4−27 64
Eigenvalue5.693.481.471.15
Explained variance (%)33.4520.468.646.75

PC2 can be labeled as “Femoral cross-sectional geometry” component (explaining 20.5% variance in sexes-combined and women, and 20.2% variance in men), because this PC correlated with femoral neck length, narrow neck, and shaft outer diameters and section moduli, and shaft buckling ratio.

PC3, explaining 8.15% total variance, was labeled a “Heel ultrasound” component, because it was strongly correlated only with the two measures of QUS (and slightly inversely, with LLM in women). PC4, explaining 6.75% variance, was correlated with NSA and LLM in the combined sample (less strongly with narrow neck buckling ratio, NN BR) and was thus deemed “Femoral shaft stability” factor. This PC was characterized with most sex-specificity: thus, in women, it was strongly correlated with NN BR, wheres in men, it was strongly correlated with leg lean mass and shaft CSA, but not with NSA. Finally, there is an additional PC5 in men, explaining 6.0% of the total variance, which can be labeled “Femoral neck geometry” (because it correlated with FNL and NSA).

In the ensuing genetic analysis we decided to focus on PC1, PC2, and PC4, because these components explained a large proportion of the total variability in multiple musculoskeletal traits, whereas PC3 is only a factor of heel ultrasound. Also, we performed GWAS in both sexes-combined and women-only analyses, given a smaller size and weaker power of men-only sample.

GWAS

Because some resulting musculoskeletal PCs showed correlation with the ancestry principal components (EIGENSTRAT-PCs), we adjusted PC1 for EIGENSTRAT-PCs 2, 3, and 4 in the SNP association analyses; similarly, PC4 was adjusted for EIGENSTRAT-PCs 1, 2, and 3. After this adjustment the inflation factors (λGC) were ≤1.015 for GWAS of all musculoskeletal PCs.

GWAS of the PCs 1, 2, and 4 in the sexes-combined sample did not produce genome-wide significant associations (p < 5 × 10−8). The best signal was between SNP rs7158720 within the A kinase anchor protein 6 (AKAP6) gene on chr. 14 and PC2, with p-value 1.40 × 10−7. Notably, rs7158720 within AKAP6 gene was also associated with PC2 in women, with p = 4.1 × 10−5 (Table 3). In the sample of women, genome-wide significant associations (p < 5 × 10−8) were observed for PC2 with two SNPs in linkage disequilibrium (r2 = 0.935) on chr. 6: rs9351097 and rs9362321, both in the 5-hydroxytryptamine (serotonin) receptor 1E (HTR1E) gene. There are several other SNPs, some in high LD, in the vicinity of the HTR1E gene, among which two SNPs were associated with PC4 in sexes-combined analysis at p < 3.2 × 10−5. Finally, two SNPs in the COL4A2 gene were highly significantly associated with PC4 in women (rs4773155, p = 2.50 × 10−8, and rs4447275, p = 5.30 × 10−8). These SNPs are in LD (r2 ∼ 0.5); there was no suggestive association of this region within the sexes-combined sample.

Table 3. Top SNPs Associated With PCs in Combined-Sexes and Women-Only Sample
PC, sampleSNPChrPositionCoded alleleNoncoded alleleCoded allele frequencyLDpvalBetah2qIn/near genea
  • LD = linkage disequilibrium with the most significant SNP; h2q = variance explained by an SNP.

  • a

    Distance from the nearest gene was assumed not to exceed 500 kb.

PC2, womenrs9351097687194834 T G0.175 3.40*10 −8 0.2520.0164HTR1E
PC2, womenrs9362321687192966TC0.1740.838 4.30*10 −8 0.2460.0162HTR1E
PC2, womenrs9362322687197144AT0.1960.838 9.30*10 −8 0.2420.0154HTR1E
PC2, womenrs9362320687192883TC0.0180.096 6.00*10 −7 0.6480.0135HTR1E
PC2, womenrs6919366687169891GT0.3370.336 9.30*10 −7 0.1760.0130HTR1E
PC4, combinedrs6911565687557585AC0.1370.1781.10*10 −5 −0.190.0081HTR1E
PC4, combinedrs12665525687553199CT0.1170.1783.20*10 −5 −0.180.0073HTR1E
PC2, combinedrs71587201432347622AC0.209 1.40*10 −7 0.1610.0086AKAP6
PC2, womenrs71587201432347622AC0.2084.10*10 −5 0.1670.0091AKAP6
PC2, combinedrs101486941432343262TC0.3580.4642.40*10 −5 0.1120.0055AKAP6
PC4, womenrs477315513109780420AC0.432 2.50*10 −8 0.3560.0204COL4A2
PC4, womenrs444727513109769067GA0.4620.533 5.30*10 −7 −0.2720.0166COL4A2

At the suggestively significant threshold p < 5 × 10−5, there were 186 and 182 SNPs associated with PC1 in the sexes combined and women-only analyses, respectively: 161 and 121 SNPs associated with PC2, and 115 and 177 SNPs with PC4. SNP associations with the most significant p-values are provided in Table 3, and regional plots are shown in Figure 1. Table 3 also shows the variance explained in PCs by each of the top SNPs, which ranged from 0.55% to 2.04%.

thumbnail image

Figure 1. Regional plots of SNP-PC phenotype association (index SNPs are marked). (A) Combined PC2 (rs7158720). (B) Women PC2 (rs9351097). (C) Women PC4 (rs4773156).

Download figure to PowerPoint

Further, each GWS SNP was tested for association with nonvertebral fracture outcomes, in sex-combined sample and in female-only sample (n = 1984 and 1208, respectively), using logistic regression. SNP rs6919366 in HTR1E was significantly associated with nonvertebral fractures in women, even after Bonferroni correction for number of test (Table 4).

Table 4. Top SNPs Associated With PCs in Combined-Sexes and Women-Only Sample: Association with the Nonvertebral Fractures
SNPChrPositionCoded alleleNoncoded alleleIn/near geneSexes combinedaWomen-onlyb
ORp-ValueORp-Value
  • a

    Sample size = 1984;

  • b

    Sample size = 1208.

rs9351097687194834TGHTR1E1.12770.28831.20890.1417
rs9362321687192966TCHTR1E1.12570.28681.20840.1359
rs9362322687197144ATHTR1E1.05410.64871.13100.3392
rs9362320687192883TCHTR1E0.86040.67380.82540.6523
rs6919366687169891GTHTR1E1.20840.04461.3451 0.0051
rs6911565687557585ACHTR1E0.86860.29030.97460.8617
rs12665525687553199CTHTR1E0.84160.19710.92690.6081
rs71587201432347622ACAKAP61.04060.70511.02140.8585
rs101486941432343262TCAKAP61.08640.36601.10200.3561
rs477315513109780420ACCOL4A21.05950.69421.03630.8366
rs444727513109769067GACOL4A20.90530.42740.91450.5371

Bioinformatics analyses

In the sexes-combined sample, 186, 161, and 115 SNPs suggestively associated with PC1, PC2, and PC4 were mapped and annotated to 48, 36, and 41 genes, respectively. All of these gene lists were significantly associated with the skeletal and muscular system development and function (“high-level function”; p < 0.05) (Table 5). For example, PC1 was significantly associated specifically with the functional annotation of osteopenia and osteoporosis (p < 0.05). PC2 was significantly associated with the functional annotation of ossification, fracture of bone, development of osteoblast, and formation of osteophyte (p < 0.05). PC4 was significantly associated with the function annotation of development of skeleton, arrest in growth and differentiation of bone cell lines (p < 0.05). In a sample of women, 182, 121, and 177 SNPs associated with PC1, PC2, and PC4 were mapped and annotated to 58, 32, and 43 genes, respectively. Similarly to the sexes-combined results, all of these gene lists were significantly associated with the skeletal and muscular system development and function (p < 0.05) in women.

Table 5. Gene Networks Associated With Skeletal and Muscular System Development and Function Inferred From GWAS of Principal Components
PCp-ValueaNo. of molecules in the network
  • a

    p-value for enrichment analysis; ranges correspond to the different specific lower level functions or subcategories (eg, osteopenia, ossification, and mineralization) that are classified within the skeletal and muscular system development and function.

PC1 (combined)9.05*10 −5 –8.72*10 −3 4
PC2 (combined)2.55*10 −3 –7.54*10 −3 6
PC4 (combined)7.71*10 −5 –4.08*10 −3 8
PC1 (women)4.64*10 −4 –1.51*10 −2 6
PC2 (women)7.57*10 −4 –1.05*10 −2 8
PC4 (women)4.66*10 −3 –4.17*10 −2 4

Novel gene network inference

To better understand potential functional roles and the biological validity of the top genes on a gene level as well as a gene network level, we inferred novel gene networks for each PC using IPA. For each PC, we ranked genes by connectivity in each network, because highly connected proteins, or hub genes, are likely to be more essential to biological function and survival, and potentially represent therapeutic targets.18, 32 The novel gene network inferences are provided in supplementary Table 1 (attached at the end), and the hub genes/molecules (defined arbitrarily as those with greater than nine connections with other members in the network, that is, ∼25% of other 34 molecules, excluding self-regulatory connection) are provided in Supplementary Table 2.

Gene networks inferred from top genes in each PC were significantly associated with skeletal and muscular system development and function (all p < 0.05; Supplementary Table 1). There were in total 11 genes or molecules identified as “hubs” in the six top gene networks. Two of them, HNF4A and GPCR, appeared as hubs in more than one gene network (Supplementary Table 2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

GWAS offer an unbiased approach to identify new genetic pathways for a complex disease such as osteoporosis. Although genetic factors substantially contribute to the risk of osteoporotic fractures, the fracture phenotype presents a challenge for a genetic association study because nontraumatic fractures typically do not occur until later in life. An abundance of multiple surrogate phenotypes assessed by various methods at different skeletal sites makes a focused genetic search similarly challenging.

We hypothesized that a composite summary measure of several correlated phenotypes may provide a comprehensive outlook on the genetics of osteoporosis. One approach to extract essential nonredundant information is to apply principal component analysis to the correlation coefficient matrix of related phenotypes. PCA transforms the original phenotypes to a number of orthogonal PCs, each defined as a specific linear combination of the original phenotypes. Of note, although the PCs capture specific constructs underlying musculoskeletal traits, biological interpretation of the resulting phenotypes may not be obvious.16 In a sample of adult participants of the Framingham Osteoporosis Study, we found that independent PCs jointly explained 69.3% of the total variability of musculoskeletal characteristics in sexes-combined analysis (similarly, 76.9% of the total variability in men and 69.3% in women). The first two factors, PC1 (“Bone strength”) and PC2 (“Femoral cross-sectional geometry”), explained most of the total variance (∼33%–34% of the variance for PC1 and about 20 for PC2). PCs 3 and 4 explained ∼8.2% and ∼6.8% of the total variance. Notably, PC3 was strongly correlated only with two measures of QUS; therefore has less appeal for the study of the musculoskeletal system as a whole. It is important to emphasize that biological interpretation of the resulting PC constructs is not straightforward; therefore, the labeling we chose is an oversimplification in order to make the constructs more intuitive. Further, using bioinformatic and pathways analyses we found that the PCs could be linked to biologically meaningful functional annotations.

We screened the whole genome for associations with the integrated phenotypes represented the three PCs (PC1, PC2, and PC4), since PC3 explained only variability in heel ultrasound. Genome-wide significant associations (p < 5 × 10−8) were observed between PC2 in women and two SNPs in linkage disequilibrium (r2 = 0.935) on 6q14–q15, both in the HTR1E gene. This signal was supported by several suggestively associated SNPs in the same region. The HTR1E gene codes for one of the several receptors for 5-hydroxytryptamine (5-HT or serotonin), a biogenic hormone that functions as a neurotransmitter and a mitogen. The activity of the 5-HT1E receptor is mediated by G-coupled proteins. Recently, it has been demonstrated that gut-derived serotonin binds to the Htr1b receptor present on osteoblasts and inhibits its proliferation.33 In addition, Oury et al.34 showed that brain-derived 5-HT binds to Htr2c receptor expressed on neurons of the hypothalamic ventromedial nucleus and favors bone mass accrual by inhibiting the activity of sympathetic neurons. Our earlier work35 demonstrated that the same HTR1E SNP rs9362321 was associated with FNL in Framingham women (p = 5.57 × 10−7). Here we extended this finding to the integrated phenotype, which included measures of cross-sectional proximal femoral geometry, such as narrow neck and shaft's outer diameters and section moduli. Also, SNPs in the vicinity of HTR1E gene were suggestively associated with PC4 (regarded as “Femoral shaft stability” factor) in the sexes-combined sample. PC4 was mostly correlated with leg lean mass in both sexes, pointing to a possible effect on nongeometric properties of the femur.

Further, in order to substantiate the value of understanding the genetic contributions to the integrated phenotype constructs, we assessed associations between top SNPs identified for PCs with fracture risk over ∼8 years follow-up. Indeed, SNP rs6919366 in HTR1E was significantly associated with nonvertebral fractures in women. This finding of association with fracture risk is especially important, because it supports the value of knowing an individual's genetic predisposition to risk factors measured in midlife (the subjects were ∼63 years old on average at the baseline), for predicting a late-life event, such as osteoporotic fracture. Taken together, serotonin and its 5-HT receptor might play an important role in bone metabolism, although their precise function in bone metabolism has yet to be elucidated.

Another gene, suggestively associated with PC2 in the sexes-combined analysis (less strongly in women), was the AKAP6 gene on chr. 14, which codes for a protein kinase A (PRKA) anchor protein 6. This protein is highly expressed in various brain regions, cardiac, and skeletal muscle. Notably, both AKAP6 and 5-HT are associated with the cyclic adenosine monophosphate (cAMP) signaling pathway. Because 5-HT exerts its effect through the cAMP effector pathway,36 which is coordinated by AKAP6,37 the crosstalk between AKAP6 and HTR1E on bone metabolism warrants further investigation. The third gene, whose SNPs were highly suggestively associated with PC4 in women only, was the COL4A2 gene. This gene encodes one of the six subunits (alpha 2) of type IV collagen, the major structural component of basement membranes and extracellular matrix organization, which is expressed in muscle. However, SNPs in neither AKAP6 nor COL4A2 were associated with nonvertebral fractures.

We further examined whether candidate regions obtained by GWAS for BMD (reported by Rivadeneira et al.38 and by Kung et al.39), for association with our PCs. In Supplementary Table 4, we provide best association p-values for the 21 candidate intervals. There were suggestive associations (p < 5 × 10−5) of PC1 with WLS (GPR177) and SHFM1 and of PC2 with the MEPE locus (including SPP1 and IBSP); this is not surprising, given that PC1 is heavily loaded on BMD measures and PC2 on Femoral cross-sectional geometry.

Despite the fact that we have identified a gene HTR1E that was significantly associated with PCs and confirmed suggestive associations with other osteoporosis-related genes, it is difficult to directly replicate our findings, because we have measured a number of musculoskeletal phenotypes that are not available collectively in many cohorts. Instead, we performed a bioinformatics analysis to see if biological information supports our most significant findings. We therefore performed gene set enrichment analysis and observed that genes having the most significant associations with the PCs were significantly associated with the “skeletal and muscular system development and function.” We further inferred a gene network from the annotated genes associated with each PC. All gene networks were highly scored, suggesting there are strong and nonrandom connections between several top genes in each PC. Previous work suggested that hub genes (those abundantly connected to other members of the network18) are likely to be more essential to biological function and survival; therefore, we also identified the hub genes in each gene network. Interestingly, all identified hub genes are known to be associated with osteoblast development and differentiation (Supplementary Table 1), except for HNF4A, which encodes for hepatocyte nuclear factor-4 alpha, a transcription factor that regulates the expression of several hepatic genes. Mutations of HNF4A have been associated with monogenic autosomal dominant noninsulin-dependent diabetes mellitus type I,40 whereas recent studies demonstrated the importance of insulin signaling in osteoblast that affects both bone remodeling and glucose metabolism.41 Therefore, it is possible that HNF4a affects bone metabolism through regulation of insulin secretion.

Because musculoskeletal traits and fracture risk are characterized by a marked sexual dimorphism, our principal component analyses were performed also in men and women, separately. Minor differences between genders were evident in PCA. However, because the proximal femur can be represented as a cantilever beam, a pronounced sex difference in femoral neck length might reflect distinct biomechanical demands, and therefore, dictate a relationship between the femur shape and muscle mass. Because our sample size for men was modest, we decided not to focus on the analysis of men and to focus on analyses in women. As results of GWAS confirmed, there was some advantage in performing a women-specific analysis, because it revealed GWS associations, and further, associations of an SNP in HTR1E with nonvertebral fractures in women.

One of the merits of PCA is a potential increase in power. Notably, our simulations of individual traits revealed low statistical power to detect SNPs with effects on a trait's variance of 0.55% to 2%, for a significance level α = 5 × 10−8. Our simulations indicated that in women we had from 11% power (for an SNP explaining 1% variance) to 64% power (2% variance), whereas in the combined sample, we had power ranging from 5% to 49% (0.5% to 1% variance, respectively). Power for <0.5% effect was close to 0%. Our finding of a GWS association in a sample of 1758 women with the HTR1E gene provides some evidence for greater power with the use of the PCA method. Both theoretically and empirically, PCA is predicted to be more powerful than an analysis of multiple traits one at a time;42 it also prevents inflation of experiment-wise type I error rates by avoiding testing numerous nonindependent phenotypes.

The PCA method is suitable for the studies similar to ours, in which multiple surrogate traits, but not endophenotypes, are measured. For example, Saless et al.16 recently performed a PCA in a reciprocal intercross of the recombinant congenic mice strains HcB-8 and HcB-23 with multiple phenotypes encompassing body size, femoral diaphysis size and shape, and femoral biomechanical performance. Similar to us, they achieved substantial dimensional reduction of the data, accounting for 80% of the phenotypic variance within the first 4 PCs. Furthermore, their linkage mapping identified a QTL that was undetected in the study of individual phenotypes. Previously, biomechanical performance, anatomy, and BMD of the femur were studied by Koller et al.17 in C57BL/6J and C3H/HeJ inbred mice. They similarly revealed pleiotropic QTLs on chromosomes 4 and 14 influencing nearly all of the bone phenotypes measured, and found QTLs on chromosomes 1, 8, 13, and 17 with effects restricted to either bone density or bone structure/biomechanical phenotypes.

There is therefore a practical importance of using PCA for data reduction and “phenomic” outcomes. We analyzed many available musculoskeletal phenotypes, because none is a perfect proxy for osteoporotic fracture. Combinations of BMD measures from more than one region,43 as well as composite use of BMD with QUS3 or BMD with hip geometry,44 have been suggested as the way to improve osteoporotic fracture prediction. Here we show that there are genetic predispositions to a combination of BMD traits with hip cross-sectional properties, such as cross-sectional areas and section moduli. Interestingly, by applying a different approach, a Bayesian block clustering,45 to GWAS results of the above musculoskeletal traits, we obtained very similar results. In that study, by examining each phenotype independently and combining the results afterward, we found a strong cluster consisting of 10 traits (BMD at several skeletal sites; ultrasound measures; cross-sectional areas and section moduli of femoral neck and shaft). Gene-set enrichment analyses indicated biological pathways associated with these clustered phenotypes, similar to ones found here.45 This “confluence” of results obtained by two different approaches is encouraging. As well, loading of both LLM and proximal femoral geometric traits on the same factor (PC4) in this study supports the notion that there are shared genetic contributions to both osteoporosis and sarcopenia.13

There are shortcomings worthy to be mentioned. There is a complex relationship of bone measurements with age. In particular, the measurement error of the DXA-derived hip geometry phenotypes is higher than that of BMD, because subperiosteal bone margins may not be detected accurately in osteoporotic bone (thereby underestimating outer diameter of bone), especially in old persons with cortical porosity and very low mineral density. Further, estimates of average cortical thickness and buckling ratio in the HSA method are based on assumptions about bone shape and the assigned fractions of cortical and trabecular bone, which might change with aging (reviewed by us previously).46 Furthermore, the biological interpretations of GWAS results are limited by the information provided in Ingenuity's Knowledge Database, which is not focused around the musculoskeletal system's interactions and has preponderance of nonbone-related data sets. With the targeted bioinformatic tools,47 new biological pathways relevant to the skeleton may emerge for further pursuit. With these limitations and without replication, we consider findings of this study to be hypothesis generating rather than definitive. In particular, inferred gene networks are preliminary, until further evidence is obtained by replication and molecular study.

In conclusion, in this study we grouped osteoporosis-related traits into composite phenotypes, which were studied for genome-wide association. We were able to decrease the number of variables that could have been used individually for GWAS to focus on essential information from an ever-growing number of phenotypes that have become available for genetic study with advances of the measurement techniques. We pointed out associations between integrated musculoskeletal traits and three genes, one of which—HTR1E—was also associated with nonvertebral fractures risk. We thus provided direction for future studies to confirm functional evidence to substantiate roles of newly found genes in etiology of osteoporotic fracture. The method of multivariate combination is a potentially powerful tool for genomic discovery because it provides additional insight into the biology of a complex condition that could not always be obtained from studying the primary (directly measured) surrogate phenotypes. This knowledge may also prove helpful in prioritizing the phenotypes to be utilized in genetic studies of osteoporosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

The study was funded by grants from the US National Institute for Arthritis, Musculoskeletal and Skin Diseases and National Institute on Aging (R01 AR/AG 41398, R01 AR 050066 and R01 AR 057118), as well as from the National Human Genome Research Institute (R03 HG004946-01). The Framingham Heart Study of the National Institutes of Health and Boston University Schools of Public Health and Medicine were supported by the National Heart, Lung, and Blood Institute's Framingham Heart Study (N01-HC-25195) and its contract with Affymetrix, Inc., for genotyping services (N02-HL-6-4278). A portion of this research was conducted using the Linux Cluster for Genetic Analysis (LinGA-II) funded by the Robert Dawson Evans Endowment of the Department of Medicine at Boston University School of Medicine and Boston Medical Center.

Authors' roles: Study design: DK and SD. Study conduct: SD, YZ, and CLC. Data collection: DPK, DK, and LAC. Data analysis: SD, YZ, CLC, and LAC. Drafting of manuscript: DK and SD. Revising of manuscript: DPK, DK, and LAC. Obtained funding: DPK and DK.

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  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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jbmr_563_sm_SupplTabs.docx28KSupplementary Tables
jbmr_563_sm_chinese_translation.pdf1230KSupplementary Information: Chinese Translation

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