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1.  Genetic variants in the SOX6 gene are associated with bone mineral density in both Caucasian and Chinese populations 
Summary
Given the biological function of SOX6 and recent genome-wide association finding, we performed a fine-mapping association analyses to investigate the relationship between SOX6 and BMD both in Caucasian and Chinese populations. We identified many single-nucleotide polymorphisms (SNPs) within or near the SOX6 gene to be significantly associated with hip bone mineral density (BMD).
Introduction
SOX6 gene is an essential transcription factor in chondrogenesis and cartilage formation. Recent genome-wide association studies (GWAS) detected a SNP (rs7117858) located at the downstream of SOX6 significantly associated with hip BMD.
Methods
Given the biological function of SOX6 and the GWAS finding, we considered SOX6 as a new candidate for BMD and osteoporosis. Therefore, in this study, we performed a fine-mapping association analyses to investigate the relationship between SNPs within and near the SOX6 gene and BMD at both hip and spine. A total of 301 SNPs were tested in two independent US Caucasian populations (2,286 and 1,000 unrelated subjects, respectively) and a Chinese population (1,627 unrelated Han subjects).
Results
We confirmed that the previously reported rs7117858-A was associated with reduced hip BMD, with combined P value of 2.45×10−4. Besides this SNP, we identified another 19 SNPs within or near the SOX6 gene to be significantly associated with hip BMD after false discovery rate adjustment. The most significant SNP was rs1347677 located at the intron 3 (P=3.15×10−7). Seven additional SNPs in high linkage disequilibrium with rs1347677 were also significantly associated with hip BMD. SNPs in SOX6 showed significant skeletal site specificity since no SNP was detected to be associated with spine BMD.
Conclusion
Our study identified many SNPs in the SOX6 gene associated with hip BMD even across different ethnicities, which further highlighted the importance of the SOX6 gene influencing BMD variation and provided more information to the understanding of the genetic architecture of osteoporosis.
doi:10.1007/s00198-011-1626-x
PMCID: PMC4171834  PMID: 21625884
Association; BMD; Osteoporosis; SOX6
2.  Association Assessment of Copy Number Polymorphism and Risk of Age-Related Macular Degeneration 
Ophthalmology  2011;118(12):2442-2446.
Purpose
We previously identified a genetic copy number polymorphism (CNP147) that was statistically associated with age-related macular degeneration (AMD), and which resides downstream of the complement factor H (CFH) gene. Factor H protein is polymorphic at amino acid 402 in which the resulting histidine containing moiety has been established to impart significant risk of AMD. Here we present a method to precisely determine the exact copy number of CNP147 and examine in more detail the association with AMD.
Design
Case-control Study
Participants
421 AREDS (Age-related Eye Disease cohort Study) subjects of whom approximately 35% were diagnosed with neovascular disease, 19% with geographic atrophy, 16% with both, 30% with large drusen and 215 controls.
Methods
Using copy number assays available from Applied Biosystems Inc., we examined four loci spanning CNP147 and neighboring CNP148 in an AREDS matched case-control sample set. We analyzed these data by copy number while controlling for two high-risk CFH variants, rs1061170 (Y402H) and rs1410996. We phased the high risk CFH variants with CNP147 and analyzed haplotype frequencies in cases and controls. To further validate copy numbers, six Utah CEPH families (Centre D’etude du Polymorphism Humaine) were typed for CNP147 and the segregation assessed.
Main Outcome Measures
Increased or decreased risk of AMD from genetic loci.
Results
Having fewer than 2 copies of CNP147 is associated with an estimated 43% reduction in odds of having AMD in this sample set (adjusted odds ratio=0.57, P=0.006). CNP148 variation is rare in Caucasians and it was not statistically significant. Common haplotypes reveal that the risk alleles for rs1061170 and rs1410996 most frequently segregate with higher copy numbers for CNP147; but not exclusively, and that one haplotype that carried a deletion of CNP147 was highly protective (odds ratio=0.25 P=1.3×10−13) when compared to the reference.
Conclusions
In this matched subset of AREDS subjects, after adjusting for two known risk variants in CFH, CNP147 deletion statistically associates with diminished risk for AMD.
doi:10.1016/j.ophtha.2011.05.027
PMCID: PMC3223559  PMID: 21856016
Copy number polymorphism; age-related macular degeneration; HapMap 3; TaqMan Copy Number Assays; qPCR
3.  Suggestion of GLYAT gene underlying variation of bone size and body lean mass as revealed by a bivariate genome-wide association study 
Human genetics  2012;132(2):189-199.
Objective
Bone and muscle, two major tissue types of musculoskeletal system, have strong genetic determination. Abnormality in bone and/or muscle may cause musculoskeletal diseases such as osteoporosis and sarcopenia. Bone size phenotypes (BSPs), such as hip bone size (HBS), appendicular bone size (ABS), are genetically correlated with body lean mass (mainly muscle mass). However, the specific genes shared by these phenotypes are largely unknown. In this study, we aimed to identify the specific genes with pleiotropic effects on BSPs and appendicular lean mass (ALM).
Methods
We performed a bivariate genome-wide association study (GWAS) by analyzing ~690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) followed by a replication study in 2,286 unrelated US Caucasians (558 males and 1728 females).
Results
We identified 14 interesting single nucleotide polymorphisms (SNPs) that may contribute to variation of both BSPs and ALM, with p values <10−6 in discovery stage. Among them, the association of three SNPs (rs2507838, rs7116722, and rs11826261) in/near GLYAT (glycine-N-acyltransferase) gene was replicated in US Caucasians, with p values ranging from 1.89×10−3 to 3.71×10−4 for ALM-ABS, from 5.14×10−3 to 1.11×10−2 for ALM-HBS, respectively. Meta-analyses yielded stronger association signals for rs2507838, rs7116722, and rs11826261, with pooled p values of 1.68×10−8, 7.94×10−8, 6.80×10−8 for ALB-ABS and 1.22×10−4, 9.85×10−5, 3.96×10−4 for ALM-HBS, respectively. Haplotype allele ATA based on these three SNPs were also associated with ALM-HBS and ALM-ABS in both discovery and replication samples. Interestingly, GLYAT was previously found to be essential to glucose metabolism and energy metabolism, suggesting the gene’s dual role in both bone development and muscle growth.
Conclusions
Our findings, together with the prior biological evidence, suggest the importance of GLYAT gene in co-regulation of bone phenotypes and body lean mass.
doi:10.1007/s00439-012-1236-5
PMCID: PMC3682481  PMID: 23108985
Bivariate GWAS; Bone size; Lean mass; GLYAT
4.  Genome-Wide Association Study Identified CNP12587 Region Underlying Height Variation in Chinese Females 
PLoS ONE  2012;7(9):e44292.
Introduction
Human height is a highly heritable trait considered as an important factor for health. There has been limited success in identifying the genetic factors underlying height variation. We aim to identify sequence variants associated with adult height by a genome-wide association study of copy number variants (CNVs) in Chinese.
Methods
Genome-wide CNV association analyses were conducted in 1,625 unrelated Chinese adults and sex specific subgroup for height variation, respectively. Height was measured with a stadiometer. Affymetrix SNP6.0 genotyping platform was used to identify copy number polymorphisms (CNPs). We constructed a genomic map containing 1,009 CNPs in Chinese individuals and performed a genome-wide association study of CNPs with height.
Results
We detected 10 significant association signals for height (p<0.05) in the whole population, 9 and 11 association signals for Chinese female and male population, respectively. A copy number polymorphism (CNP12587, chr18:54081842-54086942, p = 2.41×10−4) was found to be significantly associated with height variation in Chinese females even after strict Bonferroni correction (p = 0.048). Confirmatory real time PCR experiments lent further support for CNV validation. Compared to female subjects with two copies of the CNP, carriers of three copies had an average of 8.1% decrease in height. An important candidate gene, ubiquitin-protein ligase NEDD4-like (NEDD4L), was detected at this region, which plays important roles in bone metabolism by binding to bone formation regulators.
Conclusions
Our findings suggest the important genetic variants underlying height variation in Chinese.
doi:10.1371/journal.pone.0044292
PMCID: PMC3434125  PMID: 22957059
5.  Adaptive Copy Number Evolution in Malaria Parasites 
PLoS Genetics  2008;4(10):e1000243.
Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2–11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.
Author Summary
Recent comparative genomic hybridization studies have revealed extensive copy number variation in eukaryotic genomes. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive copy number polymorphism (CNP). We provide compelling evidence that gch1 CNP is adaptive and most likely results from selection by antifolate drugs, which target enzymes downstream in this pathway. Gch1 CNP shows extreme geographical differentiation; hitchhiking reduces diversity and increases LD in flanking sequence, indicating recent rapid spread within Thailand, while amplicon structure reveals multiple origins and parallel evolution. Furthermore, strong association between elevated copy number and a critical mutation dhfr-I164L that underlies high-level antifolate resistance indicates functional linkage and fitness epistasis between genes on different chromosomes. These data reveal hidden complexity in the evolutionary response to antifolate treatment and demonstrate that analysis of structural variation can provide a fast-track to locating genes that underlie adaptation.
doi:10.1371/journal.pgen.1000243
PMCID: PMC2570623  PMID: 18974876
6.  Genome-Wide Association Study Identifies ALDH7A1 as a Novel Susceptibility Gene for Osteoporosis 
PLoS Genetics  2010;6(1):e1000806.
Osteoporosis is a major public health problem. It is mainly characterized by low bone mineral density (BMD) and/or low-trauma osteoporotic fractures (OF), both of which have strong genetic determination. The specific genes influencing these phenotypic traits, however, are largely unknown. Using the Affymetrix 500K array set, we performed a case-control genome-wide association study (GWAS) in 700 elderly Chinese Han subjects (350 with hip OF and 350 healthy matched controls). A follow-up replication study was conducted to validate our major GWAS findings in an independent Chinese sample containing 390 cases with hip OF and 516 controls. We found that a SNP, rs13182402 within the ALDH7A1 gene on chromosome 5q31, was strongly associated with OF with evidence combined GWAS and replication studies (P = 2.08×10−9, odds ratio = 2.25). In order to explore the target risk factors and potential mechanism underlying hip OF risk, we further examined this candidate SNP's relevance to hip BMD both in Chinese and Caucasian populations involving 9,962 additional subjects. This SNP was confirmed as consistently associated with hip BMD even across ethnic boundaries, in both Chinese and Caucasians (combined P = 6.39×10−6), further attesting to its potential effect on osteoporosis. ALDH7A1 degrades and detoxifies acetaldehyde, which inhibits osteoblast proliferation and results in decreased bone formation. Our findings may provide new insights into the pathogenesis of osteoporosis.
Author Summary
Osteoporosis is a major health concern worldwide. It is a highly heritable disease characterized mainly by low bone mineral density (BMD) and/or osteoporotic fractures. However, the specific genetic variants determining risk for low BMD or OF are largely unknown. Here, taking advantage of recent technological advances in human genetics, we performed a genome-wide association study and follow-up validation studies to identify genetic variants for osteoporosis. By examining a total of 11,568 individuals from Chinese and Caucasian populations, we discovered a susceptibility gene, ALDH7A1, which is associated with hip osteoporotic fracture and BMD. ALDH7A1 might inhibit osteoblast proliferation and decrease bone formation. Our finding opens a new avenue for exploring the pathophysiology of osteoporosis.
doi:10.1371/journal.pgen.1000806
PMCID: PMC2794362  PMID: 20072603
7.  The Fat Mass and Obesity Associated Gene, FTO, Is Also Associated with Osteoporosis Phenotypes 
PLoS ONE  2011;6(11):e27312.
Obesity and osteoporosis are closely correlated genetically. FTO gene has been consistently identified to be associated with obesity phenotypes. A recent study reported that the mice lacking Fto could result in lower bone mineral density (BMD). Thus, we hypothesize that the FTO gene might be also important for osteoporosis phenotypes. To test for such a hypothesis, we performed an association analyses to investigate the relationship between SNPs in FTO and BMD at both hip and spine. A total of 141 SNPs were tested in two independent Chinese populations (818 and 809 unrelated Han subjects, respectively) and a Caucasian population (2,286 unrelated subjects). Combining the two Chinese samples, we identified 6 SNPs in FTO to be significantly associated with hip BMD after multiple testing adjustments, with the combined P values ranged from 4.99×10−4–1.47×10−4. These 6 SNPs are all located at the intron 8 of FTO and in high linkage disequilibrium. Each copy of the minor allele of each SNP was associated with increased hip BMD values with the effect size (beta) of ∼0.025 and ∼0.015 in the Chinese sample 1 and 2, respectively. However, none of these 6 SNPs showed significant association signal in the Caucasian sample, by presenting some extent of ethnic difference. Our findings, together with the prior biological evidence, suggest that the FTO gene might be a new candidate for BMD variation and osteoporosis in Chinese populations.
doi:10.1371/journal.pone.0027312
PMCID: PMC3220685  PMID: 22125610
8.  Genome-Wide Association Study Identified UQCC Locus for Spine Bone Size in Humans 
Bone  2012;53(1):129-133.
Bone size (BS) contributes significantly to the risk of osteoporotic fracture. Osteoporotic spine fracture is one of the most disabling outcomes of osteoporosis. This study aims to identify genomic loci underlying spine BS variation in humans.
We performed a genome-wide association scan in 2,286 unrelated Caucasians using Affymetrix 6.0 SNP arrays. Areal BS (cm2) at lumbar spine was measured using dual energy X-ray absorptiometry scanners. SNPs of interest were subjected to replication analyses and meta-analyses with additional two independent Caucasian populations (N = 1,000 and 2,503) and one Chinese population (N = 1,627).
In the initial GWAS, 91 SNPs were associated with spine BS (P<1.0E-4). Eight contiguous SNPs were found clustering in a haplotype block within UQCC gene (ubiquinol-cytochrome creductase complex chaperone). Association of the above eight SNPs with spine BS were replicated in one Caucasian and one Chinese populations. Meta-analyses (N = 7,416) generated much stronger association signals for these SNPs (e.g., P = 1.86E-07 for SNP rs6060373), supporting association of UQCC with spine BS across ethnicities.
This study identified a novel locus, i.e., the UQCC gene, for spine BS variation in humans. Future functional studies will contribute to elucidating the mechanisms by which UQCC regulates bone growth and development.
doi:10.1016/j.bone.2012.11.028
PMCID: PMC3682469  PMID: 23207799
Spine bone size; GWAS; UQCC
9.  Identification of PLCL1 Gene for Hip Bone Size Variation in Females in a Genome-Wide Association Study 
PLoS ONE  2008;3(9):e3160.
Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating ∼380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72×10−7. The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62×10−3 and 2.44×10−3, respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10−5 in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only ∼0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66×10−3 (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF.
doi:10.1371/journal.pone.0003160
PMCID: PMC2522269  PMID: 18776929
10.  Copy number polymorphisms near SLC2A9 are associated with serum uric acid concentrations 
BMC Genetics  2014;15:81.
Background
Hyperuricemia is associated with multiple diseases, including gout, cardiovascular disease, and renal disease. Serum urate is highly heritable, yet association studies of single nucleotide polymorphisms (SNPs) and serum uric acid explain a small fraction of the heritability. Whether copy number polymorphisms (CNPs) contribute to uric acid levels is unknown.
Results
We assessed copy number on a genome-wide scale among 8,411 individuals of European ancestry (EA) who participated in the Atherosclerosis Risk in Communities (ARIC) study. CNPs upstream of the urate transporter SLC2A9 on chromosome 4p16.1 are associated with uric acid (χ2df2=3545, p=3.19×10-23). Effect sizes, expressed as the percentage change in uric acid per deleted copy, are most pronounced among women (3.974.935.87 [ 2.55097.5 denoting percentiles], p=4.57×10-23) and independent of previously reported SNPs in SLC2A9 as assessed by SNP and CNP regression models and the phasing SNP and CNP haplotypes (χ2df2=3190,p=7.23×10-08). Our finding is replicated in the Framingham Heart Study (FHS), where the effect size estimated from 4,089 women is comparable to ARIC in direction and magnitude (1.414.707.88, p=5.46×10-03).
Conclusions
This is the first study to characterize CNPs in ARIC and the first genome-wide analysis of CNPs and uric acid. Our findings suggests a novel, non-coding regulatory mechanism for SLC2A9-mediated modulation of serum uric acid, and detail a bioinformatic approach for assessing the contribution of CNPs to heritable traits in large population-based studies where technical sources of variation are substantial.
doi:10.1186/1471-2156-15-81
PMCID: PMC4118309  PMID: 25007794
Copy number polymorphism; Hyperuricemia; Genomewide association study
11.  Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes 
Background
Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias) – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components.
Methods
Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX.
Results
We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor).
Conclusion
Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors.
doi:10.1186/1471-2474-7-87
PMCID: PMC1660540  PMID: 17116261
12.  A 32 kb Critical Region Excluding Y402H in CFH Mediates Risk for Age-Related Macular Degeneration 
PLoS ONE  2011;6(10):e25598.
Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10−109); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10−9) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.
doi:10.1371/journal.pone.0025598
PMCID: PMC3192039  PMID: 22022419
13.  Genetic Analysis Identifies DDR2 as a Novel Gene Affecting Bone Mineral Density and Osteoporotic Fractures in Chinese Population 
PLoS ONE  2015;10(2):e0117102.
DDR2 gene, playing an essential role in regulating osteoblast differentiation and chondrocyte maturation, may influence bone mineral density (BMD) and osteoporosis, but the genetic variations actually leading to the association remain to be elucidated. Therefore, the aim of this study was to investigate whether the genetic variants in DDR2 are associated with BMD and fracture risk. This study was performed in three samples from two ethnicities, including 1,300 Chinese Han subjects, 700 Chinese Han subjects (350 with osteoporotic hip fractures and 350 healthy controls) and 2,286 US white subjects. Twenty-eight SNPs in DDR2 were genotyped and tested for associations with hip BMD and fractures. We identified 3 SNPs in DDR2 significantly associated with hip BMD in the Chinese population after multiple testing adjustments, which were rs7521233 (P = 1.06×10−4, β: −0.018 for allele C), rs7553831 (P = 1.30×10−4, β: −0.018 for allele T), and rs6697469 (P = 1.59×10−3, β: −0.015 for allele C), separately. These three SNPs were in high linkage disequilibrium. Haplotype analyses detected two significantly associated haplotypes, including one haplotype in block 2 (P = 9.54×10−4, β: −0.016) where these three SNPs located. SNP rs6697469 was also associated with hip fractures (P = 0.043, OR: 1.42) in the Chinese population. The effect on fracture risk was consistent with its association with lower BMD. However, in the white population, we didn’t observe significant associations with hip BMD. eQTL analyses revealed that SNPs associated with BMD also affected DDR2 mRNA expression levels in Chinese. Our findings, together with the prior biological evidence, suggest that DDR2 could be a new candidate for osteoporosis in Chinese population. Our results also reveal an ethnic difference, which highlights the need for further genetic studies in each ethnic group.
doi:10.1371/journal.pone.0117102
PMCID: PMC4319719  PMID: 25658585
14.  Utilization of DXA Bone Mineral Densitometry in Ontario 
Executive Summary
Issue
Systematic reviews and analyses of administrative data were performed to determine the appropriate use of bone mineral density (BMD) assessments using dual energy x-ray absorptiometry (DXA), and the associated trends in wrist and hip fractures in Ontario.
Background
Dual Energy X-ray Absorptiometry Bone Mineral Density Assessment
Dual energy x-ray absorptiometry bone densitometers measure bone density based on differential absorption of 2 x-ray beams by bone and soft tissues. It is the gold standard for detecting and diagnosing osteoporosis, a systemic disease characterized by low bone density and altered bone structure, resulting in low bone strength and increased risk of fractures. The test is fast (approximately 10 minutes) and accurate (exceeds 90% at the hip), with low radiation (1/3 to 1/5 of that from a chest x-ray). DXA densitometers are licensed as Class 3 medical devices in Canada. The World Health Organization has established criteria for osteoporosis and osteopenia based on DXA BMD measurements: osteoporosis is defined as a BMD that is >2.5 standard deviations below the mean BMD for normal young adults (i.e. T-score <–2.5), while osteopenia is defined as BMD that is more than 1 standard deviation but less than 2.5 standard deviation below the mean for normal young adults (i.e. T-score< –1 & ≥–2.5). DXA densitometry is presently an insured health service in Ontario.
Clinical Need
 
Burden of Disease
The Canadian Multicenter Osteoporosis Study (CaMos) found that 16% of Canadian women and 6.6% of Canadian men have osteoporosis based on the WHO criteria, with prevalence increasing with age. Osteopenia was found in 49.6% of Canadian women and 39% of Canadian men. In Ontario, it is estimated that nearly 530,000 Ontarians have some degrees of osteoporosis. Osteoporosis-related fragility fractures occur most often in the wrist, femur and pelvis. These fractures, particularly those in the hip, are associated with increased mortality, and decreased functional capacity and quality of life. A Canadian study showed that at 1 year after a hip fracture, the mortality rate was 20%. Another 20% required institutional care, 40% were unable to walk independently, and there was lower health-related quality of life due to attributes such as pain, decreased mobility and decreased ability to self-care. The cost of osteoporosis and osteoporotic fractures in Canada was estimated to be $1.3 billion in 1993.
Guidelines for Bone Mineral Density Testing
With 2 exceptions, almost all guidelines address only women. None of the guidelines recommend blanket population-based BMD testing. Instead, all guidelines recommend BMD testing in people at risk of osteoporosis, predominantly women aged 65 years or older. For women under 65 years of age, BMD testing is recommended only if one major or two minor risk factors for osteoporosis exist. Osteoporosis Canada did not restrict its recommendations to women, and thus their guidelines apply to both sexes. Major risk factors are age greater than or equal to 65 years, a history of previous fractures, family history (especially parental history) of fracture, and medication or disease conditions that affect bone metabolism (such as long-term glucocorticoid therapy). Minor risk factors include low body mass index, low calcium intake, alcohol consumption, and smoking.
Current Funding for Bone Mineral Density Testing
The Ontario Health Insurance Program (OHIP) Schedule presently reimburses DXA BMD at the hip and spine. Measurements at both sites are required if feasible. Patients at low risk of accelerated bone loss are limited to one BMD test within any 24-month period, but there are no restrictions on people at high risk. The total fee including the professional and technical components for a test involving 2 or more sites is $106.00 (Cdn).
Method of Review
This review consisted of 2 parts. The first part was an analysis of Ontario administrative data relating to DXA BMD, wrist and hip fractures, and use of antiresorptive drugs in people aged 65 years and older. The Institute for Clinical Evaluative Sciences extracted data from the OHIP claims database, the Canadian Institute for Health Information hospital discharge abstract database, the National Ambulatory Care Reporting System, and the Ontario Drug Benefit database using OHIP and ICD-10 codes. The data was analyzed to examine the trends in DXA BMD use from 1992 to 2005, and to identify areas requiring improvement.
The second part included systematic reviews and analyses of evidence relating to issues identified in the analyses of utilization data. Altogether, 8 reviews and qualitative syntheses were performed, consisting of 28 published systematic reviews and/or meta-analyses, 34 randomized controlled trials, and 63 observational studies.
Findings of Utilization Analysis
Analysis of administrative data showed a 10-fold increase in the number of BMD tests in Ontario between 1993 and 2005.
OHIP claims for BMD tests are presently increasing at a rate of 6 to 7% per year. Approximately 500,000 tests were performed in 2005/06 with an age-adjusted rate of 8,600 tests per 100,000 population.
Women accounted for 90 % of all BMD tests performed in the province.
In 2005/06, there was a 2-fold variation in the rate of DXA BMD tests across local integrated health networks, but a 10-fold variation between the county with the highest rate (Toronto) and that with the lowest rate (Kenora). The analysis also showed that:
With the increased use of BMD, there was a concomitant increase in the use of antiresorptive drugs (as shown in people 65 years and older) and a decrease in the rate of hip fractures in people age 50 years and older.
Repeat BMD made up approximately 41% of all tests. Most of the people (>90%) who had annual BMD tests in a 2-year or 3-year period were coded as being at high risk for osteoporosis.
18% (20,865) of the people who had a repeat BMD within a 24-month period and 34% (98,058) of the people who had one BMD test in a 3-year period were under 65 years, had no fracture in the year, and coded as low-risk.
Only 19% of people age greater than 65 years underwent BMD testing and 41% received osteoporosis treatment during the year following a fracture.
Men accounted for 24% of all hip fractures and 21 % of all wrist fractures, but only 10% of BMD tests. The rates of BMD tests and treatment in men after a fracture were only half of those in women.
In both men and women, the rate of hip and wrist fractures mainly increased after age 65 with the sharpest increase occurring after age 80 years.
Findings of Systematic Review and Analysis
Serial Bone Mineral Density Testing for People Not Receiving Osteoporosis Treatment
A systematic review showed that the mean rate of bone loss in people not receiving osteoporosis treatment (including postmenopausal women) is generally less than 1% per year. Higher rates of bone loss were reported for people with disease conditions or on medications that affect bone metabolism. In order to be considered a genuine biological change, the change in BMD between serial measurements must exceed the least significant change (variability) of the testing, ranging from 2.77% to 8% for precisions ranging from 1% to 3% respectively. Progression in BMD was analyzed, using different rates of baseline BMD values, rates of bone loss, precision, and BMD value for initiating treatment. The analyses showed that serial BMD measurements every 24 months (as per OHIP policy for low-risk individuals) is not necessary for people with no major risk factors for osteoporosis, provided that the baseline BMD is normal (T-score ≥ –1), and the rate of bone loss is less than or equal to 1% per year. The analyses showed that for someone with a normal baseline BMD and a rate of bone loss of less than 1% per year, the change in BMD is not likely to exceed least significant change (even for a 1% precision) in less than 3 years after the baseline test, and is not likely to drop to a BMD level that requires initiation of treatment in less than 16 years after the baseline test.
Serial Bone Mineral Density Testing in People Receiving Osteoporosis Therapy
Seven published meta-analysis of randomized controlled trials (RCTs) and 2 recent RCTs on BMD monitoring during osteoporosis therapy showed that although higher increases in BMD were generally associated with reduced risk of fracture, the change in BMD only explained a small percentage of the fracture risk reduction.
Studies showed that some people with small or no increase in BMD during treatment experienced significant fracture risk reduction, indicating that other factors such as improved bone microarchitecture might have contributed to fracture risk reduction.
There is conflicting evidence relating to the role of BMD testing in improving patient compliance with osteoporosis therapy.
Even though BMD may not be a perfect surrogate for reduction in fracture risk when monitoring responses to osteoporosis therapy, experts advised that it is still the only reliable test available for this purpose.
A systematic review conducted by the Medical Advisory Secretariat showed that the magnitude of increases in BMD during osteoporosis drug therapy varied among medications. Although most of the studies yielded mean percentage increases in BMD from baseline that did not exceed the least significant change for a 2% precision after 1 year of treatment, there were some exceptions.
Bone Mineral Density Testing and Treatment After a Fragility Fracture
A review of 3 published pooled analyses of observational studies and 12 prospective population-based observational studies showed that the presence of any prevalent fracture increases the relative risk for future fractures by approximately 2-fold or more. A review of 10 systematic reviews of RCTs and 3 additional RCTs showed that therapy with antiresorptive drugs significantly reduced the risk of vertebral fractures by 40 to 50% in postmenopausal osteoporotic women and osteoporotic men, and 2 antiresorptive drugs also reduced the risk of nonvertebral fractures by 30 to 50%. Evidence from observational studies in Canada and other jurisdictions suggests that patients who had undergone BMD measurements, particularly if a diagnosis of osteoporosis is made, were more likely to be given pharmacologic bone-sparing therapy. Despite these findings, the rate of BMD investigation and osteoporosis treatment after a fracture remained low (<20%) in Ontario as well as in other jurisdictions.
Bone Mineral Density Testing in Men
There are presently no specific Canadian guidelines for BMD screening in men. A review of the literature suggests that risk factors for fracture and the rate of vertebral deformity are similar for men and women, but the mortality rate after a hip fracture is higher in men compared with women. Two bisphosphonates had been shown to reduce the risk of vertebral and hip fractures in men. However, BMD testing and osteoporosis treatment were proportionately low in Ontario men in general, and particularly after a fracture, even though men accounted for 25% of the hip and wrist fractures. The Ontario data also showed that the rates of wrist fracture and hip fracture in men rose sharply in the 75- to 80-year age group.
Ontario-Based Economic Analysis
The economic analysis focused on analyzing the economic impact of decreasing future hip fractures by increasing the rate of BMD testing in men and women age greater than or equal to 65 years following a hip or wrist fracture. A decision analysis showed the above strategy, especially when enhanced by improved reporting of BMD tests, to be cost-effective, resulting in a cost-effectiveness ratio ranging from $2,285 (Cdn) per fracture avoided (worst-case scenario) to $1,981 (Cdn) per fracture avoided (best-case scenario). A budget impact analysis estimated that shifting utilization of BMD testing from the low risk population to high risk populations within Ontario would result in a saving of $0.85 million to $1.5 million (Cdn) to the health system. The potential net saving was estimated at $1.2 million to $5 million (Cdn) when the downstream cost-avoidance due to prevention of future hip fractures was factored into the analysis.
Other Factors for Consideration
There is a lack of standardization for BMD testing in Ontario. Two different standards are presently being used and experts suggest that variability in results from different facilities may lead to unnecessary testing. There is also no requirement for standardized equipment, procedure or reporting format. The current reimbursement policy for BMD testing encourages serial testing in people at low risk of accelerated bone loss. This review showed that biannual testing is not necessary for all cases. The lack of a database to collect clinical data on BMD testing makes it difficult to evaluate the clinical profiles of patients tested and outcomes of the BMD tests. There are ministry initiatives in progress under the Osteoporosis Program to address the development of a mandatory standardized requisition form for BMD tests to facilitate data collection and clinical decision-making. Work is also underway for developing guidelines for BMD testing in men and in perimenopausal women.
Conclusion
Increased use of BMD in Ontario since 1996 appears to be associated with increased use of antiresorptive medication and a decrease in hip and wrist fractures.
Data suggest that as many as 20% (98,000) of the DXA BMD tests in Ontario in 2005/06 were performed in people aged less than 65 years, with no fracture in the current year, and coded as being at low risk for accelerated bone loss; this is not consistent with current guidelines. Even though some of these people might have been incorrectly coded as low-risk, the number of tests in people truly at low risk could still be substantial.
Approximately 4% (21,000) of the DXA BMD tests in 2005/06 were repeat BMDs in low-risk individuals within a 24-month period. Even though this is in compliance with current OHIP reimbursement policies, evidence showed that biannual serial BMD testing is not necessary in individuals without major risk factors for fractures, provided that the baseline BMD is normal (T-score < –1). In this population, BMD measurements may be repeated in 3 to 5 years after the baseline test to establish the rate of bone loss, and further serial BMD tests may not be necessary for another 7 to 10 years if the rate of bone loss is no more than 1% per year. Precision of the test needs to be considered when interpreting serial BMD results.
Although changes in BMD may not be the perfect surrogate for reduction in fracture risk as a measure of response to osteoporosis treatment, experts advised that it is presently the only reliable test for monitoring response to treatment and to help motivate patients to continue treatment. Patients should not discontinue treatment if there is no increase in BMD after the first year of treatment. Lack of response or bone loss during treatment should prompt the physician to examine whether the patient is taking the medication appropriately.
Men and women who have had a fragility fracture at the hip, spine, wrist or shoulder are at increased risk of having a future fracture, but this population is presently under investigated and under treated. Additional efforts have to be made to communicate to physicians (particularly orthopaedic surgeons and family physicians) and the public about the need for a BMD test after fracture, and for initiating treatment if low BMD is found.
Men had a disproportionately low rate of BMD tests and osteoporosis treatment, especially after a fracture. Evidence and fracture data showed that the risk of hip and wrist fractures in men rises sharply at age 70 years.
Some counties had BMD utilization rates that were only 10% of that of the county with the highest utilization. The reasons for low utilization need to be explored and addressed.
Initiatives such as aligning reimbursement policy with current guidelines, developing specific guidelines for BMD testing in men and perimenopausal women, improving BMD reports to assist in clinical decision making, developing a registry to track BMD tests, improving access to BMD tests in remote/rural counties, establishing mechanisms to alert family physicians of fractures, and educating physicians and the public, will improve the appropriate utilization of BMD tests, and further decrease the rate of fractures in Ontario. Some of these initiatives such as developing guidelines for perimenopausal women and men, and developing a standardized requisition form for BMD testing, are currently in progress under the Ontario Osteoporosis Strategy.
PMCID: PMC3379167  PMID: 23074491
15.  Plasma CNP Forms and Thyroid Status in Prepubertal Children with Acquired Thyroid Disease 
Clinical Endocrinology  2012;76(2):228-235.
Objective
C-type natriuretic peptide (CNP) and thyroid hormone (TH) are essential for normal skeletal growth. Plasma CNP peptides correlate with growth velocity, but the relationship between thyroid status and CNP production is unknown. This study examined the impact of restoring normal TH levels on CNP and height velocity (HV) in children with acquired hypo- and hyperthyroidism.
Design
We performed a prospective, observational study in prepubertal children with acquired hypothyroidism (n=15) and hyperthyroidism (n=12).
Measurements
Blood levels of CNP, amino-terminal proCNP (NTproCNP), bone-specific alkaline phosphatase (BSAP), IGF-I, and TH levels were measured before and during the first six months of standard treatment for hypo- and hyperthyroidism, and correlations were determined.
Results
At baseline, HV, CNP, NTproCNP, and BSAP were significantly higher in hyper- than in hypothyroid subjects. Changes in TH after treatment were closely coupled to change in CNP and NTproCNP in hyper-, but not in hypothyroid children. In addition, a positive association of HV with CNP peptides was found during treatment of hyperthyroidism. Normalizing TH did not correlate with changes in BSAP or IGF-I in either group.
Conclusions
Plasma CNP peptides are higher in children with hyper- than in hypothyroidism at diagnosis, and, in hyperthyroid children, change concordantly with TH and HV during treatment. Differential responses of CNP in the two groups suggest CNP production is dependent on growth plate activity and not a direct effect of TH on CNP gene expression. Our findings suggest novel mechanisms underlying changes in skeletal response during treatment in children with acquired thyroid disease.
doi:10.1111/j.1365-2265.2011.04187.x
PMCID: PMC3243819  PMID: 21815902
C-type natriuretic peptide; amino-terminal proCNP; bone-specific alkaline phosphatase; hypothyroidism; hyperthyroidism; height velocity
16.  Haplotypes with Copy Number and Single Nucleotide Polymorphisms in CYP2A6 Locus Are Associated with Smoking Quantity in a Japanese Population 
PLoS ONE  2012;7(9):e44507.
Smoking is a major public health problem, but the genetic factors associated with smoking behaviors are not fully elucidated. Here, we have conducted an integrated genome-wide association study to identify common copy number polymorphisms (CNPs) and single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) in Japanese smokers ( = 17,158). Our analysis identified a common CNP with a strong effect on CPD (rs8102683; ) in the 19q13 region, encompassing the CYP2A6 locus. After adjustment for the associated CNP, we found an additional associated SNP (rs11878604; ) located 30 kb downstream of the CYP2A6 gene. Imputation of the CYP2A6 locus revealed that haplotypes underlying the CNP and the SNP corresponded to classical, functional alleles of CYP2A6 gene that regulate nicotine metabolism and explained 2% of the phenotypic variance of CPD (ANOVA -test ). These haplotypes were also associated with smoking-related diseases, including lung cancer, chronic obstructive pulmonary disease and arteriosclerosis obliterans.
doi:10.1371/journal.pone.0044507
PMCID: PMC3458030  PMID: 23049750
17.  Genome-Wide Association Study Identified Copy Number Variants Important for Appendicular Lean Mass 
PLoS ONE  2014;9(3):e89776.
Skeletal muscle is a major component of the human body. Age-related loss of muscle mass and function contributes to some public health problems such as sarcopenia and osteoporosis. Skeletal muscle, mainly composed of appendicular lean mass (ALM), is a heritable trait. Copy number variation (CNV) is a common type of human genome variant which may play an important role in the etiology of many human diseases. In this study, we performed genome-wide association analyses of CNV for ALM in 2,286 Caucasian subjects. We then replicated the major findings in 1,627 Chinese subjects. Two CNVs, CNV1191 and CNV2580, were detected to be associated with ALM (p = 2.26×10−2 and 3.34×10−3, respectively). In the Chinese replication sample, the two CNVs achieved p-values of 3.26×10−2 and 0.107, respectively. CNV1191 covers a gene, GTPase of the immunity-associated protein family (GIMAP1), which is important for skeletal muscle cell survival/death in humans. CNV2580 is located in the Serine hydrolase-like protein (SERHL) gene, which plays an important role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli. In summary, our study suggested two novel CNVs and the related genes that may contribute to variation in ALM.
doi:10.1371/journal.pone.0089776
PMCID: PMC3953533  PMID: 24626161
18.  Active Shape Modeling of the Hip in the Prediction of Incident Hip Fracture 
The objective of this study was to evaluate right proximal femur shape as a risk factor for incident hip fracture using active shape modeling (ASM). A nested case-control study of white women 65 years of age and older enrolled in the Study of Osteoporotic Fractures (SOF) was performed. Subjects (n = 168) were randomly selected from study participants who experienced hip fracture during the follow-up period (mean 8.3 years). Controls (n = 231) had no fracture during follow-up. Subjects with baseline radiographic hip osteoarthritis were excluded. ASM of digitized right hip radiographs generated 10 independent modes of variation in proximal femur shape that together accounted for 95% of the variance in proximal femur shape. The association of ASM modes with incident hip fracture was analyzed by logistic regression. Together, the 10 ASM modes demonstrated good discrimination of incident hip fracture. In models controlling for age and body mass index (BMI), the area under receiver operating characteristic (AUROC) curve for hip shape was 0.813, 95% confidence interval (CI) 0.771–0.854 compared with models containing femoral neck bone mineral density (AUROC = 0.675, 95% CI 0.620–0.730), intertrochanteric bone mineral density (AUROC = 0.645, 95% CI 0.589–0.701), femoral neck length (AUROC = 0.631, 95% CI 0.573–0.690), or femoral neck width (AUROC = 0.633, 95% CI 0.574–0.691). The accuracy of fracture discrimination was improved by combining ASM modes with femoral neck bone mineral density (AUROC = 0.835, 95% CI 0.795–0.875) or with intertrochanteric bone mineral density (AUROC = 0.834, 95% CI 0.794–0.875). Hips with positive standard deviations of ASM mode 4 had the highest risk of incident hip fracture (odds ratio = 2.48, 95% CI 1.68–3.31, p < .001). We conclude that variations in the relative size of the femoral head and neck are important determinants of incident hip fracture. The addition of hip shape to fracture-prediction tools may improve the risk assessment for osteoporotic hip fractures. © 2011 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.254
PMCID: PMC3179295  PMID: 20878772
ACTIVE SHAPE MODELING; HIP SHAPE; HIP FRACTURE; OSTEOPOROSIS; BONE
19.  Inhibition of HIV-1 particle assembly by 2',3'-cyclic-nucleotide 3'-phosphodiesterase 
Cell host & microbe  2012;12(4):585-597.
Summary
The expression of hundreds of interferon-stimulated genes (ISGs) causes the cellular ‘antiviral state’ in which the replication of many viruses, including HIV-1, is attenuated. We conducted a screen for ISGs that inhibit HIV-1 virion production and found that 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP), a membrane-associated protein with unknown function in mammals has this property. CNP binds to the structural protein Gag and blocks HIV-1 particle assembly after Gag and viral RNA have associated with the plasma membrane. Several primate lentiviruses are CNP-sensitive, and CNP sensitivity/resistance is determined by a single, naturally dimorphic, codon (E/K40) in the matrix domain of Gag. Like other antiretroviral proteins, CNP displays interspecies variation in antiviral activity. Mice encode an inactive CNP variant and a single amino-acid difference in murine versus human CNP determines Gag binding and antiviral activity. Some cell types express high levels of CNP and we speculate that CNP evolved to restrict lentivirus replication therein.
doi:10.1016/j.chom.2012.08.012
PMCID: PMC3498451  PMID: 23084924
20.  Computing Power and Sample Size for Case-Control Association Studies with Copy Number Polymorphism: Application of Mixture-Based Likelihood Ratio Test 
PLoS ONE  2008;3(10):e3475.
Recent studies suggest that copy number polymorphisms (CNPs) may play an important role in disease susceptibility and onset. Currently, the detection of CNPs mainly depends on microarray technology. For case-control studies, conventionally, subjects are assigned to a specific CNP category based on the continuous quantitative measure produced by microarray experiments, and cases and controls are then compared using a chi-square test of independence. The purpose of this work is to specify the likelihood ratio test statistic (LRTS) for case-control sampling design based on the underlying continuous quantitative measurement, and to assess its power and relative efficiency (as compared to the chi-square test of independence on CNP counts). The sample size and power formulas of both methods are given. For the latter, the CNPs are classified using the Bayesian classification rule. The LRTS is more powerful than this chi-square test for the alternatives considered, especially alternatives in which the at-risk CNP categories have low frequencies. An example of the application of the LRTS is given for a comparison of CNP distributions in individuals of Caucasian or Taiwanese ethnicity, where the LRTS appears to be more powerful than the chi-square test, possibly due to misclassification of the most common CNP category into a less common category.
doi:10.1371/journal.pone.0003475
PMCID: PMC2566806  PMID: 18941524
21.  Genome-wide Copy Number Variation Association Study Suggested VPS13B Gene for Osteoporosis in Caucasian 
Introduction
Bone mineral density (BMD) and femoral neck cross-sectional geometric parameters (FNCSGPs) are under strong genetic control. DNA copy number variation (CNV) is an important source of genetic diversity for human diseases. This study aims to identify CNVs associated with BMD and FNCSGPs.
Methods
Genome-wide CNV association analyses were conducted in 1,000 unrelated Caucasian subjects for BMD at the spine, hip, femoral neck, and for three FNCSGPs - cortical thickness (CT), cross section area (CSA), and buckling ratio (BR). BMD was measured by dual energy X-ray absorptiometry (DEXA). CT, CSA, and BR were estimated using DEXA measurements. Affymetrix 500K arrays and copy number analysis tool was used to identify CNVs.
Results
A CNV in VPS13B gene was significantly associated with spine, hip and FN BMDs, and CT, CSA, and BR (p < 0.05). Compared to subjects with 2 copies of the CNV, carriers of one copy had an average of 14.6%, 12.4%, and 13.6% higher spine, hip, and FN BMD, 20.0% thicker CT, 10.6% larger CSA, and 12.4% lower BR. Thus a decrease of the CNV consistently produced stronger bone, thereby reducing osteoporotic fracture risk.
Conclusions
VPS13B gene, via affecting BMD and FNCSGPs, is a novel osteoporosis risk gene
doi:10.1007/s00198-009-0998-7
PMCID: PMC2924432  PMID: 19680589
copy number variation; bone mineral density; bone geometry; osteoporosis
22.  2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication 
PLoS ONE  2013;8(11):e80769.
2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.
doi:10.1371/journal.pone.0080769
PMCID: PMC3832489  PMID: 24260477
23.  Genome-Wide Association Study of Copy Number Variants Suggests LTBP1 and FGD4 Are Important for Alcohol Drinking 
PLoS ONE  2012;7(1):e30860.
Alcohol dependence (AD) is a complex disorder characterized by psychiatric and physiological dependence on alcohol. AD is reflected by regular alcohol drinking, which is highly inheritable. In this study, to identify susceptibility genes associated with alcohol drinking, we performed a genome-wide association study of copy number variants (CNVs) in 2,286 Caucasian subjects with Affymetrix SNP6.0 genotyping array. We replicated our findings in 1,627 Chinese subjects with the same genotyping array. We identified two CNVs, CNV207 (combined p-value 1.91E-03) and CNV1836 (combined p-value 3.05E-03) that were associated with alcohol drinking. CNV207 and CNV1836 are located at the downstream of genes LTBP1 (870 kb) and FGD4 (400 kb), respectively. LTBP1, by interacting TGFB1, may down-regulate enzymes directly participating in alcohol metabolism. FGD4 plays a role in clustering and trafficking GABAA receptor and subsequently influence alcohol drinking through activating CDC42. Our results provide suggestive evidence that the newly identified CNV regions and relevant genes may contribute to the genetic mechanism of alcohol dependence.
doi:10.1371/journal.pone.0030860
PMCID: PMC3266269  PMID: 22295116
24.  Mitochondria-wide Association Study of Common Variants in Osteoporosis 
Annals of human genetics  2011;75(5):569-574.
Many lines of evidence suggest that mitochondrial DNA (mtDNA) variants are involved in the pathogenesis of human complex diseases, especially for age-related disorders. Osteoporosis is a typical age-related complex disease. However, the role of mtDNA variants in the susceptibility of osteoporosis is largely unknown. In this study, we performed a mitochondria-wide association study for osteoporosis in Caucasians. A total of 445 mitochondrial single nucleotide polymorphisms (mtSNPs) were genotyped in a large sample of 2,286 unrelated Caucasian subjects by using the Affymetrix Genome-Wide SNP Array 6.0, and 72 mtSNPs survived the quality control. We first tested for association between single-mtSNP and bone mineral density (BMD), and identified that, a mtSNP within the NADH dehydrogenase 2 gene (ND2), mt4823 C/A polymorphism, was strongly associated with hip BMD (P = 2.05 × 10−4), even after conservative Bonferroni correction‥ The C allele of mt4823 was associated with reduced hip BMD and the effect size (β) was estimated to be ~0.044. Another SNP mt15885 within the Cytochrome b gene (Cytb) was found to be associated both with spine (P = 1.66×10−3) and hip BMD (P = 0.023). The T allele of mt15885 had a protective effect on spine (β = 0.064) and hip BMD (β = 0.038). Next, we classified subjects into the nine common European haplogroups and conducted association analyses. Subjects classified as haplogroup X had significantly lower mean hip BMD values than others (P = 0.040). Our results highlighted the importance of mtDNA variants in influencing BMD variation and risk to osteoporosis.
doi:10.1111/j.1469-1809.2011.00663.x
PMCID: PMC3155639  PMID: 21762117
mtSNP; haplogroup; osteoporosis; BMD; association
25.  Copy number variation of the APC gene is associated with regulation of bone mineral density☆ 
Bone  2012;51(5):939-943.
Introduction
Genetic studies of osteoporosis have commonly examined SNPs in candidate genes or whole genome analyses, but insertions and deletions of DNA, collectively called copy number variations (CNVs), also comprise a large amount of the genetic variability between individuals. Previously, SNPs in the APC gene have been strongly associated with femoral neck and lumbar spine volumetric bone mineral density in older men. In addition, familial adenomatous polyposis patients carrying heterozygous mutations in the APC gene have been shown to have significantly higher mean bone mineral density than age- and sex-matched controls suggesting the importance of this gene in regulating bone mineral density. We examined CNV within the APC gene region to test for association with bone mineral density.
Methods
DNA was extracted from venous blood, genotyped using the Human Hap610 arrays and CNV determined from the fluorescence intensity data in 2070 Caucasian men and women aged 47.0 ± 13.0 (mean ± SD) years, to assess the effects of the CNV on bone mineral density at the forearm, spine and total hip sites.
Results
Data for covariate adjusted bone mineral density from subjects grouped by APC CNV genotype showed significant difference (P = 0.02–0.002). Subjects with a single copy loss of APC had a 7.95%, 13.10% and 13.36% increase in bone mineral density at the forearm, spine and total hip sites respectively, compared to subjects with two copies of the APC gene.
Conclusions
These data support previous findings of APC regulating bone mineral density and demonstrate that a novel CNV of the APC gene is significantly associated with bone mineral density in Caucasian men and women.
Highlights
► Previously, SNPs in APC gene have been associated with volumetric BMD in older men. ► We examined CNV in the APC gene and found significant association with BMD in the TwinsUK cohort. ► The maximum difference between subjects with APC CNV and wild type was 13.4% at the total hip site. ► APC plays an important role in bone regulation via the Wnt signaling pathway.
doi:10.1016/j.bone.2012.07.022
PMCID: PMC3918860  PMID: 22884971
Bone mineral density; Osteoporosis; Copy number variation; APC; Association

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