Osteoporos Int DOI 10.1007/s00198-015-3195-x
ORIGINAL ARTICLE
Evaluation of common variants in CNR2 gene for bone mineral density and osteoporosis susceptibility in postmenopausal women of Han Chinese C. Zhang 1 & J. Ma 1 & G. Chen 2 & D. Fu 2 & L. Li 2 & M. Li 3
Received: 2 March 2015 / Accepted: 28 May 2015 # International Osteoporosis Foundation and National Osteoporosis Foundation 2015
Abstract Summary Postmenopausal osteoporosis is a major health problem with important genetic factors in postmenopausal women. We thoroughly evaluated the relationship of CNR2 polymorphisms with osteoporosis in a cohort of 1032 osteoporosis patients and 2089 healthy controls from Han Chinese postmenopausal women. Statistically significant differences, depending on different genotypes, were presented. Introduction Osteoporosis is a major health problem in postmenopausal women, which is a multifactorial disease in which genetic determinants are modulated by hormonal, environmental, and nutritional factors. An important clinical risk factor in the pathogenesis of osteoporosis is the presence of genetic polymorphism in susceptibility genes. The aim of our study was to investigate whether CNR2 gene, which attributes to osteoporosis susceptibility in some populations, is associated with bone mineral density (BMD) or osteoporosis in Han Chinese postmenopausal women. Methods We examine 39 SNPs covering the region of CNR2 gene in 3121 Han Chinese postmenopausal women,
consisting of 1032 osteoporosis patients and 2089 healthy controls, to evaluate the association with BMD and osteoporosis. Results We found that rs4237 and rs2501431 were significantly associated with BMD and osteoporosis (corrected p= 0.020085 and 0.017199) in our sample, and the TT genotype of rs2501431 and the AA genotype of rs4237 had lower lumbar spine BMD and femoral neck BMD compared with the other genotypes. Additionally, analyses by haplotypes indicated that two haplotype blocks, containing rs4237 and rs2501431 respectively, in the CNR2 gene significantly associated with BMD and osteoporosis (both global permutation p < 0.001), and a risk haplotype (ATTT) in the block of rs3003336-rs2501431-rs2502992-rs2501432 had almost 4fold increase in the cases. Conclusions Our results provide further supportive evidence for an important role of CNR2 gene in the etiology of osteoporosis and suggest that it may be a genetic risk factor for BMD and osteoporosis in Han Chinese postmenopausal women.
Electronic supplementary material The online version of this article (doi:10.1007/s00198-015-3195-x) contains supplementary material, which is available to authorized users.
Keywords Bone mineral density . CNR2 gene . Osteoporosis . Postmenopausal women . Single-nucleotide polymorphisms
* M. Li [email protected]
Introduction 1
The First Department of Orthopedics, the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, 157 Xiwu road, Xi’an 710061, China
2
College of Medicine & Forensics, Xi’an Jiaotong University, 76 West Yanta road, Xi’an 710061, China
3
Department of Ultrasound, the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, 157 Xiwu road, Xi’an 710061, China
Osteoporosis is one of the commonest metabolic bone diseases worldwide, which is characterized by bone strength weak and reduced bone mineral density (BMD) and is associated with chronic pain, disability, and mortality [1]. Some research suggested that osteoporosis shows the distinct age and gender characteristics [2], and it is more common in postmenopausal women
Osteoporos Int
for a consequent increase of bone fracture risk [3–6]. As a major risk factor for osteoporosis, 60–90 % of BMD variation in the population is genetically determined [7]. In fact, increasing evidence suggest that genetic factors are important risk factors for predicting the susceptibility to osteoporosis [6, 8–12], and twin and family studies have emphasized the heritability of BMD [13]. While association studies provide a promising approach for studying the genetics of complex diseases, the discoveries of genetic mechanism of them, such as schizophrenia, hypertension, and osteoporosis, lead to the suggestion that the additive effect of many minor genetic effects contributes to the disease occurrence [14–17]. Based on the recent genome-wide association studies (GWAS), meta-analysis studies, and largescale association studies, many genes have been identified as being associated with osteoporosis and/or fracture risk, and several genomic regions, such as 1p36, 1q21-25, 2p22-24, 3p14-25, 4q25-34, 6p21, 7p14-21, 11q14-25, 12q23-24, 13q14-34, and 20p12, have been well validated [5, 11, 18, 19]. Among these candidate genes, CNR2 gene has gained much attention. CNR2 gene contains six exons spanning a 90-kb region located on 1p36.11, which encodes nonneuronal CB2 receptor mainly expressed in immune cells [20]. Because the chromosomal region of 1p36 containing CNR2 gene has been implicated in osteoporosis [21–23], and a previous study showed CNR2 knockout mice had a decreased bone mass, reminiscent of human osteoporosis [24]. Some studies have investigated the association between a few CNR2 polymorphisms and BMD [25–29]. However, they were with conflicting results. Recently, it has been reported that CNR2 gene was associated with BMD in Korean postmenopausal women [30]. Despite evidence of strongly significant association in Koreans, the underlying biological mechanisms are largely unknown and the genetic loci that contribute to osteoporosis remain to be elucidated. In addition, it has been reported in various populations, with conflicting data depending on the ethnicity examined. Therefore, it is still necessary to explore the potential association between CNR2 polymorphisms and osteoporosis in other distinct populations. So far, the association between CNR2 gene and osteoporosis has not been systematically investigated in Han Chinese population. To confirm and replicate the association between CNR2 gene and osteoporosis, further studies with larger samples may be required. In the current study, we conducted the first large-scale systematic association study of CNR2 gene containing preciously reported all single nucleotide polymorphisms (SNPs), which are significantly associated with osteoporosis in Koreans as well as other populations, to determine whether or not CNR2 gene is associated with osteoporosis in postmenopausal women of Han Chinese based on a case–control study. Additionally, this study provides further information regarding the use of CNR2 polymorphisms as markers of
susceptibility to BMD and osteoporosis in postmenopausal women.
Materials and methods Subjects All subjects recruited in this study were random genetically unrelated Han Chinese postmenopausal women from the city of Xi’an in Shaanxi Province, with no migration history within the previous three generations. None of those women had a history of taking medicines for the treatment of osteoporosis, and subjects with diseases or medications known to affect bone metabolism were excluded in the controls. Additionally, given the possibility of osteoporosis resulting from obesity and the overall level of Asian body mass index (BMI), we ruled out subjects of BMI ≥27 in our study. In total, 1032 women (aged 48–68 years) with primary postmenopausal osteoporosis and 2089 healthy age-matched women (aged 48– 68 years) were recruited from the Second Affiliated Hospital of Xi’an Jiaotong University and Xi’an Honghui Hospital from April 2013 to August 2014. All participants have completed written informed consent forms. The protocol of this study was approved by the Medical Ethics Committee of Xi’an Jiaotong University. Measurement of bone mineral density Dual-energy X-Ray absorptiometry (Lunar Expert 1313, Lunar Corp., USA) was used to measure BMD at the lumbar spine (L2–4) and femoral neck. Bone mineral density was determined according to standard Lunar protocols. Osteoporosis was defined according to the conventional World Health Organization (WHO) definition. Participants with T-score< −1.0 SD were grouped into patients as having low bone mass, and those with T-score>−1.0 SD were classified into normals. The anthropometric baseline data of all subjects were obtained by measurement and questionnaire. The continuous parameters were presented in Table 1. Independent sample t test for continuous variables was performed in comparisons of anthropometric characteristics of the participants (Table 1). SNP selection and genotyping Firstly, we searched for all SNPs with minor allele frequencies (MAF) ≥0.05 between 10 kb upstream and 10 kb downstream (20 kb window) of CNR2 gene in the HapMap HCB database by Haploview v4.2. We found 19 SNPs. Then, we selected other 20 SNPs from previous reports [25–30]. Therefore, based on the above criteria, 39 SNPs were included in the further analyses (rs10917411, rs3753270, rs10917414, rs622770, rs4237, rs4649119, rs3003328, rs3123554,
Osteoporos Int Table 1 Comparisons of anthropometric characteristics of the case and control groups
Parameters
Assessment method
Case
Control
P value
Age (years) Range of age (years) YSM (years) Weight (kg) Height (cm)
Questionnaire Questionnaire Questionnaire Measured Measured
61.2±5.77 48–68 9.8±3.65 62.1±2.38 163.4±4.99
61.4±5.52 48–68 10.0±4.11 62.2±2.36 163.2±4.83
0.368 NA 0.167 0.293 0.305
BMI (kg/m2) LS BMD (g/cm2) FN BMD (g/cm2)
Measured Measured Measured
23.3±1.72 0.818±0.072 0.705±0.053
23.4±1.65 1.001±0.050 0.859±0.066
0.192
ORIGINAL ARTICLE
Evaluation of common variants in CNR2 gene for bone mineral density and osteoporosis susceptibility in postmenopausal women of Han Chinese C. Zhang 1 & J. Ma 1 & G. Chen 2 & D. Fu 2 & L. Li 2 & M. Li 3
Received: 2 March 2015 / Accepted: 28 May 2015 # International Osteoporosis Foundation and National Osteoporosis Foundation 2015
Abstract Summary Postmenopausal osteoporosis is a major health problem with important genetic factors in postmenopausal women. We thoroughly evaluated the relationship of CNR2 polymorphisms with osteoporosis in a cohort of 1032 osteoporosis patients and 2089 healthy controls from Han Chinese postmenopausal women. Statistically significant differences, depending on different genotypes, were presented. Introduction Osteoporosis is a major health problem in postmenopausal women, which is a multifactorial disease in which genetic determinants are modulated by hormonal, environmental, and nutritional factors. An important clinical risk factor in the pathogenesis of osteoporosis is the presence of genetic polymorphism in susceptibility genes. The aim of our study was to investigate whether CNR2 gene, which attributes to osteoporosis susceptibility in some populations, is associated with bone mineral density (BMD) or osteoporosis in Han Chinese postmenopausal women. Methods We examine 39 SNPs covering the region of CNR2 gene in 3121 Han Chinese postmenopausal women,
consisting of 1032 osteoporosis patients and 2089 healthy controls, to evaluate the association with BMD and osteoporosis. Results We found that rs4237 and rs2501431 were significantly associated with BMD and osteoporosis (corrected p= 0.020085 and 0.017199) in our sample, and the TT genotype of rs2501431 and the AA genotype of rs4237 had lower lumbar spine BMD and femoral neck BMD compared with the other genotypes. Additionally, analyses by haplotypes indicated that two haplotype blocks, containing rs4237 and rs2501431 respectively, in the CNR2 gene significantly associated with BMD and osteoporosis (both global permutation p < 0.001), and a risk haplotype (ATTT) in the block of rs3003336-rs2501431-rs2502992-rs2501432 had almost 4fold increase in the cases. Conclusions Our results provide further supportive evidence for an important role of CNR2 gene in the etiology of osteoporosis and suggest that it may be a genetic risk factor for BMD and osteoporosis in Han Chinese postmenopausal women.
Electronic supplementary material The online version of this article (doi:10.1007/s00198-015-3195-x) contains supplementary material, which is available to authorized users.
Keywords Bone mineral density . CNR2 gene . Osteoporosis . Postmenopausal women . Single-nucleotide polymorphisms
* M. Li [email protected]
Introduction 1
The First Department of Orthopedics, the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, 157 Xiwu road, Xi’an 710061, China
2
College of Medicine & Forensics, Xi’an Jiaotong University, 76 West Yanta road, Xi’an 710061, China
3
Department of Ultrasound, the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, 157 Xiwu road, Xi’an 710061, China
Osteoporosis is one of the commonest metabolic bone diseases worldwide, which is characterized by bone strength weak and reduced bone mineral density (BMD) and is associated with chronic pain, disability, and mortality [1]. Some research suggested that osteoporosis shows the distinct age and gender characteristics [2], and it is more common in postmenopausal women
Osteoporos Int
for a consequent increase of bone fracture risk [3–6]. As a major risk factor for osteoporosis, 60–90 % of BMD variation in the population is genetically determined [7]. In fact, increasing evidence suggest that genetic factors are important risk factors for predicting the susceptibility to osteoporosis [6, 8–12], and twin and family studies have emphasized the heritability of BMD [13]. While association studies provide a promising approach for studying the genetics of complex diseases, the discoveries of genetic mechanism of them, such as schizophrenia, hypertension, and osteoporosis, lead to the suggestion that the additive effect of many minor genetic effects contributes to the disease occurrence [14–17]. Based on the recent genome-wide association studies (GWAS), meta-analysis studies, and largescale association studies, many genes have been identified as being associated with osteoporosis and/or fracture risk, and several genomic regions, such as 1p36, 1q21-25, 2p22-24, 3p14-25, 4q25-34, 6p21, 7p14-21, 11q14-25, 12q23-24, 13q14-34, and 20p12, have been well validated [5, 11, 18, 19]. Among these candidate genes, CNR2 gene has gained much attention. CNR2 gene contains six exons spanning a 90-kb region located on 1p36.11, which encodes nonneuronal CB2 receptor mainly expressed in immune cells [20]. Because the chromosomal region of 1p36 containing CNR2 gene has been implicated in osteoporosis [21–23], and a previous study showed CNR2 knockout mice had a decreased bone mass, reminiscent of human osteoporosis [24]. Some studies have investigated the association between a few CNR2 polymorphisms and BMD [25–29]. However, they were with conflicting results. Recently, it has been reported that CNR2 gene was associated with BMD in Korean postmenopausal women [30]. Despite evidence of strongly significant association in Koreans, the underlying biological mechanisms are largely unknown and the genetic loci that contribute to osteoporosis remain to be elucidated. In addition, it has been reported in various populations, with conflicting data depending on the ethnicity examined. Therefore, it is still necessary to explore the potential association between CNR2 polymorphisms and osteoporosis in other distinct populations. So far, the association between CNR2 gene and osteoporosis has not been systematically investigated in Han Chinese population. To confirm and replicate the association between CNR2 gene and osteoporosis, further studies with larger samples may be required. In the current study, we conducted the first large-scale systematic association study of CNR2 gene containing preciously reported all single nucleotide polymorphisms (SNPs), which are significantly associated with osteoporosis in Koreans as well as other populations, to determine whether or not CNR2 gene is associated with osteoporosis in postmenopausal women of Han Chinese based on a case–control study. Additionally, this study provides further information regarding the use of CNR2 polymorphisms as markers of
susceptibility to BMD and osteoporosis in postmenopausal women.
Materials and methods Subjects All subjects recruited in this study were random genetically unrelated Han Chinese postmenopausal women from the city of Xi’an in Shaanxi Province, with no migration history within the previous three generations. None of those women had a history of taking medicines for the treatment of osteoporosis, and subjects with diseases or medications known to affect bone metabolism were excluded in the controls. Additionally, given the possibility of osteoporosis resulting from obesity and the overall level of Asian body mass index (BMI), we ruled out subjects of BMI ≥27 in our study. In total, 1032 women (aged 48–68 years) with primary postmenopausal osteoporosis and 2089 healthy age-matched women (aged 48– 68 years) were recruited from the Second Affiliated Hospital of Xi’an Jiaotong University and Xi’an Honghui Hospital from April 2013 to August 2014. All participants have completed written informed consent forms. The protocol of this study was approved by the Medical Ethics Committee of Xi’an Jiaotong University. Measurement of bone mineral density Dual-energy X-Ray absorptiometry (Lunar Expert 1313, Lunar Corp., USA) was used to measure BMD at the lumbar spine (L2–4) and femoral neck. Bone mineral density was determined according to standard Lunar protocols. Osteoporosis was defined according to the conventional World Health Organization (WHO) definition. Participants with T-score< −1.0 SD were grouped into patients as having low bone mass, and those with T-score>−1.0 SD were classified into normals. The anthropometric baseline data of all subjects were obtained by measurement and questionnaire. The continuous parameters were presented in Table 1. Independent sample t test for continuous variables was performed in comparisons of anthropometric characteristics of the participants (Table 1). SNP selection and genotyping Firstly, we searched for all SNPs with minor allele frequencies (MAF) ≥0.05 between 10 kb upstream and 10 kb downstream (20 kb window) of CNR2 gene in the HapMap HCB database by Haploview v4.2. We found 19 SNPs. Then, we selected other 20 SNPs from previous reports [25–30]. Therefore, based on the above criteria, 39 SNPs were included in the further analyses (rs10917411, rs3753270, rs10917414, rs622770, rs4237, rs4649119, rs3003328, rs3123554,
Osteoporos Int Table 1 Comparisons of anthropometric characteristics of the case and control groups
Parameters
Assessment method
Case
Control
P value
Age (years) Range of age (years) YSM (years) Weight (kg) Height (cm)
Questionnaire Questionnaire Questionnaire Measured Measured
61.2±5.77 48–68 9.8±3.65 62.1±2.38 163.4±4.99
61.4±5.52 48–68 10.0±4.11 62.2±2.36 163.2±4.83
0.368 NA 0.167 0.293 0.305
BMI (kg/m2) LS BMD (g/cm2) FN BMD (g/cm2)
Measured Measured Measured
23.3±1.72 0.818±0.072 0.705±0.053
23.4±1.65 1.001±0.050 0.859±0.066
0.192
