doi: 10.1111/iji.12130
Associations between polymorphisms of HLA-B gene and postmenopausal osteoporosis in Chinese Han population S.-m. Li*, D.-x. Zhou†,‡ & M.-Y. Liu†
Summary Osteoporosis is a systemic skeletal disease, which is more prevalent in postmenopausal women. Osteoporosis likely develops beginning with genetic risk. This study explored the relationships between polymorphisms of HLA-B gene and postmenopausal osteoporosis in a Chinese Han population. Polymerase chain reaction sequence-based typing (PCR-SBT) method was used for DNA typing at HLA-B locus in 70 patients with postmenopausal osteoporosis and 73 healthy controls in female Han population of Shaanxi Province, situated in north-western China. We found that 40 HLA-B alleles in postmenopausal osteoporosis patients and control subjects, respectively. Furthermore, the frequency of HLA-B* 3501 allele was significantly higher in postmenopausal osteoporosis patients than in the control group (P = 0.033), and the relative risk was 7.632 (95% CI: 0.927–62.850). Our results suggest that HLA-B* 3501 was likely an important risk factor for postmenopausal osteoporosis. As different populations have different HLA polymorphisms, further investigation of the relationship of various HLA genes and osteoporosis with larger sample size is still necessary in the future.
Introduction Osteoporosis is a systemic skeletal disease characterized by low bone mass and micro-architectural changes of the bone tissue, which leads to increased bone fragility and susceptibility to nontraumatic fracture (Dodd & Rowe, 2013). It is among the most prevalent metabolic bone disease in a rapidly ageing
* Department of Orthopedics, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China, † Department of Pathology, Medical School, Xi’an Jiaotong University, Xi’an, China and ‡ Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China Received 11 February 2014; revised NA; accepted 27 April 2014 Correspondence: Dang-xia Zhou, Pathology Department, Medical School, Xi’an Jiaotong University, Xi’an, 710061, China. Tel./Fax: +86 2982655189; E-mail: [email protected]
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population, especially in postmenopausal women (Esfahanian et al., 2012). Osteoporosis-induced fractures further lead to an increased risk of death, long-term nursing home care or permanent limitations in mobility and performance of daily-living activities in postmenopausal women (Esfahanian et al., 2012; Lai et al., 2013; Yang et al., 2013). Although osteoporosis has been described decades ago, its exact mechanisms remain poorly understood (Martınez-Maestre et al., 2013). Therefore, it is urgent to understand the aetiology and pathogenesis of postmenopausal osteoporosis. Currently, there is growing evidence that genetic factors play an important role in the pathogenesis of osteoporosis (Prockop, 1998; Theoleyre et al., 2004; Ozbas et al., 2012; Athanasiadis et al., 2013; Yang et al., 2013; Oei et al., 2014). Human leucocyte antigen (HLA) system, also known as the major histocompatibility complex (MHC), is the most polymorphic immunogenetic systems, which located on the chromosome 6p21.3 (Schreuder et al., 2005). Currently, more than 9946 alleles were identified in HLA system (http://www.ebi.ac.uk/imgt/hla/stats.html). High polymorphism of HLA complex has made it valuable for disease association studies. Studies have linked HLA allele to this susceptibility to more than 100 diseases (Carrington & O Brien, 2003; Noble et al., 2010; Zhou et al., 2011). The HLA system has been reported to be involved in several bone metabolic disorders including rheumatoid arthritis and ankylosing spondylitis (Vignal et al., 2009; Cauli et al., 2013; Cojocaru & Chicosß, 2013; Djidjik et al., 2014). Moreover, HLA genes have been associated with peak bone mass (PBM) and bone mineral density (BMD). For instance, Tsuji et al. (1998) investigated the regulation of HLA polymorphism and peak bone mass (PBM) to elucidate the genetic backgrounds of bone metabolism in young Japanese women, and they concluded that the HLA-A*24-B*07DRB*01 haplotype can be considered a new genetic marker implicated with low PBM in healthy young Japanese women. Douroudis et al. (2007) found that HLA-B7, -DR15 and -DQ6 (P = 0.026) were associated with a lower BMD measured at the forearm, and their study showed a significant association between HLA alleles and bone mass loss in the postmenopausal
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 324–329
HLA and postmenopausal osteoporosis
osteoporosis of Greek population. Due to the distributions of HLA alleles, haplotypes show high variation in different ethnic groups or even the same ethnic group living in different geographic areas (Zhou et al., 2012). China is one of the largest and most populous areas in the world. Han nationality constitutes about 92% of population of China. However, to the best of our knowledge, there are no studies concerning the association of HLA genes and osteoporosis in this region. Therefore, this study was designed to investigate a possible association of HLA-B gene with susceptibility or resistance to postmenopausal osteoporosis in Chinese Han population.
Materials and methods Subjects
The protocol was fully approved by the Institutional Medical Ethics Committee of Xi’an Jiaotong University. The risks and benefits of participating in the study were explained to all subjects. Written informed consent forms were signed by all subjects. Participants were random unrelated Chinese Han nationality men whose ancestors have lived in the Shaanxi Province for at least three generations. Detailed medical history was obtained from all subjects. Women with a history of bone disease, metabolic or endocrine disorders such as diabetes mellitus, hyperthyroidism or any systemic illness known to affect bone metabolism were excluded from the study. In addition, women taking drugs that influence bone metabolism, including calcium supplements or under treatment with drugs known to affect BMD were also ruled out from the study. Finally, 70 women (aged 50–65 years) with primary postmenopausal osteoporosis and 73 healthy age-matched women (aged 51– 65 years) patients were recruited. Osteoporosis assessment
Bone mineral density of all subjects was measured using dual-energy X-ray absorptiometry (Lunar Corp, Madison, WI, USA) at the lumbar spine (L1-L4) and femoral neck by two qualified radiologists who were blinded to other medical data. BMD was expressed in grams per square centimetre (g cm 2) and as peak bone mass percentage in normal subjects (T-score). According to the criteria of World Health Organization, osteoporosis in postmenopausal women was diagnosed when T-score < 2.5 SD. Peripheral blood DNA extraction
After informed consent, peripheral blood was drawn into a sterile tube containing noncoagulated ethylenediaminetetraacetic acid (EDTA) and stored at 20°C before DNA extraction. Genomic DNA was extracted from peripheral blood leucocytes according to the
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manufacturer’s protocol (A004-1; Dinguo, Beijing, China). Genotyping for HLA-B gene
According to the methods previously described (Zhou et al., 2011), all subjects were typed for HLA-B locus using polymerase chain reaction sequence-based typing (PCR-SBT) method. At first, polymerase chain reaction amplifications were accomplished on GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA), and then, amplified DNA fragments were purified and sequenced using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems) in an ABI 3730XL DNA Sequencer (Applied Biosystems) according to the manufacturer’s instructions. Ambiguous types were resolved to four digits according to the updated database (IMGT release 2.21.0; European Bioinformatics Institute, London, UK, http://www.ebi.ac.uk/imgt/hla/stats.html). Statistical analysis
At first, Hardy–Weinberg equilibrium (HWE) for HLA-B locus was assessed using the Arlequin software package version 3.1 (Laurent Excoffier, CMPG, Zoological Institute, University of Bern, Switzerland), and P < 0.05 was regarded as statistically significant. Then, genetic parameters including homozygotes (Ho), heterozygosity (He), power of discrimination (PD), polymorphism information content (PIC) and probability of paternity exclusion (PPE) were estimated using the PowerStat version 1.2 spreadsheet (Promega Corp, Madison, WI, USA). Allele frequencies of HLAB locus were calculated by SPSS13.0 software (SPSS Inc., Chicago, IL, USA). Genotypic associations were performed using SPSS13.0 (SPSS Inc.). The frequencies of HLA alleles were compared between controls and patients using the chi-square test. Fisher’s exact test was applied for expected numbers less than 5. Relative risk was calculated as an odds ratio (OR) with 95% confidence interval (CI) using Cornfield’s approximation. P < 0.05 was accepted as statistically significant.
Results Hardy–Weinberg Equilibrium
The alleles in HLA-B locus were comply with Hardy– Weinberg equilibrium in both postmenopausal osteoporosis (P = 0.82892485) and control groups (P = 0.3702555). Genetic parameters
The genetic parameters of HLA-B locus in postmenopausal osteoporosis patients and controls were summarized in Table 1. Ho, He, PD, PIC and PPE are genetic
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Table 1. Genetic parameters of HLA-B locus in postmenopausal osteoporosis patients and controls Locus
Group
Ho
He
PD
PIC
PPE
HLA-B
Patients Controls
4.3% 1.4%
95.7% 98.6%
0.984 0.983
0.95 0.96
0.913 0.972
Ho, homozygotes; He, heterozygotes; PD, power of discrimination; PIC, polymorphism information content; PPE, probability of paternity exclusion.
parameters for evaluating the polymorphisms of gene. A gene locus is considered highly polymorphic when its PIC is higher than 0.5, or PD value higher than 0.8 or PPE value higher than 0.5, respectively. As shown in Table 1, all alleles in HLA-B locus were highly polymorphic in both patients and control groups. HLA-B allelic frequencies
Totally, we detected 40 high-resolution HLA-B alleles in 70 postmenopausal osteoporosis patients. The allele frequencies of HLA-B locus were shown in Table 2. At the HLA-B loci, the six most common alleles with a frequency >5% were HLA-B*1302 (8.6%), B*5101 (8.6%), B*4601 (7.9%), B*1301 (6.4%), B*3501 (5%) and B*5801 (5%), respectively. On the other hand, we found 40 high-resolution HLA-B alleles in 73 control subjects (Table 2). At the HLA-B loci, the five most common alleles with a frequency larger than 5% were HLA-B*1302 (6.8%), B*4001 (6.8%), B*1501 (6.2%), B*4601 (5.5%) and B*5101 (5.5%), respectively. Association of HLA-B allele with postmenopausal osteoporosis
The distribution of high-resolution HLA-B alleles between the postmenopausal osteoporosis patients and controls was compared. As shown in Table 2, we observed that the frequency of HLA-B* 3501 allele was significantly higher in patients than in the control group (P = 0.033) and the relative risk was 7.632 (95% CI: 0.927–62.850). As most previous association studies of HLA-B and male infertility were based on low resolution (serotypes) of HLA. To be compatible with previous studies, we grouped our sequence-based high-resolution HLA genotype to their associated serotype according to the classification of Holdsworth et al. (2009). As B7, B27 was ever reported to be associated with bone diseases in previous population, and we considered B7, B27, B35 as candidate alleles in this study (Table 3). The results showed no significant increase of B7 in patients group, with an odds ratio (OR) 1.590 (95%CI: 0.439–5.757; P = 0.534). Moreover, B27 allele showed no significant difference in patients and that in the controls, with an odds ratio 0.777 (95%CI: 0.171–3.537; P = 1.000). Nevertheless, the
frequency of B35 allele was significant higher in patients than in the control group (P = 0.039), and the relative risk was 3.328 (95% CI: 1.047–10.580).
Discussion and conclusions Osteoporosis is a common disorder characterized by low bone mass and micro-architectural deterioration of bone tissue, resulting in an increase in bone fragility and in susceptibility to fractures (Dodd & Rowe, 2013). It has been proved that osteoporosis showed the distinct age and gender characteristics, and it was more prevalent in postmenopausal women (Pietschmann et al., 2009; Gammage et al., 2012). Therefore, the aim of the present study was designed to found the genetic marker in the population of postmenopausal osteoporosis. Bone homeostasis is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. It is also regulated by immune system, and its imbalance often results in osteoporosis (Jianbo et al., 2013; Tanaka, 2013). Recently, substantial evidences also supported that immunological factors play an important role in the pathogenesis of osteoporosis. For instance, Moln ar reported that increased IL-17A levels are involved in postmenopausal osteoporosis, and it plays a important role in the bone-resorpting processes (Moln ar et al., 2014). The study of Zhao demonstrated that T helper (Th) 17 cells are critical modulator in the pathogenesis of oestrogen-deficient osteoporosis, and therapeutic strategies targeting IL-17 networks may be clinically useful in treatment for postmenopausal osteoporosis (Zhao, 2013). The study of Kim showed that leucocyte common antigen-related (LAR) tyrosine phosphatase positively regulates osteoblast differentiation by modulating extracellular signal-regulated kinase (ERK) activation. LAR phosphatase could be used as a novel regulatory target protein in many bone-associated diseases, including osteoporosis (Kim et al., 2010). The HLA is one of the most important immunoregulatory genes. Therefore, investigating HLA polymorphism in postmenopausal osteoporosis is an open field of research with great potentials. The present study is the first to explore the association of HLA-B alleles with postmenopausal osteoporosis in Chinese Han population using the PCR-SBT method. In this study, we found the frequency of HLA-B* 3501 allele was significantly higher in postmenopausal osteoporosis patients than in the control group, and the relative risk was 7.632 (95% CI: 0.927–62.850), indicating HLA-B* 3501 might be a major risk gene of postmenopausal osteoporosis patients in Chinese Han population. Human leucocyte antigen genes in humans include three groups: class I, class II and class III. The HLA-B gene was one of the most often studied genes in HLA class I genes. One of the particular strengths in our present study is that PCR-SBT method was used to
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Table 2. High-resolution genotyping distributions of HLA-B alleles in postmenopausal osteoporosis patients and controls
Control (n = 146 alleles)
Patients (n = 140 alleles)
HLA-B
n
AF (%)
n
AF (%)
P
OR (95% CI)
B*0702 B*0705 B*0801 B*1301 B*1302 B*1402 B*1501 B*1502 B*1503 B*1505 B*1511 B*1512 B*1518 B*1525 B*1527 B*1529 B*1532 B*1558 B*1801 B*2704 B*2705 B*2706 B*3501 B*3502 B*3503 B*3701 B*3801 B*3802 B*3901 B*4001 B*4002 B*4003 B*4006 B*4402 B*4403 B*4601 B*4701 B*4801 B*4901 B*5001 B*5101 B*5102 B*5201 B*5401 B*5502 B*5601 B*5701 B*5801
3 1 1 6 10 1 9 1 1 0 5 1 1 0 3 0 2 1 3 1 2 1 1 0 3 4 5 3 0 10 5 0 6 1 3 8 0 2 2 1 8 2 5 7 6 0 5 6
2.1 0.7 0.7 4.1 6.8 0.7 6.2 0.7 0.7 0 3.4 0.7 0.7 0 2.1 0 1.4 0.7 2.1 0.7 1.4 0.7 0.7 0 2.1 2.7 3.4 2.1 0 6.8 3.4 0 4.1 0.7 2.1 5.5 0 1.4 1.4 0.7 5.5 1.4 3.4 4.8 4.1 0 3.4 4.1
6 0 0 9 12 0 5 2 0 1 4 0 2 1 1 1 0 0 3 1 1 1 7 1 4 1 3 1 1 6 5 1 3 2 2 11 1 4 0 2 12 1 3 6 2 2 3 7
4.3 0 0 6.4 8.6 0 3.6 1.4 0 0.7 2.9 0 1.4 0.7 0.7 0.7 0 0 2.1 0.7 0.7 0.7 5 0.7 2.9 0.7 2.1 0.7 0.7 4.3 3.6 0.7 2.1 1.4 1.4 7.9 0.7 2.9 0 1.4 8.6 0.7 2.1 4.3 1.4 1.4 2.1 5
0.327 – – 0.379 0.585 – 0.310 0.616 – – 0.783 – 0.616 – 0.623 – – – 1.000 1.000 1.000 1.000 0.033* – 0.718 0.371 0.723 0.623 – 0.443 1.000 – 0.502 0.616 1.000 0.481 – 0.438 – 0.616 0.358 1.000 0.723 0.836 0.283 – 0.723 0.718
2.134 – – 1.603 1.275 – 0.564 2.101 – – 0.829 – 2.101 – 0.343 – – – 1.044 1.043 0.518 1.043 7.632 – 1.402 0.255 0.618 0.343 – 0.609 1.044 – 0.511 2.101 0.691 1.471 – 2.118 – 2.101 1.617 0.518 0.618 0.889 0.338 – 0.618 1.228
(0.523–8.705)
(0.555–4.628) (0.532–3.053) (0.184–1.726) (0.188–23.438)
(0.218–3.154) (0.188–23.438) (0.035–3.337)
(0.207–5.261) (0.065–16.841) (0.046–5.777) (0.065–16.841) (0.927–62.850) (0.308–6.380) (0.028–2.314) (0.145–2.634) (0.035–3.337) (0.215–1.732) (0.296–3.689) (0.125–2.084) (0.188–23.438) (0.114–4.198) (0.574–3.772) (0.382–11.749) (0.188–23.438) (0.640–4.084) (0.046–5.777) (0.145–2.634) (0.291–2.714) (0.067–1.704) (0.145–2.634) (0.402–3.749)
AF, allele frequency; OR, odds ratio; CI, confidence interval; *P < 0.05.
Table 3. Serotype (low-resolution typing) distributions of HLA-B alleles in postmenopausal osteoporosis patients and controls Serotype
DNA-based genotype
Control (n = 146 alleles, %)
Patients (n = 140 alleles, %)
P
OR (95%CI)
B7 B27 B35
B*0702, B*0705 B*2704, B*2705, B*2706 B*3501, B*3503, B*3508
4 (2.7) 4 (2.7) 4 (2.7)
6 (4.3) 3 (2.1) 12 (8.6)
0.534 1.000 0.039*
1.590 (0.439–5.757) 0.777 (0.171–3.537) 3.328 (1.047–10.580)
OR, odds ratio; CI, confidence interval; *P < 0.05.
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confirm the association between postmenopausal osteoporosis and HLA alleles. PCR-SBT method is considered the gold standard for high-resolution definition of HLA (Woo et al., 2012). Previous HLA association studies in osteoporosis have been carried out with low-resolution HLA serologic typing by PCRSSP. PCR-SBT method has much higher accuracy and reliability than simply serological typing methods and also facilitates the standardization of HLA typing processes. Ethnic background and geographic variations are very important factors affecting the study results. China has 56 officially identified ethnic groups, and Han nationality constitutes about 92% of population of Chinese. Shaanxi Province, located in the northwest of China, is a major Han population residual region. It is not only the birthplace of the Chinese nation, but also the birthplace of human civilization in Asia and the cultural centre of prehistoric civilization. In present study, we selected subjects from the same ethnic background and geographic background to avoid the possible occurrence of sampling errors. In addition, women with conditions potentially causing secondary osteoporosis or women over the age of 65 years were ruled out from the present study to avoid the impact of senile osteoporosis. Moreover, all samples in patients and control groups were in Hardy–Weinberg equilibrium at HLAB locus. Ho, He, PD, PIC and PPE are genetic parameters for evaluating the polymorphisms of gene. A gene locus is considered highly polymorphic when its PIC is higher than 0.5, or PD value higher than 0.8 or PPE value higher than 0.5, respectively. In our present study, the PIC values are 0.950 and 0.950, and PD values are 0.984 and 0.983, and PPE values are 0.913 and 0.972, respectively, in both patients and control groups. These values were higher than the cut-off values mentioned above. Therefore, the HLA-B data obtained from our sample population are highly polymorphism, which was valuable for further genetic science research. The association between HLA-B antigens and postmenopausal osteoporosis has also been examined in several populations. Douroudis et al. (2007) found that HLA-B7 was associated with a lower BMD in the postmenopausal osteoporosis patients of Greek origin. Tsuji et al. (1998) found that the haplotype HLAA*24-B*07-DRB*01 is a new genetic marker implicated with low PBM in healthy young Japanese women. However, we did not detect significant differences of HLA-B7 between patients and control groups in our Chinese population. Nevertheless, interestingly, we found the frequency of HLA-B* 3501 allele was significantly higher in postmenopausal osteoporosis patients than in the control group, and the relative risk was 7.632 (95% CI: 0.927–62.850), indicating HLAB* 3501 might be a major risk gene of postmenopausal osteoporosis patients in Chinese Han population, which was not reported in other different
population. We speculated that these conflicting results might be attributed to differences in HLA typing methodology, sample size, ethnic background and geographic variations. Due to the distributions of HLA alleles, haplotypes show high variation in different ethnic groups or even the same ethnic group living in different geographic areas (Zhou et al., 2012). In conclusion, our present study explored the association of the HLA-B alleles with postmenopausal osteoporosis in Chinese Han population using PCR-SBT high-resolution HLA genotyping method, which provides a more accurate and credible resolution than the previous methods. Our results suggest that HLA-B* 3501 was likely an important risk factor for postmenopausal osteoporosis, and our results would add some valuable evidences for osteoporosis study.
Acknowledgements This research was financially supported by National Natural Science Fundings of China (No. 81273018; 30700654), Science Funding of Health Department, Shaanxi Province (2012D58) and the Fundamental Research Funds for the Central University (XJJ 2011024).
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Associations between polymorphisms of HLA-B gene and postmenopausal osteoporosis in Chinese Han population S.-m. Li*, D.-x. Zhou†,‡ & M.-Y. Liu†
Summary Osteoporosis is a systemic skeletal disease, which is more prevalent in postmenopausal women. Osteoporosis likely develops beginning with genetic risk. This study explored the relationships between polymorphisms of HLA-B gene and postmenopausal osteoporosis in a Chinese Han population. Polymerase chain reaction sequence-based typing (PCR-SBT) method was used for DNA typing at HLA-B locus in 70 patients with postmenopausal osteoporosis and 73 healthy controls in female Han population of Shaanxi Province, situated in north-western China. We found that 40 HLA-B alleles in postmenopausal osteoporosis patients and control subjects, respectively. Furthermore, the frequency of HLA-B* 3501 allele was significantly higher in postmenopausal osteoporosis patients than in the control group (P = 0.033), and the relative risk was 7.632 (95% CI: 0.927–62.850). Our results suggest that HLA-B* 3501 was likely an important risk factor for postmenopausal osteoporosis. As different populations have different HLA polymorphisms, further investigation of the relationship of various HLA genes and osteoporosis with larger sample size is still necessary in the future.
Introduction Osteoporosis is a systemic skeletal disease characterized by low bone mass and micro-architectural changes of the bone tissue, which leads to increased bone fragility and susceptibility to nontraumatic fracture (Dodd & Rowe, 2013). It is among the most prevalent metabolic bone disease in a rapidly ageing
* Department of Orthopedics, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China, † Department of Pathology, Medical School, Xi’an Jiaotong University, Xi’an, China and ‡ Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an, China Received 11 February 2014; revised NA; accepted 27 April 2014 Correspondence: Dang-xia Zhou, Pathology Department, Medical School, Xi’an Jiaotong University, Xi’an, 710061, China. Tel./Fax: +86 2982655189; E-mail: [email protected]
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population, especially in postmenopausal women (Esfahanian et al., 2012). Osteoporosis-induced fractures further lead to an increased risk of death, long-term nursing home care or permanent limitations in mobility and performance of daily-living activities in postmenopausal women (Esfahanian et al., 2012; Lai et al., 2013; Yang et al., 2013). Although osteoporosis has been described decades ago, its exact mechanisms remain poorly understood (Martınez-Maestre et al., 2013). Therefore, it is urgent to understand the aetiology and pathogenesis of postmenopausal osteoporosis. Currently, there is growing evidence that genetic factors play an important role in the pathogenesis of osteoporosis (Prockop, 1998; Theoleyre et al., 2004; Ozbas et al., 2012; Athanasiadis et al., 2013; Yang et al., 2013; Oei et al., 2014). Human leucocyte antigen (HLA) system, also known as the major histocompatibility complex (MHC), is the most polymorphic immunogenetic systems, which located on the chromosome 6p21.3 (Schreuder et al., 2005). Currently, more than 9946 alleles were identified in HLA system (http://www.ebi.ac.uk/imgt/hla/stats.html). High polymorphism of HLA complex has made it valuable for disease association studies. Studies have linked HLA allele to this susceptibility to more than 100 diseases (Carrington & O Brien, 2003; Noble et al., 2010; Zhou et al., 2011). The HLA system has been reported to be involved in several bone metabolic disorders including rheumatoid arthritis and ankylosing spondylitis (Vignal et al., 2009; Cauli et al., 2013; Cojocaru & Chicosß, 2013; Djidjik et al., 2014). Moreover, HLA genes have been associated with peak bone mass (PBM) and bone mineral density (BMD). For instance, Tsuji et al. (1998) investigated the regulation of HLA polymorphism and peak bone mass (PBM) to elucidate the genetic backgrounds of bone metabolism in young Japanese women, and they concluded that the HLA-A*24-B*07DRB*01 haplotype can be considered a new genetic marker implicated with low PBM in healthy young Japanese women. Douroudis et al. (2007) found that HLA-B7, -DR15 and -DQ6 (P = 0.026) were associated with a lower BMD measured at the forearm, and their study showed a significant association between HLA alleles and bone mass loss in the postmenopausal
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osteoporosis of Greek population. Due to the distributions of HLA alleles, haplotypes show high variation in different ethnic groups or even the same ethnic group living in different geographic areas (Zhou et al., 2012). China is one of the largest and most populous areas in the world. Han nationality constitutes about 92% of population of China. However, to the best of our knowledge, there are no studies concerning the association of HLA genes and osteoporosis in this region. Therefore, this study was designed to investigate a possible association of HLA-B gene with susceptibility or resistance to postmenopausal osteoporosis in Chinese Han population.
Materials and methods Subjects
The protocol was fully approved by the Institutional Medical Ethics Committee of Xi’an Jiaotong University. The risks and benefits of participating in the study were explained to all subjects. Written informed consent forms were signed by all subjects. Participants were random unrelated Chinese Han nationality men whose ancestors have lived in the Shaanxi Province for at least three generations. Detailed medical history was obtained from all subjects. Women with a history of bone disease, metabolic or endocrine disorders such as diabetes mellitus, hyperthyroidism or any systemic illness known to affect bone metabolism were excluded from the study. In addition, women taking drugs that influence bone metabolism, including calcium supplements or under treatment with drugs known to affect BMD were also ruled out from the study. Finally, 70 women (aged 50–65 years) with primary postmenopausal osteoporosis and 73 healthy age-matched women (aged 51– 65 years) patients were recruited. Osteoporosis assessment
Bone mineral density of all subjects was measured using dual-energy X-ray absorptiometry (Lunar Corp, Madison, WI, USA) at the lumbar spine (L1-L4) and femoral neck by two qualified radiologists who were blinded to other medical data. BMD was expressed in grams per square centimetre (g cm 2) and as peak bone mass percentage in normal subjects (T-score). According to the criteria of World Health Organization, osteoporosis in postmenopausal women was diagnosed when T-score < 2.5 SD. Peripheral blood DNA extraction
After informed consent, peripheral blood was drawn into a sterile tube containing noncoagulated ethylenediaminetetraacetic acid (EDTA) and stored at 20°C before DNA extraction. Genomic DNA was extracted from peripheral blood leucocytes according to the
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manufacturer’s protocol (A004-1; Dinguo, Beijing, China). Genotyping for HLA-B gene
According to the methods previously described (Zhou et al., 2011), all subjects were typed for HLA-B locus using polymerase chain reaction sequence-based typing (PCR-SBT) method. At first, polymerase chain reaction amplifications were accomplished on GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA), and then, amplified DNA fragments were purified and sequenced using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems) in an ABI 3730XL DNA Sequencer (Applied Biosystems) according to the manufacturer’s instructions. Ambiguous types were resolved to four digits according to the updated database (IMGT release 2.21.0; European Bioinformatics Institute, London, UK, http://www.ebi.ac.uk/imgt/hla/stats.html). Statistical analysis
At first, Hardy–Weinberg equilibrium (HWE) for HLA-B locus was assessed using the Arlequin software package version 3.1 (Laurent Excoffier, CMPG, Zoological Institute, University of Bern, Switzerland), and P < 0.05 was regarded as statistically significant. Then, genetic parameters including homozygotes (Ho), heterozygosity (He), power of discrimination (PD), polymorphism information content (PIC) and probability of paternity exclusion (PPE) were estimated using the PowerStat version 1.2 spreadsheet (Promega Corp, Madison, WI, USA). Allele frequencies of HLAB locus were calculated by SPSS13.0 software (SPSS Inc., Chicago, IL, USA). Genotypic associations were performed using SPSS13.0 (SPSS Inc.). The frequencies of HLA alleles were compared between controls and patients using the chi-square test. Fisher’s exact test was applied for expected numbers less than 5. Relative risk was calculated as an odds ratio (OR) with 95% confidence interval (CI) using Cornfield’s approximation. P < 0.05 was accepted as statistically significant.
Results Hardy–Weinberg Equilibrium
The alleles in HLA-B locus were comply with Hardy– Weinberg equilibrium in both postmenopausal osteoporosis (P = 0.82892485) and control groups (P = 0.3702555). Genetic parameters
The genetic parameters of HLA-B locus in postmenopausal osteoporosis patients and controls were summarized in Table 1. Ho, He, PD, PIC and PPE are genetic
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Table 1. Genetic parameters of HLA-B locus in postmenopausal osteoporosis patients and controls Locus
Group
Ho
He
PD
PIC
PPE
HLA-B
Patients Controls
4.3% 1.4%
95.7% 98.6%
0.984 0.983
0.95 0.96
0.913 0.972
Ho, homozygotes; He, heterozygotes; PD, power of discrimination; PIC, polymorphism information content; PPE, probability of paternity exclusion.
parameters for evaluating the polymorphisms of gene. A gene locus is considered highly polymorphic when its PIC is higher than 0.5, or PD value higher than 0.8 or PPE value higher than 0.5, respectively. As shown in Table 1, all alleles in HLA-B locus were highly polymorphic in both patients and control groups. HLA-B allelic frequencies
Totally, we detected 40 high-resolution HLA-B alleles in 70 postmenopausal osteoporosis patients. The allele frequencies of HLA-B locus were shown in Table 2. At the HLA-B loci, the six most common alleles with a frequency >5% were HLA-B*1302 (8.6%), B*5101 (8.6%), B*4601 (7.9%), B*1301 (6.4%), B*3501 (5%) and B*5801 (5%), respectively. On the other hand, we found 40 high-resolution HLA-B alleles in 73 control subjects (Table 2). At the HLA-B loci, the five most common alleles with a frequency larger than 5% were HLA-B*1302 (6.8%), B*4001 (6.8%), B*1501 (6.2%), B*4601 (5.5%) and B*5101 (5.5%), respectively. Association of HLA-B allele with postmenopausal osteoporosis
The distribution of high-resolution HLA-B alleles between the postmenopausal osteoporosis patients and controls was compared. As shown in Table 2, we observed that the frequency of HLA-B* 3501 allele was significantly higher in patients than in the control group (P = 0.033) and the relative risk was 7.632 (95% CI: 0.927–62.850). As most previous association studies of HLA-B and male infertility were based on low resolution (serotypes) of HLA. To be compatible with previous studies, we grouped our sequence-based high-resolution HLA genotype to their associated serotype according to the classification of Holdsworth et al. (2009). As B7, B27 was ever reported to be associated with bone diseases in previous population, and we considered B7, B27, B35 as candidate alleles in this study (Table 3). The results showed no significant increase of B7 in patients group, with an odds ratio (OR) 1.590 (95%CI: 0.439–5.757; P = 0.534). Moreover, B27 allele showed no significant difference in patients and that in the controls, with an odds ratio 0.777 (95%CI: 0.171–3.537; P = 1.000). Nevertheless, the
frequency of B35 allele was significant higher in patients than in the control group (P = 0.039), and the relative risk was 3.328 (95% CI: 1.047–10.580).
Discussion and conclusions Osteoporosis is a common disorder characterized by low bone mass and micro-architectural deterioration of bone tissue, resulting in an increase in bone fragility and in susceptibility to fractures (Dodd & Rowe, 2013). It has been proved that osteoporosis showed the distinct age and gender characteristics, and it was more prevalent in postmenopausal women (Pietschmann et al., 2009; Gammage et al., 2012). Therefore, the aim of the present study was designed to found the genetic marker in the population of postmenopausal osteoporosis. Bone homeostasis is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. It is also regulated by immune system, and its imbalance often results in osteoporosis (Jianbo et al., 2013; Tanaka, 2013). Recently, substantial evidences also supported that immunological factors play an important role in the pathogenesis of osteoporosis. For instance, Moln ar reported that increased IL-17A levels are involved in postmenopausal osteoporosis, and it plays a important role in the bone-resorpting processes (Moln ar et al., 2014). The study of Zhao demonstrated that T helper (Th) 17 cells are critical modulator in the pathogenesis of oestrogen-deficient osteoporosis, and therapeutic strategies targeting IL-17 networks may be clinically useful in treatment for postmenopausal osteoporosis (Zhao, 2013). The study of Kim showed that leucocyte common antigen-related (LAR) tyrosine phosphatase positively regulates osteoblast differentiation by modulating extracellular signal-regulated kinase (ERK) activation. LAR phosphatase could be used as a novel regulatory target protein in many bone-associated diseases, including osteoporosis (Kim et al., 2010). The HLA is one of the most important immunoregulatory genes. Therefore, investigating HLA polymorphism in postmenopausal osteoporosis is an open field of research with great potentials. The present study is the first to explore the association of HLA-B alleles with postmenopausal osteoporosis in Chinese Han population using the PCR-SBT method. In this study, we found the frequency of HLA-B* 3501 allele was significantly higher in postmenopausal osteoporosis patients than in the control group, and the relative risk was 7.632 (95% CI: 0.927–62.850), indicating HLA-B* 3501 might be a major risk gene of postmenopausal osteoporosis patients in Chinese Han population. Human leucocyte antigen genes in humans include three groups: class I, class II and class III. The HLA-B gene was one of the most often studied genes in HLA class I genes. One of the particular strengths in our present study is that PCR-SBT method was used to
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Table 2. High-resolution genotyping distributions of HLA-B alleles in postmenopausal osteoporosis patients and controls
Control (n = 146 alleles)
Patients (n = 140 alleles)
HLA-B
n
AF (%)
n
AF (%)
P
OR (95% CI)
B*0702 B*0705 B*0801 B*1301 B*1302 B*1402 B*1501 B*1502 B*1503 B*1505 B*1511 B*1512 B*1518 B*1525 B*1527 B*1529 B*1532 B*1558 B*1801 B*2704 B*2705 B*2706 B*3501 B*3502 B*3503 B*3701 B*3801 B*3802 B*3901 B*4001 B*4002 B*4003 B*4006 B*4402 B*4403 B*4601 B*4701 B*4801 B*4901 B*5001 B*5101 B*5102 B*5201 B*5401 B*5502 B*5601 B*5701 B*5801
3 1 1 6 10 1 9 1 1 0 5 1 1 0 3 0 2 1 3 1 2 1 1 0 3 4 5 3 0 10 5 0 6 1 3 8 0 2 2 1 8 2 5 7 6 0 5 6
2.1 0.7 0.7 4.1 6.8 0.7 6.2 0.7 0.7 0 3.4 0.7 0.7 0 2.1 0 1.4 0.7 2.1 0.7 1.4 0.7 0.7 0 2.1 2.7 3.4 2.1 0 6.8 3.4 0 4.1 0.7 2.1 5.5 0 1.4 1.4 0.7 5.5 1.4 3.4 4.8 4.1 0 3.4 4.1
6 0 0 9 12 0 5 2 0 1 4 0 2 1 1 1 0 0 3 1 1 1 7 1 4 1 3 1 1 6 5 1 3 2 2 11 1 4 0 2 12 1 3 6 2 2 3 7
4.3 0 0 6.4 8.6 0 3.6 1.4 0 0.7 2.9 0 1.4 0.7 0.7 0.7 0 0 2.1 0.7 0.7 0.7 5 0.7 2.9 0.7 2.1 0.7 0.7 4.3 3.6 0.7 2.1 1.4 1.4 7.9 0.7 2.9 0 1.4 8.6 0.7 2.1 4.3 1.4 1.4 2.1 5
0.327 – – 0.379 0.585 – 0.310 0.616 – – 0.783 – 0.616 – 0.623 – – – 1.000 1.000 1.000 1.000 0.033* – 0.718 0.371 0.723 0.623 – 0.443 1.000 – 0.502 0.616 1.000 0.481 – 0.438 – 0.616 0.358 1.000 0.723 0.836 0.283 – 0.723 0.718
2.134 – – 1.603 1.275 – 0.564 2.101 – – 0.829 – 2.101 – 0.343 – – – 1.044 1.043 0.518 1.043 7.632 – 1.402 0.255 0.618 0.343 – 0.609 1.044 – 0.511 2.101 0.691 1.471 – 2.118 – 2.101 1.617 0.518 0.618 0.889 0.338 – 0.618 1.228
(0.523–8.705)
(0.555–4.628) (0.532–3.053) (0.184–1.726) (0.188–23.438)
(0.218–3.154) (0.188–23.438) (0.035–3.337)
(0.207–5.261) (0.065–16.841) (0.046–5.777) (0.065–16.841) (0.927–62.850) (0.308–6.380) (0.028–2.314) (0.145–2.634) (0.035–3.337) (0.215–1.732) (0.296–3.689) (0.125–2.084) (0.188–23.438) (0.114–4.198) (0.574–3.772) (0.382–11.749) (0.188–23.438) (0.640–4.084) (0.046–5.777) (0.145–2.634) (0.291–2.714) (0.067–1.704) (0.145–2.634) (0.402–3.749)
AF, allele frequency; OR, odds ratio; CI, confidence interval; *P < 0.05.
Table 3. Serotype (low-resolution typing) distributions of HLA-B alleles in postmenopausal osteoporosis patients and controls Serotype
DNA-based genotype
Control (n = 146 alleles, %)
Patients (n = 140 alleles, %)
P
OR (95%CI)
B7 B27 B35
B*0702, B*0705 B*2704, B*2705, B*2706 B*3501, B*3503, B*3508
4 (2.7) 4 (2.7) 4 (2.7)
6 (4.3) 3 (2.1) 12 (8.6)
0.534 1.000 0.039*
1.590 (0.439–5.757) 0.777 (0.171–3.537) 3.328 (1.047–10.580)
OR, odds ratio; CI, confidence interval; *P < 0.05.
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confirm the association between postmenopausal osteoporosis and HLA alleles. PCR-SBT method is considered the gold standard for high-resolution definition of HLA (Woo et al., 2012). Previous HLA association studies in osteoporosis have been carried out with low-resolution HLA serologic typing by PCRSSP. PCR-SBT method has much higher accuracy and reliability than simply serological typing methods and also facilitates the standardization of HLA typing processes. Ethnic background and geographic variations are very important factors affecting the study results. China has 56 officially identified ethnic groups, and Han nationality constitutes about 92% of population of Chinese. Shaanxi Province, located in the northwest of China, is a major Han population residual region. It is not only the birthplace of the Chinese nation, but also the birthplace of human civilization in Asia and the cultural centre of prehistoric civilization. In present study, we selected subjects from the same ethnic background and geographic background to avoid the possible occurrence of sampling errors. In addition, women with conditions potentially causing secondary osteoporosis or women over the age of 65 years were ruled out from the present study to avoid the impact of senile osteoporosis. Moreover, all samples in patients and control groups were in Hardy–Weinberg equilibrium at HLAB locus. Ho, He, PD, PIC and PPE are genetic parameters for evaluating the polymorphisms of gene. A gene locus is considered highly polymorphic when its PIC is higher than 0.5, or PD value higher than 0.8 or PPE value higher than 0.5, respectively. In our present study, the PIC values are 0.950 and 0.950, and PD values are 0.984 and 0.983, and PPE values are 0.913 and 0.972, respectively, in both patients and control groups. These values were higher than the cut-off values mentioned above. Therefore, the HLA-B data obtained from our sample population are highly polymorphism, which was valuable for further genetic science research. The association between HLA-B antigens and postmenopausal osteoporosis has also been examined in several populations. Douroudis et al. (2007) found that HLA-B7 was associated with a lower BMD in the postmenopausal osteoporosis patients of Greek origin. Tsuji et al. (1998) found that the haplotype HLAA*24-B*07-DRB*01 is a new genetic marker implicated with low PBM in healthy young Japanese women. However, we did not detect significant differences of HLA-B7 between patients and control groups in our Chinese population. Nevertheless, interestingly, we found the frequency of HLA-B* 3501 allele was significantly higher in postmenopausal osteoporosis patients than in the control group, and the relative risk was 7.632 (95% CI: 0.927–62.850), indicating HLAB* 3501 might be a major risk gene of postmenopausal osteoporosis patients in Chinese Han population, which was not reported in other different
population. We speculated that these conflicting results might be attributed to differences in HLA typing methodology, sample size, ethnic background and geographic variations. Due to the distributions of HLA alleles, haplotypes show high variation in different ethnic groups or even the same ethnic group living in different geographic areas (Zhou et al., 2012). In conclusion, our present study explored the association of the HLA-B alleles with postmenopausal osteoporosis in Chinese Han population using PCR-SBT high-resolution HLA genotyping method, which provides a more accurate and credible resolution than the previous methods. Our results suggest that HLA-B* 3501 was likely an important risk factor for postmenopausal osteoporosis, and our results would add some valuable evidences for osteoporosis study.
Acknowledgements This research was financially supported by National Natural Science Fundings of China (No. 81273018; 30700654), Science Funding of Health Department, Shaanxi Province (2012D58) and the Fundamental Research Funds for the Central University (XJJ 2011024).
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