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Mar 7, 2010 Dear Editors On behalf of my co-authors, I am submitting the manuscript entitled “Linkage of three polymorphisms on chromosome 20p12 to ossification of the posterior longitudinal ligament of spine and its severity in Han Chinese patients” for possible publication in your journal. We certify that we have participated sufficiently in the work to take public responsibility for the appropriateness of the experimental design and method, and the collection, analysis, and interpretation of the data. We have reviewed the final version of the manuscript and approve it for publication. To the best of our knowledge and belief, this manuscript has not been published in whole or in part nor is it being considered for publication elsewhere. Best Regards Yours Sincerely, Liang Yan Linkage of three polymorphisms on chromosome 20p12 to ossification of the posterior longitudinal ligament of spine and its severity in Han Chinese patients YAN Liang1, ZHAO Wei-guang1, LI Jin-jun2, YANG Hui3, WANG Hao1and LIN Xin1 1Department of Orthopaedics, Beijing Tiantan Hospital, Capital Medical University (CMU), Beijing 100050, China 2Department of Orthopaedics, Beijing Friendship Hospital, Capital Medical University (CMU), Beijing, 100050, China 3Beijing Institute for Neuroscience, Capital Medical University (CMU), Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing 100069, China First Author: YAN Liang Beijing Tiantan Hospital Capital Medical University (CMU) Beijing 100050, China E-mail: [email protected] Corresponding Author: LIN Xin, MD, PhD Beijing Tiantan Hospital Capital Medical University (CMU) Beijing 100050, China Tel: +86 10 67098437 E-mail address: [email protected] Co-Corresponding Author: WANG Hao, MD, PhD Beijing Tiantan Hospital Capital Medical University (CMU) Beijing 100050, China Tel: +86 10 67098437 E-mail address: [email protected] Acknowledgments The authors thank the DNA donors for making this study possible. This research was supported by grants from the National Nature Science Foundation of China (No. 30872599), and the Beijing Nature Science Foundation of China (No. 7092028). Keywords ossification of the posterior longitudinal ligament; single nucleotide polymorphisms; susceptibility; polymerase chain reaction Background Ossification of the posterior longitudinal ligament (OPLL) is characterized by replacement of ligamentous tissue with new ectopic bone formation, and has a strong genetic background. Because of the abnormal bone metabolic features and the strong genetic component, osteoporosis is a related disorder with OPLL. Three polymorphisms on chromosome 20p12 were identified associated with the risk of osteoporosis and osteoporotic fracture. The rs996544 (C/T) “TT” and rs965291 (G/A) “AA” genotypes conferred higher risks for vertebral and hip fractures. The osteoporosis haplotype is defined by two polymorphisms, rs1116867 (A) and D35548 (T). However, it remains unknown whether these three polymorphisms predispose to an increased frequency and severity of OPLL in Han Chinese patients. Methods A total of 420 OPLL patients and 506 age- and sex-matched controls were studied. Three single nucleotide polymorphisms (SNPs), rs996544 (C/T), rs965291 (G/A) and rs1116867 (A/G), were analyzed by direct sequencing. Associations between these SNPs with the occurrence and extent of OPLL were statistically evaluated. Results There was no significant association between the rs996544 (C/T) polymorphism and the prevalence of OPLL. The rs1116867 (A/G) polymorphism “AG” genotype was associated with the occurrence of OPLL. The rs1116867 (A/G) polymorphism “G” allele was associated with the occurrence of OPLL, but not with the extent of OPLL. The rs965291 (G/A) polymorphism in female patients was statistically different between cases and controls (P<0.05). The rs965291 (G/A) polymorphism “A” allele was associated with the occurrence of OPLL in female patients. For the rs965291 (G/A) polymorphism, patients with the “A” allele (genotype, “AG” or “AA”) showed a significantly greater number of ossified cervical vertebrae than those without the “A” allele (genotype, “GG” P<0.05), particularly in female patients. Conclusions The rs1116867 (A/G) and rs965291 (G/A) polymorphisms on chromosome 20p12 are associated with the occurrence and the extent of OPLL, at least in Han Chinese subjects. Our data should advance our understanding of the molecular etiology of OPLL and may guide approaches to prevent the onset of OPLL. Ossification of the posterior longitudinal ligament (OPLL) is pathological ectopic ossification of this ligament at the cervical and thoracic spine, causing myeloradiculopathy as a result of chronic pressure on the spinal cord and nerve roots [1, 2]. OPLL of the spine was first reported in Japan and has even been called “a Japanese disease,” because many more cases have been reported in Japanese and other Asian populations than in non-Asiatic populations. The prevalence of OPLL in Japan is 1.9–4.3% of the general population aged >30 years old [3, 4]. Among Chinese individuals, the prevalence of OPLL ranges from 0.44 to 8.92%, with a mean prevalence of 3.08% [5]. Many clinical studies conducted in Japan have suggested that OPLL is a multifactorial disease in which complex genetic and environmental factors interact [1, 6–9]. Studies have shown that OPLL is prevalent with a high concordance in twins and families, and is associated with human leukocyte antigen haplotypes [10, 11]. Because genetic factors appear to play a crucial role in OPLL, molecular genetic studies are important to better understand the molecular etiologies of OPLL and will lead to the development of new therapeutic approaches. On the other hand, osteoporosis is a bone metabolic disorder that has been extensively investigated [12-15]. Osteoporosis is a common disease that is characterized by a reduction in bone mass, microarchitectural deterioration of bone tissue and an increased risk of fracture [16-18]. Recently, a compound osteoporosis phenotype was reported to be linked to chromosome 20p12. Furthermore, three single nucleotide polymorphisms (SNPs) of rs996544 (C/T), rs965291 (G/A) and rs1116867 (A/G) were found to be associated with osteoporosis and increase the risk of osteoporosis and osteoporotic fracture [19-21]. Because abnormal bone metabolic features and strong genetic components of OPLL and osteoporosis have been reported, but with limited information about the extent of the overlap, OPLL could be widespread among individuals with osteoporosis. The purpose of this study was to investigate the associations between three SNPs [rs996544 (C/T), rs965291 (G/A), rs1116867 (A/G)] with susceptibility to OPLL of the spine and its severity in Han Chinese patients. To our knowledge, we are the first to report an association between these three SNPs and OPLL. METHODS Disease criteria and subjects This study was approved by the ethical committee at Beijing Tiantan Hospital Capital Medical University. Informed consent was obtained from all participants in this study. Overall, 420 patients with OPLL and 506 age- and sex-matched healthy control subjects without OPLL participated in this study. All the participants are from third grade class A hospitals in Beijing and live in the Beijing region. The characteristics of the OPLL patients and controls are shown in Table 1. The diagnosis of OPLL was based on radiologic findings including radiographs, computed tomography (CT) and magnetic resonance imaging (MRI) of the cervical spine according to the criteria reported by Tsuyama [22]. The severity of OPLL was determined based on the number of ossified cervical vertebrae on lateral radiograph films and CT images. Patients were also stratified according to the extent of ossification. The patients with ankylosing spondylitis and metabolic diseases associated rickets/osteomalacia, with OPLL, osteoporosis, such diffuse as hypophosphate idiopathic skeletal hyperostosis (DISH) and hyperparathyroidism were excluded according to radiographic and biochemical examinations. Women who had taken drugs such as estrogen, progesterone, glucocorticoids, bisphosphonates, alfacalcidol and calcitriol were also excluded. Genomic DNA analysis SNPs for genotyping were obtained from the two public databases: NCBI dbSNP (http://www.ncbi.nlm.nib.gov/SNP/) and Ensembl Genome (http://www.ensembl.org/index.html). Venous blood (5 ml) was collected in tubes containing EDTA (50 mmol/l of disodium salt). The blood samples were stored at –20°C until use for genomic DNA extraction and SNP genotyping. Genomic DNA was isolated using Wizard Genomic DNA Purification Kits (Promega Corporation, USA) and polymerase chain reaction (PCR) was performed with a standard protocol using sense and antisense primers for each factor (Table 2). Reactions were performed in a total volume of 50 μl containing 0.5 μg of genomic DNA; 1 μl of each primer (20 μM); 8 μl of dCTP, dTTP, dGTP and dATP mixture (each 2.5 μM); 0.5 μl TAKARA LA Taq DNA polymerase (TakaRa Biotechnology Dalian Co., Ltd); 5 μl MgCl2 (25 mM); 5 μl 10× LA PCR Buffer Ⅱ (Mg2+ Free); and double distilled H2O (ddH2O) to 50 μl. The PCR products (including the three SNPs) were analyzed by direct sequencing using BigDye Terminator cycle sequencing on an ABI 3730XL POP7 DNA sequencing analysis 5.2 (Applied Biosystems). Statistical analysis The two groups were compared using Student unpaired t test. Hardy-Weinberg equilibrium and the genotypic and allelic distribution were evaluated using χ2 tests. The nonparametric Mann-Whitney U test was used to compare the number of ossified cervical vertebrae between the two groups. Analysis of variance was used to compare group means. A P value less than 0.05 was considered statistically significant. RESULTS There were no significant differences in age, height, body weight, or personal history between the case and control subjects. Within the OPLL cases, there were significantly more men than women compared with the control group. The distributions of genotype and allele types of the three SNPs among OPLL cases and controls are shown in Table 3. The genotype distributions in the case and control subjects were in Hardy-Weinberg equilibrium, which suggests that the study population was genetically homogenous, and that selection bias was avoided. The number of subjects in the total study population with the rs996544 (C/T) “CC,” “CT” and “TT” genotypes was 779, 147 and 0, respectively, with frequencies of 84, 16 and 0%, respectively. The number of subjects with the rs965291 (G/A) “AA,” “AG” and “GG” genotypes was 67, 320 and 539, with frequencies of 7, 35 and 58%, respectively. The number of subjects with the rs1116867 (A/G) “AA,” “AG,” “GG” genotypes was 228, 464 and 234, with frequencies of 24, 50 and 26%, respectively. Comparing the distributions of the genotype of these three SNPs, there was a statistical difference among the rs1116867 (A/G) “AA,” “AG” and “GG” genotypes (P<0.05) (Table 3). The “AG” genotype was associated with the occurrence of OPLL (Figure 1). However, no association was found between male and female cases in terms of the rs1116867 (A/G) genotypes (data was not shown). Furthermore, there was no statistical difference among the rs965291 (G/A) “AA,” “AG,” and “GG” genotypes (P=0.898). In addition, the rs965291 (G/A) in the male (P=0.779) and female (P=0.742) cases was not significantly associated with controls. The rs965291 (G/A) polymorphism in females was statistically different between cases and controls (P<0.05) (Figure 2), whereas the difference in males was not significant (P=0.099) (Table 4). These results suggest that the genetic background of this polymorphism affects the initiation of OPLL more strongly in women than in men. There was no statistical difference among the rs996544 (C/T) “CC,” “CT” and “TT” genotypes (P=0.548). Comparing the allelic distributions of the three SNPs, there was a significant association between the rs1116867 (A/G) polymorphism and the occurrence of OPLL in the cervical spine. In addition, the “G” allele was significantly more frequent in OPLL cases than in control subjects (P<0.05). Among females with OPLL, the rs965291 (G/A) “A” allele was significantly associated with the occurrence of OPLL (P<0.05) (Table 5). To study the contribution of these polymorphisms to the severity or promotion of OPLL, we investigated the association between the three SNPs with the number of ossified cervical vertebrae in cases with OPLL. Regarding the rs996544 (C/T) polymorphism, patients with the “T” allele (genotype, “CT” or “TT”) showed no increase in the number of ossified cervical vertebrae than those without the “T” allele (genotype, “CC” P=0.362). Similarly, for the rs1116867 (A/G) polymorphism, patients with the “G” allele (genotype, “AG” or “GG”) showed no increase in the number of ossified cervical vertebrae than those without the “G” allele (genotype, “AA” P=0.423). However, for the rs965291 (G/A) polymorphism, patients with the “A” allele (genotype, “AG” or “AA”) had a significantly greater number of ossified cervical vertebrae compared with patients without the “A” allele (genotype, “GG” P<0.05). Female patients with the “A” allele (genotype, “AG” or “AA”) had a greater number of ossified vertebrae than those without the “A” allele (genotype, “GG” P<0.05). This finding was not observed among males (Figure 3). The present results indicate that the “A” allele promotes ectopic ossification in the cervical spine among patients with OPLL, particularly among females. DISCUSSION OPLL is characterized by replacement of ligamentous tissue by ectopic new bone formation in the spine and often causes narrowing of the spinal canal. OPLL is a common disorder among elderly populations and is the leading cause of spinal myelopathy in Asian populations. Although various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging and hormonal disturbances have been suggested as causes of OPLL, the etiology of OPLL is not fully understood. However, the possible involvement of genetic factors has been proposed based on the high incidence of OPLL in families and among twins [22, 23]. According to a study of probands and their relatives within the second degree of consanguinity, OPLL was present in 23% of relatives, exhibiting autosomal dominant inheritance. The rarity of the disease among white populations of Europe and the United States, despite the relatively high prevalence among Japanese populations, also suggests a genetic predisposition. Therefore, the development of OPLL may be affected by an interaction between genetic and environmental factors. Previous studies have shown that restriction fragment length polymorphisms of the estrogen receptor and interleukin-1 (IL-1) genes, and SNPs of pyrophosphatase the transforming (NPPS), leptin growth receptor, factor-β, COL11A2 nucleotide and bone morphogenetic protein-2 (BMP-2) genes are associated with the development of OPLL [24-28]. Our research group has also previously shown that the Ser37Ala (T/G) and exon 3 (–726) T/C polymorphisms are associated with the occurrence of OPLL, but not with extensive OPLL in the cervical spine. Meanwhile, the Ser87Ser (A/G) polymorphism “G” allele was reported to promote the extent of OPLL [29, 30]. In the present study, one of the three SNPs on chromosome 20p12, the rs965291 (G/A) polymorphism (20:6491074), is located upstream of the BMP-2 gene, and span 25 kb. The population genotypes and allele frequencies of rs965291 (G/A) supplied by the NCBI dbSNP database showed that these were the only “AG” or “GG” genotypes in Han Chinese people. However, the current study also identified 43 subjects with the “AA” genotype. The rs996544 (C/T) polymorphism (20:6796219) is located downstream of the BMP-2 gene and spans 20 kb. Similarly, the rs1116867 (A/G) polymorphism (20:6778164) is located downstream of the BMP-2 gene and spans 18 kb. Previous studies have shown that these three polymorphisms on chromosome 20p12 were associated with the risk of osteoporosis and osteoporotic fracture [19, 20]. The rs996544 (C/T) “TT” genotype and rs965291 (G/A) “AA” genotype were associated with higher risk of vertebral and hip fractures. The osteoporosis haplotype is defined by two polymorphisms, rs1116867 (A) and D35548 (T). BMP-2 was suggested as a candidate gene involved in the predisposition to OPLL; these three SNPs are located near the BMP-2 gene and may affect the initiation or severity of OPLL by regulating the expression of BMP-2 gene. Our study showed that the rs1116867 (A/G) polymorphism is associated with the occurrence of OPLL, but not with more extensive OPLL in the cervical spine. Furthermore, the “AG” genotype is associated with the occurrence of OPLL. The results indicate that the “G” allele is a risk factor for genetic susceptibility to OPLL, but is not associated with the extent of heterotopic ossification in the cervical spine. The rs965291 (G/A) polymorphism in female patients is associated with the occurrence of OPLL and that the genetic background of this polymorphism may affect the initiation of OPLL more strongly in women than in men. The present results show that the rs965291 (G/A) polymorphism is associated with the severity or promotion of OPLL, but not with its rate of occurrence in the cervical spine, while the rs965291 (G/A) “A” allele promotes the extent of OPLL. Patients not carrying the rs965291 (G/A) “G” allele had a greater number of ossified vertebrae than those carrying the “G” allele. Furthermore, the analysis adjusted for sex showed that the female patients with the rs965291 (G/A) “A” allele (genotype, “AG” or “AA”) had a greater number of ossified vertebrae than those without the “A” allele (genotype, “GG”). These results indicate that the “A” allele promotes the extent of OPLL, while the “G” allele restricts ectopic ossification in the cervical spine in OPLL patients, particularly in female patients. The functional impact of the three SNPs neighboring the BMP-2 gene is uncertain. However, because the three SNPs are located upstream or downstream of the BMP-2 gene, they may yield alternative transcript or result in gene products with abnormal function, or induce overexpression of BMP-2 protein in the posterior longitudinal ligament matrix. Overexpression of the BMP-2 protein can augment the signaling activity of endogenously produced BMPs. Furthermore, the posterior longitudinal ligament matrix shows an imbalance between the expression of BMP-2 agonists and the potential existence of antagonists. Thus, the BMP-2 protein may be refractory to inhibitors such as noggin. Further augmentation of the BMP-2 signal via the SMADs and p38 mitogen activated protein kinase signaling pathways may promote ectopic ossification in the posterior longitudinal ligament of the cervical spine [31-35]. In conclusion, the present results demonstrate that the rs1116867 (A/G) “AG” genotype is associated with the occurrence of OPLL. The rs1116867 (A/G) “G” allele is associated with the occurrence of OPLL, but not with the extent of OPLL. The rs965291 (G/A) “A” allele promotes the extent of OPLL. Among female patients with OPLL, the rs965291 (G/A) “A” allele is not only associated with the occurrence of OPLL, but is also associated with the extent of OPLL, at least in Han Chinese subjects. Our data should advance our understanding of the molecular etiology of OPLL and may guide approaches to prevent the onset of OPLL. Acknowledgments The authors thank the DNA donors for making this study possible. This research was supported by grants from the National Nature Science Foundation of China (No. 30872599) and the Beijing Nature Science Foundation of China (No. 7092028). REFERENCES 1. Sakou T, Matsunaga S, Koga H. Recent progress in the study of pathogenesis of ossification of the posterior longitudinal ligament. J Orthop Sci 2000; 5: 310-315. 2. Schmidt MH, Quinones-Hinojosa A, Rosenberg WS. Cervical myelopathy associated with degenerative spine disease and ossification of the posterior longitudinal ligament. Semin Neurol 2002; 22: 143-148. 3. Matsunaga S, Sakou T. Epidemiology of ossification of the posterior longitudinal ligament. In: Yonenobu K, Sakou T, Ono K (eds) OPLL: ossification of the posterior longitudinal ligament. Springer-Verlag, Tokyo 1997; pp: 11-17. 4. 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Characteristics of OPLL cases and healthy control subjects ALL Number Age (years) Body weight (kg) Height (cm) Personal history of: Smoking Alcohol use Food preference Type of OPLL Continuous Mixed Segmental localized Male Female OPLL Controls OPLL Controls OPLL Controls 420 32-77 (55.2±9.9) 51-84 (59.7±6.4) 150-178 (163.1±6.6) 506 31-79 (54.8±7.6) 50-85 (59.1±8.0) 152-180 (164.1±9.8) 232 32-77 (56.9±10.2) 63-84 (60.1±9.4) 165-178 (168.6±6.3) 288 31-79 (55.7±5.7) 61-85 (61.3±6.3) 167-180 (169.3±3.6) 188 34-75 (52.7±9.0) 51-71 (57.7±9.6) 150-170 (158.6±3.5) 218 32-78 (51.6±7.2) 50-72 (56.9±7.8) 152-169 (159.0±4.4) 167 68 28 221 94 47 151 52 16 204 81 22 16 16 12 17 13 25 155 88 139 38 78 52 81 21 77 36 58 17 Age, body weight and height are expressed as means ± SD Table 2. Primers for each SNP SNP rs965291 rs99654 rs1116867 Sense CAGTCTATGAACATGGGATA CAGACACCGGGAAACCAT CTGATCTTTGAATTGGGTAA Antisense Product size (bp) Annealing temperature (°C) TAAGTTGGGCAAAGTGAA CAGACCGACAGCAGCAAC AGTGGAGGCAGAGTGGAA 500 505 486 54 60 55 Table 3. Genotypic and allelic distributions of the three SNPs in OPLL cases and healthy control subjects SNPs Genotype, n (%) OPLL (n=420) Control (n=506) Allele n (%) OPLL (n=420) Control (n=506) rs996544 CC 350 (83) 429 (85) P=0.548 C 770 (92) 935 (92) P=0.566 CT 70 (17) 77 (15) rs965291 TT 0 (0) 0 (0) T 70 (8) 77 (8) AA 32 (8) 35 (7) P=0.898 A 207 (25) 247 (24) P=0.907 AG 143 (34) 177 (35) rs1116867 GG 245 (58) 294 (58) G 633 (75) 765 (76) AA 80 (19) 148 (29) P=0.002 A 387 (46) 533 (53) P=0.005 AG 227 (54) 237 (47) GG 113 (27) 121 (24) G 453 (54) 479 (47) Table 4. Genotypic distributions of rs965291 (G/A) classified by sex in OPLL cases and healthy control subjects SNP Genotype OPLL cases (n=420) Male (n=232) Female (n=188) Control subjects (n=506) Male (n=288) Female (n=218) rs965291 (G/A) AA 32 19 13 35 19 16 AG 143 83 60 177 81 96 GG 245 130 115 294 188 106 Note: Differences in genotypic and allelic distribution classified by sex between OPLL cases and controls subjects were analyzed using χ2-tests Male of OPLL and control, P=0.779 Female of OPLL and control, P=0.742 Male of OPLL and male of control, P=0.099 Female of OPLL and female of control, P=0.033 Table 5. Allelic distributions of rs965291 (G/A) in female OPLL cases and female control subjects SNP Allele n (%) OPLL cases Controls rs965291 (G/A) A 86(23) 128(29) G 290(77) 308(71) P=0.036 Figure 1. Two-dimensional computed tomography (2D-CT) and sequencing results of a 62-year-old man with OPLL. A) The 2D-CT scan revealed ossification of the posterior longitudinal ligament in the cervical spine (C4–6, Continuous type). B) Direct sequencing of the PCR products showed an AG heterozygotic mutation in the rs1116867 (A/G) polymorphism. Green line: A allele; black line: G allele; blue line: C allele; red line: T allele. Number of ossified vertebrae Figure 2. Two-dimensional computed tomography (2D-CT) and sequencing results of a 59-year-old woman with OPLL. A) The 2D-CT scan revealed ossification of the posterior longitudinal ligament in the cervical spine (C2-7, Mixed type). B) Direct sequencing of the PCR products showed an AG heterozygotic mutation in the rs965291 (G/A) polymorphism. Green line: A allele; black line: G allele; blue line: C allele; red line: T allele. 10 * 8 * 6 N.S. A allele(+) A allele(-) 4 2 0 All Male rs965291(G/A) Female Figure 3. The number of ossified vertebrae of OPLL patients in subjects classified by sex and rs965291 (G/A) genotype. Data are expressed as means ± SEM. The difference in the number of ossified vertebrae between carriers (closed bar) and non-carriers (open bar) of each allele was statistically analyzed. *: P<0.05; N.S.: not significant.