Abstract
Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common spinal disorder among the elderly that causes myelopathy and radiculopathy. To identify genetic factors for OPLL, we performed a genome-wide association study (GWAS) in ∼8,000 individuals followed by a replication study using an additional ∼7,000 individuals. We identified six susceptibility loci for OPLL: 20p12.3 (rs2423294: P = 1.10 × 10−13), 8q23.1 (rs374810: P = 1.88 × 10−13), 12p11.22 (rs1979679: P = 4.34 × 10−12), 12p12.2 (rs11045000: P = 2.95 × 10−11), 8q23.3 (rs13279799: P = 1.28 × 10−10) and 6p21.1 (rs927485: P = 9.40 × 10−9). Analyses of gene expression in and around the loci suggested that several genes are involved in OPLL etiology through membranous and/or endochondral ossification processes. Our results bring new insight to the etiology of OPLL.
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Acknowledgements
We thank the individuals with OPLL and their families who participated in this study and the Japan OPLL Network (Zen-Sekichu-Ren), including Ishikawa prefecture OPLL Tomo-no-kai. We also thank Y. Takahashi and T. Oguma for technical assistance. The work reported in this article was supported by grants from the Ministry of Health, Labour and Welfare of Japan: Committee for Study of Ossification of Spinal Ligament (H23-Nanchi-Ippan-032) and Committee for Research and Development of Therapies for Ossification of Posterior Longitudinal Ligament (H26-Itaku(Nan)-Ippan-053).
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S.I. designed the project and provided overall project management. M.N. and S.I. drafted the manuscript. M.N. and M. Kubo performed the genotyping for the GWAS. A.T. analyzed the GWAS data. T. Tsuji, T. Karasugi, H.B., K.U., S. Kawabata, A.O., S.S., K.T., Y. Taniguchi, S.M., M. Kashii, A.S., H.N., Y.K., S.F., M. Takahata, T. Tanaka, K.W., K.K., T. Kanchiku, Z.I., K.M., T. Kaito, S. Kobayashi, K.Y., M. Takahashi, K.C., M.M., K.-I.F. and Y. Toyama managed the DNA samples and clinical data.
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Supplementary Figure 1 Evaluation of the GWAS for OPLL.
(a,b) Principal-component analysis of the GWAS samples with (a) and without (b) four HapMap populations. YRI, Yoruba in Ibadan, Nigeria; CEU, Utah residents with ancestry from northern and western Europe; CHB, Han Chinese in Beijing, China; JPT, Japanese in Tokyo, Japan. (c) A quantile-quantile plot with Cochran-Armitage trend P values from the GWAS. Horizontal and vertical lines represent expected P values under a null distribution and observed P values, respectively. The genetic inflation factor λ is 1.01, indicating that population stratification is unlikely.
Supplementary Figure 2 Conditional logistic regression analysis of six susceptibility loci for OPLL.
Each plot shows the –log10 (P value) against the chromosomal position of each SNP with (red) and without (blue) adjustments for each primary associated SNP indicated in the panel. (a) 20p12.3. (b) 8q23.1. (c) 12p11.22. (d) 12p12.2. (e) 8q23.3. (f) 6p21.1. Each region consisted of a single locus.
Supplementary Figure 3 Regional association plots for the previously reported linkage regions.
Each plot shows the –log10 (P value) against the chromosomal position of each genotyped SNP in the previously reported linkage regions. Each top SNP at five loci (labeled by rs number) is shown with its P value. (a) 1p21. (b) 2p22-2p24. (c) 7q22. (d) 16q24. (e) 20p12. The 20p12 linkage region contained a significantly associated (P < 5 × 10−8) SNP in the GWAS.
Supplementary Figure 4 Expression of genes within the LD blocks of associated SNPs in osteoblasts and fibroblasts.
Gene expression was examined by RT-PCR on mRNA obtained from bone and skin samples from three individuals each. EIF3E, EMC2 and CCDC91 were abundantly expressed in both osteoblasts and fibroblasts. NC, negative control; PC, positive control.
Supplementary Figure 5 Expression of genes within the LD blocks of the associated SNPs during chondrogenic differentiation of ATDC5 cells.
ATDC5 cells were cultured in the presence (filled box) or absence (open box) of chondrogenic differentiation medium (insulin-transferrin-sodium selenite) for the indicated time periods. Expression of Hao1 (a), Rspo2 (b), Eif3e (c), Emc2 (d), Ccdc91 (e), Acan (f), Col2a1 (g) and Sox9 (h) was examined by quantitative PCR. Expression of Hao1, Rspo2 and Ccdc91 was decreased by the induction of chondrogenesis. Data represent mean ± s.d. (n = 3). *P < 0.05 at the same time point.
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Nakajima, M., Takahashi, A., Tsuji, T. et al. A genome-wide association study identifies susceptibility loci for ossification of the posterior longitudinal ligament of the spine. Nat Genet 46, 1012–1016 (2014). https://doi.org/10.1038/ng.3045
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DOI: https://doi.org/10.1038/ng.3045
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