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A genome-wide association study identifies susceptibility loci for ossification of the posterior longitudinal ligament of the spine

Nature Genetics volume 46, pages 1012–1016 (2014)Cite this article

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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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Figure 1: Manhattan plot showing the −log10 P value for each SNP in the GWAS.
Figure 2: Regional association plots at six susceptibility loci for 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).

Author information

Author notes

  1. Takashi Tsuji & Kazuhiro Chiba

    Present address: Present address: Department of Orthopaedic Surgery, Kitasato University Kitasato Institute Hospital, Tokyo, Japan.,

  2. Masahiro Nakajima and Atsushi Takahashi: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory for Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, Japan

    Masahiro Nakajima, Tatsuki Karasugi & Shiro Ikegawa

  2. Laboratory for Statistical Analysis, Center for Integrative Medical Sciences, RIKEN, Yokohama, Japan

    Atsushi Takahashi

  3. Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan

    Takashi Tsuji, Kazuhiro Chiba, Morio Matsumoto & Yoshiaki Toyama

  4. Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

    Tatsuki Karasugi

  5. Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan

    Hisatoshi Baba, Kenzo Uchida & Hideaki Nakajima

  6. Department of Orthopaedic Surgery, Tokyo Medical and Dental University, Tokyo, Japan

    Shigenori Kawabata & Atsushi Okawa

  7. Department of Orthopedics, Kudanzaka Hospital, Tokyo, Japan

    Shigeo Shindo

  8. Department of Orthopaedic Surgery, National Okayama Medical Center, Okayama, Japan

    Kazuhiro Takeuchi

  9. Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

    Yuki Taniguchi

  10. Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

    Shingo Maeda

  11. Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

    Masafumi Kashii

  12. Department of Orthopedics, Jichi Medical University, Shimotsuke, Japan

    Atsushi Seichi

  13. Department of Orthopaedic Surgery, Toyama University, Toyama, Japan

    Yoshiharu Kawaguchi

  14. Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Shunsuke Fujibayashi

  15. Department of Orthopedic Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan

    Masahiko Takahata

  16. Department of Orthopaedic Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

    Toshihiro Tanaka

  17. Department of Orthopaedic Surgery, Niigata University Medical and Dental General Hospital, Niigata, Japan

    Kei Watanabe

  18. Department of Orthopaedic Surgery, Kochi Medical School, Kochi, Japan

    Kazunobu Kida

  19. Department of Orthopedic Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan

    Tsukasa Kanchiku

  20. Department of Orthopedics, Nagoya University Graduate School of Medicine, Nagoya, Japan

    Zenya Ito

  21. Department of Orthopaedic Surgery, Shiga University of Medical Science, Otsu, Japan

    Kanji Mori

  22. Department of Orthopaedic Surgery, National Hospital Organization Osaka Minami Medical Center, Osaka, Japan

    Takashi Kaito

  23. Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Sho Kobayashi

  24. Department of Orthopaedic Surgery, Kurume University School of Medicine, Kurume, Japan

    Kei Yamada

  25. Department of Orthopaedic Surgery, Kyorin University School of Medicine, Tokyo, Japan

    Masahito Takahashi

  26. Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

    Ken-Ichi Furukawa

  27. Laboratory for Genotyping Development, Center for Integrative Medical Sciences, RIKEN, Yokohama, Japan

    Michiaki Kubo

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  1. Masahiro Nakajima
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Consortia

Genetic Study Group of Investigation Committee on Ossification of the Spinal Ligaments

Contributions

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.

Corresponding author

Correspondence to Shiro Ikegawa.

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Competing interests

The authors declare no competing financial interests.

Additional information

A full list of members and affiliations appears in the Supplementary Note.

Integrated supplementary information

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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Supplementary Figures 1–5, Supplementary Tables 1–7 and Supplementary Note. (PDF 3107 kb)

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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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