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Am J Hum Genet. 2003 Oct; 73(4): 812–822.
Published online 2003 Sep 4.
PMCID: PMC1180604

Genomewide Linkage and Linkage Disequilibrium Analyses Identify COL6A1, on Chromosome 21, as the Locus for Ossification of the Posterior Longitudinal Ligament of the Spine


Ossification of the posterior longitudinal ligament (OPLL) of the spine is a subset of “bone-forming” diseases, characterized by ectopic ossification in the spinal ligaments. OPLL is a common disorder among elderly populations in eastern Asia and is the leading cause of spinal myelopathy in Japan. We performed a genomewide linkage study with 142 affected sib pairs, to identify genetic loci related to OPLL. In multipoint linkage analysis using GENEHUNTER-PLUS, evidence of linkage to OPLL was detected on chromosomes 1p, 6p, 11q, 14q, 16q, and 21q. The best evidence of linkage was detected near D21S1903 on chromosome 21q22.3 (maximum Zlr=3.97); therefore, the linkage region was extensively investigated for linkage disequilibrium with single-nucleotide polymorphisms (SNPs) covering 20 Mb. One hundred fifty positional candidate genes lie in the region, and 600 gene-based SNPs were genotyped. There were positive allelic associations with seven genes (P<.01) in 280 patients and 210 controls, and four of the seven genes were clustered within a region of 750 kb, ∼1.2 Mb telomeric to D21S1903. Extensive linkage disequilibrium and association studies of the four genes indicated that SNPs in the collagen 6A1 gene (COL6A1) were strongly associated with OPLL (P=.000003 for the SNP in intron 32 [−29]). Haplotype analysis with three SNPs in COL6A1 gave a single-point P value of .0000007. Identification of the locus of susceptibility to OPLL by genomewide linkage and linkage disequilibrium studies permits us to investigate the pathogenesis of the disease, which may lead to the development of novel therapeutic tools.


Ossification of the posterior longitudinal ligament (OPLL [MIM 602475]) of the spine was first reported in Japan and is a common disorder among Japanese and other Asian populations (Matsunaga and Sakou 1997). The incidence of OPLL in Japan is 2%–4% of the general population >30 years of age, with a male predominance of 2:1 (Matsunaga and Sakou 1997). Heterotopic ossification of the spinal ligament is the specific feature of OPLL that causes compression of the spinal cord and leads to various degrees of myelopathy. Typical symptoms of OPLL are sensory and motor disturbance of the upper and lower extremities, abnormal reflexes, hyperresponsive deep reflexes, and bladder-bowel dysfunction. Various degrees of dysfunction, such as precise action and gait disturbance, lead to the restriction of activities involved in daily living and the deterioration of quality of life. Multiple etiologies for OPLL need to be considered because of the late-onset nature of the disease; however, the disease is, to some extent, genetically determined, as is demonstrated by the classic epidemiologic study and by the estimated relative risk of 10 for siblings of affected individuals (Terayama 1989; Sakou et al. 1991). OPLL is a high-bone-mass disease (or systemic hyperostosis), and patients with OPLL, especially women >60 years of age, have increased systemic bone mineral density (Yamauchi et al. 1999). Another ossification disorder, diffuse idiopathic skeletal hyperostosis (DISH), appears to be related to OPLL (Trojan et al. 1992). DISH is a common skeletal disease in middle-aged and elderly patients (with frequencies in individuals >50 years of age of 25% in men and 15% in women) in Western countries. The disease is characterized by ligamentous ossification of the anterolateral aspect of the spinal column, sometimes leading to bony ankylosis (Resnick et al. 1978). The extent of overlap of OPLL with DISH is uncertain; however, the pathophysiological mechanism of DISH may be similar to that of OPLL (Trojan et al. 1992; Weinfeld et al. 1997).

Because genetic factors appear to have a crucial role in OPLL, the use of molecular genetic studies is important to the understanding of the molecular etiologies of OPLL and will lead to the development of new therapeutics. Elsewhere, we reported linkage evidence in the HLA region of chromosome 6 in 91 affected sib pairs and identified the collagen 11A2 gene (COL11A2 [MIM 120290]) as a possible candidate (Koga et al. 1998). Allelic association studies between OPLL and molecular variants in COL11A2 demonstrated that a nucleotide substitution at intron 6 (−4), a T→A substitution, is significantly associated with OPLL, and the functional variant results in altered splicing, which is protective in the pathogenesis of OPLL (Maeda et al. 2001). Nakamura et al. (1999) reported that the nucleotide pyrophosphatase gene (NPPS [MIM 173335]), which has been identified as causal in a mouse model of OPLL (Okawa et al. 1998), is also associated with OPLL.

To understand the whole picture of OPLL susceptibility, we conducted a genomewide linkage study in 70 Japanese nuclear families, comprising 169 subjects and 142 affected sib pairs. At least six potential loci were linked to OPLL, with evidence for linkage being particularly strong on chromosome 21. Linkage analysis alone cannot provide the necessary resolution to identify the underlying genee, especially in complex diseases. Fine mapping of the linked regions can be attempted using linkage disequilibrium analysis with SNPs. Because a recent study of asthma susceptibility found that the 1-LOD decrease in the support interval of the linkage likely contains the susceptibility locus (Van Eerdewegh et al. 2002), the interval of the 1-LOD decrease on chromosome 21, ∼20 cM, was extensively studied by use of linkage disequilibrium mapping to pinpoint the locus of OPLL. When 600 SNPs of 150 genes were screened, significant associations were revealed between OPLL and molecular variants in collagen 6A1 (COL6A1 [MIM 120220]). The disease-related linkage disequilibrium was also assessed by haplotype-based association study.

Subjects and Methods

Disease Criteria and Subjects

The ethics committees of Hirosaki University and Kagoshima University approved the study, and all patients gave written informed consent. OPLL was diagnosed by the observation of ectopic bone formation in the posterior longitudinal ligament during x-ray or computed tomography examinations of the cervical, thoracic, or lumbar portions of the spine. The disease status of OPLL in radiograms was classified into four types, according to criteria of the Investigation Committee on the Ossification of the Spinal Ligaments, the Ministry of Health, Labor, and Welfare, Japan: (i) segmental, (ii) continuous, (iii) mixed, and (iv) localized, circumscribed, or bridged. All four types of OPLL were examined in the present study.

The siblings of patients with OPLL were screened for excessive spinal bone formation by x-ray examination, and affected siblings, including asymptomatic individuals, were recruited (Koga et al. 1998; Furushima et al. 2002). Samples for DNA analysis were collected from a total 142 affected sib pairs from 70 Japanese families; 98 pairs were from Kagoshima, 40 pairs from Hirosaki, 2 pairs from Asahikawa, and 1 pair each from Wakayama and Okinawa. The family structure was as follows: 51 affected pairs, 12 affected trios, 5 affected quartets, 1 affected quintet, and 1 affected sextet. The affected siblings included 76 women and 93 men. For the allelic association study, 342 unrelated patients with OPLL (73 familial cases, including 70 probands used for linkage study and 269 sporadic cases) and 298 unrelated subjects without OPLL were recruited in Kagoshima and Hirosaki. All the subjects without OPLL were >60 years of age and had no signs of spinal ossification when examined by standard x-ray methods, thereby excluding excluding most unmanifested diseases.

Microsatellite Genotyping

Multiplex fluorescent genotyping was performed using ABI PRISM linkage mapping set version 2 (Applied Biosystems). Because several markers were not polymorphic in the Japanese population (Ikari et al. 2001), a set of 47 markers obtained from the Genome Database were added to the original set to fill in the gap (Onda et al. 2001). An extra sequence was attached to the 5′ end of the reverse primer to promote nontemplated addition of adenine for accurate genotyping (Brownstein et al. 1996). Four markers (D6S446, D9S1678, D17S938, and DXS8067) generated incomplete data on the first round of genotyping and were excluded from further analysis. Marker positions (in Kosambi centimorgans) were obtained from the Marshfield Medical Research Foundation (Broman et al. 1998). For chromosome 21, which demonstrated significant linkage when the framework marker set was used, markers were added for dense mapping that covered <5 cM in the region (markers D21S1904, D21S1884, D21S1258, D21S262, D21S1900, D21S1893, D21S1411, and D21S1903). Microsatellite genotyping was performed as described elsewhere (Furushima et al. 2002). All marker genotypes were checked for Mendelian inheritance, using the Checkfam program (Saito et al. 2002), and genotyping inconsistencies were corrected.

Affected-Sib-Pair Linkage Analysis

GENEHUNTER-PLUS (Kong and Cox 1997) and GENEHUNTER 2.1 (Kruglyak et al. 1996) were used to analyze the data on affected sib pairs. Multipoint analysis of the data from the genomewide scan and from the fine mapping was performed by weighting each family equally with GENEHUNTER-PLUS, a modified version of GENEHUNTER. GENEHUNTER-PLUS assumes a linear model for risk and thus provides more-accurate calculations of the variance than does the original program. GENEHUNTER 2.1 was used for estimates of the mean proportion of alleles shared identical by descent (IBD) and for exclusion mapping. The linkage exclusion was set at a LOD score of −2. GENEHUNTER 2.1 was also used to calculate information content. Allele frequencies of microsatellite markers were estimated in 64 unrelated Japanese subjects (Ikari et al. 2001). For dense mapping on chromosome 21, allele frequencies of the markers were estimated with 70 probands, to perform more stringent linkage analysis.

SNP Genotyping

SNPs were obtained from the two public databases: National Center for Biotechnology Information dbSNP and Institute of Medical Science–Japan Science and Technology Corporation database of Japanese SNPs. Gene-based SNPs were selected at 3- to 10-kb intervals to cover the gene. Twelve subjects with OPLL were genotyped for each SNP, to confirm frequencies of minor alleles, and SNPs with minor allele frequencies >0.08 were subjected to further analyses. SNPs in the coding region or regulatory region that may directly affect gene function were given priority, regardless of the minor allele frequencies. PCR analysis was performed with a standard protocol, except that a biotin-labeled primer was used when the pyrosequencing method was applied. SNPs were genotyped using either the pyrosequencing method (a real-time pyrophosphate DNA sequencing, on a PSQ96 Instrument [Pyrosequencing]) or direct sequencing (using BigDye terminator cycle sequencing on an ABI PRISM 3700 DNA analyzer [Applied Biosystems]).

Other Statistical Analyses

Allelic frequencies of polymorphisms in cases and controls were compared using a contingency χ2 test. Haplotype frequencies for multiple loci were estimated using the expectation-maximization method with the Arlequin program, which is available at the Arlequin Web site or SNPAlyze program (Dynacom). Pairwise LD was estimated as D=x11-p1q1, where x11 is the frequency of haplotype A1B1, and p1 and q1 are the frequencies of alleles A1 and B1 at loci A and B, respectively. A standardized LD coefficient, r, is given by D/(p1p2q1q2)1/2, where p2 and q2 are the frequencies of the other alleles at loci A and B, respectively (Hill and Robertson 1968). Lewontin’s coefficient, D′, is given by D=D/Dmax, where Dmax=min(p1q2,p2q1) when D<0 or Dmax=min(p1q1,p2q2) when D>0 (Lewontin 1964).


Affected-Sib-Pair Linkage Analysis

A total of 70 Japanese families comprising 169 affected subjects and 142 sib pairs were included in the genome-scan data set. Multipoint Zlr scores for all chromosomes (except the Y chromosome) are displayed in figure 1. The region with the most prominent evidence of linkage was on chromosome 21q22, with a maximum Zlr score of 3.09 near marker D21S266. In addition to chromosome 21, five others—on chromosomes 1p, 6p, 11q, 14q, and 16q—showed evidence of linkage (defined as Zlr>2.2). Because the best evidence of linkage was observed with markers on chromosome 21q, high-resolution mapping was performed by adding eight markers. The fine mapping on chromosome 21 (fig. 2) indicated that the linkage peak (Zlr=3.97) was close to the q terminus near marker D21S1903. The highest two-point Zlr score (3.69) was detected at D21S1903 on the q terminus, and the highest IBD sharing of 71% (Z0=0.04; Z1=0.50; Z2=0.46) was observed at D21S262 on 21q22.

Figure  1
Genomewide linkage analysis of OPLL among 142 affected sib pairs. Summary of the framework genome scan for OPLL susceptibility loci showing the Zlr scores that were calculated by GENEHUNTER-PLUS for all chromosomes. Dotted lines indicate chromosome boundaries, ...
Figure  2
High-resolution mapping of chromosome 21. The solid line indicates the result of high-resolution linkage with 13 markers. The dotted line indicates the framework linkage result; asterisks (*) indicate the markers in the framework marker set. A Zlr score ...

Screening of Genes on the Linkage Region of Chromosome 21

The best evidence of linkage was detected with markers on chromosome 21q21; however, the linkage region (Zlr>2.2) spans ∼30 cM, and the 1-LOD decrease in the support interval was ∼20 cM and contained 200 genes. To pinpoint the locus for OPLL, linkage disequilibrium analysis was performed using gene-based SNPs in the region. We first genotyped 600 SNPs of 150 genes in the linkage region from 96 selected patients and 96 controls. The selected patients consisted of 73 unrelated patients with family history and 23 patients with age at onset <45 years. The remaining 50 genes were not investigated, because there were no SNPs in the database or SNPs were not polymorphic in Japanese populations. Allelic association was assessed by χ2 analysis with a contingency table. Seventy-four SNPs of 24 genes exhibited significant allelic associations (P<.05; data not shown). For the second screening, we genotyped the 74 SNPs in an expanded number of patients with OPLL (n=280) and in control subjects without OPLL (n=210). These patients included the initial screening sets. Fourteen SNPs of seven genes had allelic associations (P<.01) with OPLL (table 1). The most significant association with OPLL was observed with SNPs of COL6A1, which is located 1.2 Mb from the peak linkage marker, D21S1903. Because the four genes (COL18A1 [MIM 120328], PCBP3, COL6A1, and COL6A2 [MIM 120240]) that showed positive associations with OPLL are clustered within a 750-kb region in the vicinity of marker D21S1903, extensive SNP and pairwise linkage disequilibrium analyses were performed in the region. A total of 92 SNPs were analyzed for linkage disequilibrium structures in the region (fig. 3). Highly structured linkage disequilibrium blocks, as estimated with the D′ statistic, were observed; for example, COL18A1 was separated in two blocks, and the second block included SLC19A1. Because we genotyped gene-specific SNPs rather than SNPs in intergenic regions, we could not detect a broad scale of linkage disequilibrium blocks. The structures of pairwise linkage disequilibrium blocks were almost identical between patients and controls. SNPs of COL6A1 that had the strongest association with OPLL were in a gene-specific linkage disequilibrium block; therefore, COL6A1 was examined in more detail as the most likely candidate gene for OPLL. A total of 32 distinct SNPs—including the 4 SNPs used for the first screening, 25 additional dbSNPs, and 7 non-dbSNPs identified by direct sequencing—were genotyped in all the patients (n=342) and controls (n=298) (table 2). Among 7 SNPs identified in the present study, SNP31 at exon 35 led to a nonsynonymous change (serine to leucine), and other changes occurred in introns (table 2). Of 32 SNPs, 21 were statistically associated with OPLL (P<.05); of these, 4 had a particularly strong association (P<.0001 with OPLL; table 2). The most significant allelic association with OPLL was observed with a T→C substitution at intron 32 (−29) (χ2=21.99; df=1; P=.000003; odds ratio = 1.82 [95% CI = 1.60–2.35]). A T→C substitution at exon 15 (+39), which is a silent substitution, had the most significant association with OPLL among six coding SNPs (χ2=11.23; df=1; P=.0008; odds ratio = 1.49 [95% CI = 1.33–1.89]). For an explanation of numbers in parentheses that follow indications of location, see footnotes to table 2.

Figure  3
Pairwise linkage disequilibrium analysis on the 750-kb region where candidate genes are clustered. The upper figure shows six genes, including four associated genes (shown in table 1) within 750 kb of the linkage region near the marker D21S1903. The lower ...
Table 1
Genes Showing Positive Associations (P<.01) with OPLL on Chromosome 21
Table 2
Polymorphisms in COL6A1 and Their Association with OPLL

Haplotype Analysis of COL6A1

Because multiple SNPs may act in combination to increase the risk of OPLL, haplotypes were constructed on the basis of maximum likelihood. Three SNPs in COL6A1 (intron 21 [+18], intron 32 [−29], and intron 33 [+20]) that had highly significant associations with OPLL and were not in complete linkage disequilibrium with each other, as estimated using the r measure (r2<0.7; fig. 4), were selected for haplotype analysis (table 3). Estimated haplotype frequencies of the four common haplotypes (H1–H4) of subjects with OPLL were compared with those of control subjects without OPLL. There was a significant difference in haplotype frequency between cases and controls: haplotype H2, which comprises all the minor alleles at each site, occurred at a higher frequency in patients with OPLL than in control subjects (χ2=24.73, df=1, P=.0000007, odds ratio = 2.03 [95% CI = 1.76–2.70]); and haplotype H1, which comprises all the common alleles at each site, had a higher frequency in subjects without OPLL than in patients with OPLL (χ2=11.64; df=1; P=.0006; odds ratio = 0.66 [95% CI = 0.59–0.84]). The global test also gave a statistically significant result (χ2=26.73; df=4; P=.000023).

Table 3
Estimated Haplotype Frequency in Subjects with and without OPLL


The linkage results of the genomewide scan with 142 affected sib pairs revealed that six loci, on chromosomes 1p, 6p, 11q, 14q, 16q, and 21q, confer susceptibility to OPLL (Zlr>2.2), and the most promising locus was on chromosome 21q22 (Zlr=3.09) (fig. 1). Dense mapping of chromosome 21 revealed increased evidence of linkage on 21q22 near D21S1903 (Zlr=3.97) that would exceed the threshold of significant linkage (fig. 2) (Lander and Kruglyak 1995). The linkage region was studied extensively through linkage disequilibrium mapping and haplotype analysis, to narrow the susceptibility locus of OPLL.

Among five other susceptibility loci, chromosome 6 was of particular interest because previous studies reported that two candidate genes for OPLL (COL11A2 and NPPS) are on that chromosome (Koga et al. 1998; Nakamura et al. 1999). In the multipoint linkage study of chromosome 6, the highest Zlr score (2.22) and the best IBD sharing (59%; Z0=0.16; Z1=0.50; Z2=0.34) was on 6p21, where COL11A2 is located. On the other hand, the NPPS locus did not show any evidence of linkage (Zlr=0.00; mean IBD sharing 50.5%), confirming previous results of a single-point linkage analysis (Furushima et al. 2002).

The second most significant linkage (Zlr=2.94) was observed at chromosome 14, indicating that the chromosome 14q region may harbor another responsible gene. Notably, the linkage region at 14q is the widest linkage peak throughout the genome (43 cM; Zlr⩾2.2). Terwilliger et al. (1997) suggest that IBD sharing would be expected to decline gradually over adjacent markers when true linkage is present, resulting in a broad Zlr score distribution. Because the best evidence of linkage was on chromosome 21, the 14q region was not pursued in the present study.

The genomewide linkage results indicated the best evidence of linkage to OPLL on chromosome 21; therefore, we proceeded to narrow the locus through a linkage disequilibrium study. We performed an association study with ∼600 SNPs of 150 genes covering the 20-Mb linkage region of chromosome 21q. COL6A1 is the gene most significantly associated with OPLL in the case-control comparison (P=.04–.000003) and the haplotype analysis (P=.0000007) (tables 2 and and3).3). In addition, pairwise linkage disequilibrium analysis revealed that COL6A1 is in the gene-specific linkage disequilibrium block (fig. 3). Thus, susceptibility to OPLL could be accurately pinpointed to the COL6A1 locus. Mutations in the genes that encode collagen VI subunits (COL6A1, COL6A2, and COL6A3) are causal of Bethlem myopathy (MIM 158810), a dominant form of myopathy, and Ullrich syndrome (MIM 254090), a recessive form of muscular dystrophy (Jobsis et al. 1996; Lamande et al. 1998; Pape et al. 1999; Camacho Vanegas et al. 2001; Scacheri et al. 2002) characterized by early childhood onset of proximal muscle weakness and contractures of multiple joints (Bethlem and Wijngaarden 1976; Mohire et al. 1988). In the present study, we did not detect any molecular variants in the key glycine residues of the triple-helix domain that result in Bethlem myopathy. COL6A1 encodes the α1 chain of type VI collagen, which is an extracellular matrix protein consisting of a short central triple helix flanked by two large globular domains. Because the SNP (the T→C substitution at intron 32 [−29]) that is most strongly associated with OPLL is near the branch site of the intron, that SNP may affect the lariat-shaped structure, thereby causing aberrant splicing. Our previous linkage and association studies identified COL11A2 as a candidate for OPLL, and the functional intron 6 (−4A) polymorphism of COL11A2 affects splicing of exon 6 (Koga et al. 1998; Maeda et al. 2001). RT-PCR analyses of COL6A1 on cultured interspinous ligament cells from 10 patients with OPLL were performed, to study the splicing variants of COL6A1, as described in the analysis of COL11A2 published elsewhere (Maeda et al. 2001). Thus far, we have not detected a splicing variant of COL6A1 due to the intron 32 (−29) genotype (data not shown). The functional impact of the polymorphisms of COL6A1 is uncertain, but COL6A1 may lead to increased bone mass. Two genes (COL6A1 and COL11A2) encode extracellular matrix proteins, which may provide a scaffold for osteoblastic or preosteoblastic cells or chondrocytes that subsequently proceed to membranous or endochondral ossification (Zhang and Chen 2000; Gowen et al. 2003). Therefore, molecular variants of the extracellular proteins may be implicated in the ectopic ossification observed in OPLL.

The etiologies of all complex diseases potentially involve gene-gene and gene-environment interactions. In the case of OPLL, interactions of COL6A1 and COL11A2 must be taken into account in efforts to further understand the etiology of the disease. Elucidation of the molecular etiology of OPLL will lead to the development of new therapeutic approaches, not only for bone-forming diseases but also for diseases of bone loss, such as osteoporosis.


We thank DNA donors for making this study possible, and we thank Dr. Koga (Imakiire Hospital) and Dr. Sakou (Sakou Clinic) for recruiting families for the study. We are grateful to Ms. Eguchi and Ms. Nakamura for their technical efforts. This work was supported, in part, by a Research for the Future Program grant from the Japan Society for the Promotion of Science (to I.I.), a Research Grant for Specific Diseases (to I.I., S.I., and S.T.) and the Japan Foundation of Aging and Health from the Ministry of Public Health and Welfare (to I.I. and S.T), and a grant-in-aid for scientific research from the Japanese Ministry of Education, Science, Sports and Culture (to I.I.).

Electronic-Database Information

URLs for data presented herein are as follows:

Arlequin, http://anthropologie.unige.ch/arlequin (for the Arlequin program)
Center for Medical Genetics, Marshfield Medical Research Foundation http://research.marshfieldclinic.org/genetics/ (for genetic linkage maps)
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for gene sequences)
Genome Database, The, http://www.gdb.org/ (for primer design of microsatellite genotyping)
Institute of Medical Science–Japan Science and Technology Corporation database of Japanese SNPs, http://snp.ims.u-tokyo.ac.jp/ (for dbSNP selection)
NCBI dbSNP Home Page, http://www.ncbi.nlm.nih.gov/SNP/ (for dbSNP selection)
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for OPLL, Bethlem myopathy, Ullrich syndorome, COL11A2, COL6A1, COL6A2, and NPPS)
Pyrosequencing Technical Support, SNP Primer Design, http://techsupport.pyrosequencing.com/v2/index.asp (for designing sequencing primers for pyrosequencing)


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