Genomewide linkage and linkage disequilibrium analyses identify COL6A1, on chromosome 21, as the locus for ossification of the posterior longitudinal ligament of the spine

Abstract

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, approximately 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.

Figure 1

Genomewide linkage analysis of OPLL among 142 affected sib pairs. Summary of the framework genome scan for OPLL susceptibility loci showing the Z_lr scores that were calculated by GENEHUNTER-PLUS for all chromosomes. Dotted lines indicate chromosome boundaries, and the distance from the p terminus of chromosome 1 is shown on the X-axis.

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 Z_lr score of 3.97 is detected within the peak region close to the q terminus by fine mapping. The Y-axis indicates Z_lr score, and the X-axis indicates distance from the p terminus of chromosome 21.

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 figure shows pairwise linkage disequilibrium blocks in the region. Pairwise linkage disequilibrium between all pairs was evaluated by the D′ statistic, and COL6A1 linkage disequilibrium was analyzed with the r² statistic. SNPs with allele frequencies <.05 were excluded for the linkage disequilibrium estimate.