Abstract
Objective. To study whether gene variants associated with lumbar disc degeneration (LDD) phenotypes are also associated with hip osteoarthritis (OA).
Methods. Magnetic resonance imaging (MRI)-based hip OA changes for 345 twins were assessed and 99 single-nucleotide polymorphisms (SNP) were analyzed.
Results. Variants in the COL9A2 (rs7533552, p = 0.0025) and COL10A1 (rs568725, p = 0.002) genes showed association with hip OA.
Conclusion. The associating G allele in COL9A2 changes a glutamine to arginine or to tryptophan and may predispose to both hip OA and LDD, making it a candidate for degenerative connective tissue diseases.
Osteoarthritis (OA) is a degenerative joint disease common in the elderly. Based on twin studies, the heritability of hip OA is over 50%1. It is likely that there are common genetic factors behind hip OA and disc degeneration, since hip OA has been shown to predict the progression of disc space narrowing and the presence of anterior vertebral osteophytes of the discs2. Many different genes including collagens and collagen-breaking factors have been suggested to have an effect on OA as well as with other connective tissue and skeletal diseases3.
In our previous study, we monitored for association of 25 candidate genes with the lumbar disc degeneration (LDD) traits disc bulging, disc narrowing, and a quantitative measure of water content, using magnetic resonance imaging (MRI). We studied 99 single-nucleotide polymorphisms (SNP) in aggrecan 1 (AGC1, ACAN) and 12 collagen, 8 interleukin, and 4 matrix metalloproteinase genes4. In our current study, we investigated whether the same genes were also involved in another connective tissue disease, hip OA, using the same twin study material and the same SNP panel.
MATERIALS AND METHODS
The study sample was the part of the population-based Twin Spine Study material4 that has been investigated by MRI. It consists of 588 Finnish monozygotic (MZ) and dizygotic (DZ) male twins aged 35–70 years. The studied phenotype was MRI-based calcified structure changes also visible in radiographs. Imaging of the hips was done using Siemens Vision 1.5 T scanning, both hips at the same time, using a body matrix coil. Analysis of the scans was done using Siemens MRI workstation at the Puijon Magneetti Oy company, Kuopio, Finland. The grading scale was based on Li, et al5 and the Kellgren-Lawrence radiological assessment of OA6: Individuals with 2 normal MRI images were considered as healthy, and individuals with at least small local osteophytes in caput or acetabulum in 1 hip were considered affected, yielding a phenotype allowing for early-onset OA (Table 1).
Subject characteristics by hip OA status (data are mean ± SD).
Polychoric correlations for MZ and DZ twins and heritability using quantitative genetic models allowing for additive genetic, common, and unique environmental variance in the liability were estimated using Mx software (M. Neale, Virginia Commonwealth University; available from: http://www.vcu.edu/mx/). Lifestyle factors (body mass index, mean age, smoking pack-years, and exercise frequency) were compared between the healthy and affected subjects (Mann-Whitney U test).
Genotyping and ethical approval is described in Videman, et al4. One individual from each MZ twin pair was used in the SNP association analysis (n = 345, 216 unrelated). Evidence for association of the studied SNP was evaluated using the Pseudomarker program that can use various pedigree structures including combined data of individuals and sibling pairs. The program is able to separate the linkage evidence from evidence for association. The test statistic for association linkage was used with dominant and recessive modes7. Association was also calculated for unrelated individuals using Fisher’s exact test and the size of genetic effect for unrelated individuals was analyzed. The Haploview program8 was used to calculate the linkage disequilibrium (LD) between the markers.
To define variation at the codon level, we sequenced the flanking region of SNP rs7533552 in all samples (ABI3730xl DNA Analyzer, BigDye Terminator v3.1 Cycle Sequencing Kit, Life Technologies Corp., Carlsbad, CA, USA). Genotype call rate for rs7533552 was 97.5% and Hardy-Weinberg equilibrium was 0.826.
To correct for multiple testing, the SNP spectral decomposition (SpD) method9 with modifications by Li and Ji10 was used to calculate the p value threshold for 5% significance (0.00073; Genetic Epidemiology Laboratory, Queensland Institute of Medical Research; available from: http://genepi.qimr.edu.au/general/daleN/SNPSpD).
RESULTS
The differences in lifestyle factors were nonsignificant between the healthy and affected subjects (Table 1). The polychoric correlation for hip OA was 0.71 (SE 0.11) for MZ twins and 0.40 (SE 0.13) for DZ twins. A quantitative genetic model allowing for additive genetic and unique environmental variance estimated the heritability of hip OA to be 0.72 (95% CI 0.49–0.87).
One variant in the COL9A2 gene (rs7533552; location chr1:40545736, hg18, second base in the codon for Gln326), which has previously shown association with disc bulging in the lumbar spine (L1 to L4)4, also showed association with hip OA (p = 0.0025; Table 2). The G allele was the predisposing allele in both diseases, with a frequency of 30.8% in the hip OA cases and 17.5% in the healthy subjects. The G allele changes a polar negatively charged glutamine (Gln326) to a polar positively charged arginine (Arg). The same amino acid will be further changed to a nonpolar hydrophobic tryptophan (Trp), if the rare SNP in the first position of the same codon is a T allele (rs12077871; location chr1:40545737, hg18; Figure 1). Since the genotyping method used was only designed to monitor variation at rs7533552, we further sequenced the locus to define the genotypes of both the variants in order to reveal the carried amino acid for each individual. Eleven individuals carried a Trp allele as a combination of C/T heterozygote for SNP rs12077871 (first base of the codon) together with A/G heterozygote or G/G homozygote for SNP rs7533552 (second base of the codon; Table 3). This indicated that the minor alleles T and G of the 2 studied SNP were inherited together (D’ LD = 1.00, r2 LD = 0.04, calculated using the Haploview program)8.
Single-nucleotide polymorphisms in the amino acid Gln326.
Association of the studied SNP with hip OA using the Pseudomarker program.
Amino acid combinations for variants in Gln326 codon in unrelated individuals.
The size of genetic effect for unrelated individuals was analyzed by comparing amino acid counts of the wild-type Gln allele with the predisposing Arg or Trp alleles between hip OA cases (45 predisposing alleles, 101 wild-type alleles) and controls (50 predisposing alleles, 236 wild-type alleles). The disease odds ratio for carrying either of the predisposing variant alleles Arg or Trp versus the wild-type allele Gln was 2.10 (95% CI 1.66–2.67; p = 0.0021, Fisher’s exact test).
Also, 3 SNP in LD with each other (r2 > 0.63, D’ > 0.83) in the COL10A1 gene showed association with hip OA in the present study (p = 0.0015) but were not associated with any of the lumbar disc degeneration traits in our previous study (Table 2).
DISCUSSION
We monitored for association between hip OA and 99 SNP that were previously studied with MRI-based lumbar disc degeneration phenotypes4. One SNP (rs7533552, also known as rs2228564) in the COL9A2 gene was found to be associated with hip OA (p = 0.0025) and also with greater disc bulging in the L1-L4 lumbar region (p = 0.036; p value for the within-family component of multivariate regression analysis of disc bulging in L1-L4 discs using the quantitative transmission disequilibrium test program version 2.4.3, calculated with 1000 Monte Carlo permutations for correction of the skewed phenotype)4. It is possible that either one of the observed SNP associations is affected by the other trait, or that the SNP independently affects both traits.
Hip OA has previously been shown to associate with growth differentiation factor 5 (GDF5) with genome-wide significance (p = 1.8 × 10−13)11,12, while variants in SMAD family member 313 (SMAD3), acidic (leucine-rich) nuclear phosphoprotein 32 family member A14 (ANP32A), deiodinase, iodothyronine type II15 (DIO2), and frizzled-related protein16,17 (FRZP) genes have shown association in replicated studies. Our finding is, to our knowledge, the first to suggest that the change from Gln326 to Arg or Trp in COL9A2 gene predisposes to hip OA. Neither of the 2 SNP in the codon nor any SNP in high LD with them in European populations were represented on the Illumina HumanHap 610 genome-wide chip used in the genome-wide association studies. The Gln326 to Arg change has previously been reported to be more frequent in patients with severe and chronic back pain surgically treated for intervertebral disc herniation, although the association did not reach statistical significance18. The Gln326 to Trp change (“Trp2 allele”) has been shown to be associated with unilateral back and radiating leg pain19 and with degenerative lumbar spinal stenosis20.
COL9A2 encodes one of the 3 alpha chains of type IX collagen, having an important role in load-bearing cartilages stabilizing the collagen fibrils, usually found in tissues containing type II collagen. Lack of type IX collagen can increase the susceptibility of the matrix to mechanical damage21. According to the PolyPhen protein structure prediction program, the change from glutamine to arginine is predicted not to cause major structural damage for the protein, but the change from glutamine to tryptophan is predicted as possibly damaging (Division of Genetics, Department of Medicine, Brigham and Women’s Hospital – Harvard Medical School, Cambridge, MA, USA; available from: http://genetics.bwh.harvard.edu/pph/).
Association was also observed with the COL10A1 gene that encodes 3 alpha 1 chains in type X collagen homotrimer. It is expressed by hypertrophic chondrocytes in a correlation with endochondral ossification and thus can be considered as a marker for new bone formation in articular cartilage22. Additionally, Boos, et al23 found type X collagen in osteoarthritic cartilage of the hip when absent from normal adult cartilage.
We focused on a strictly narrowed measurable phenotype instead of on symptoms such as joint pain potentially caused by various biological mechanisms. The phenotype for bone changes was selected because of its known reliability/validity in OA grading on the basis of radiographic studies, but the lack of information on measurement reliability is a limitation. The high MZ correlation suggests that reliability must be high. The age range used also allows cases of early-onset hip OA to be included in the group of affected individuals. However, including the younger, seemingly healthy individuals, who may later develop the disease, in the group of unaffected individuals could decrease the power of detecting a true association. Also, the associations did not exceed the 5% significance level (p = 0.00073, according to the SNP SpD method)9 when multiple testing was taken into account, and thus the results need to be interpreted carefully. Analysis in larger materials and functional studies is needed to validate the finding by excluding the possibility of a false-positive finding.
Ninety-nine SNP were analyzed in a population-based male twin cohort to study if there were common variants affecting hip OA and lumbar disc degeneration. One SNP (rs7533552) changing Gln326 to Arg or Trp in the COL9A2 gene predisposed to hip OA and lumbar disc degeneration, making type IX collagen an interesting target for future studies.
Acknowledgment
We thank the twin pairs, and Heidi Lönnberg, Minna Suvela, Aki Salo, Eila Voipio, Kauko Heikkilä, Sanna Kouhia, Riitta Simonen, Annina Ropponen, Brad Sinclair, Kevin Gill, and Hannu Manninen.
Footnotes
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Supported by the US National Institutes of Health (grant AR-40857); the Work Environment Fund of Finland; the Academy of Finland (grants 38332 and 42044); the Alberta Heritage Foundation for Medical Research, Canada; the European Community’s Seventh Framework Programme (FP7, 2007-2013; grant HEALTH-F2-2008-201626; project GENODISC); Ministry of Education of Finland; the University of Jyväskylä; and the University of Kuopio. A. Näkki was supported by TULES Graduate School, TBGS National Graduate School of Musculoskeletal Disorders and Biomaterials, The Finnish Cultural Foundation, and The Otto A. Malm Foundation. The Finnish Twin Cohort Study is a project of the Academy of Finland Centre of Excellence in Complex Disease Genetics.
- Accepted for publication November 2, 2010.