Rheumatology Advance Access originally published online on August 5, 2006
Rheumatology 2007 46(2):250-252; doi:10.1093/rheumatology/kel265
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Genetic association analysis of LRCH1 as an osteoarthritis susceptibility locus
University of Oxford, Nuffield Department of Orthopaedic Surgery, Institute of Musculoskeletal Sciences, Botnar Research Centre, Oxford, UK and 1Departments of Human Genetics and Biomathematics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1766, USA
Correspondence to: Sarah Snelling, University of Oxford, Institute of Musculoskeletal Sciences, Botnar Research Centre, Nuffield Orthopaedic Centre, Oxford OX3 7LD, UK. E-mail: sarah.snelling{at}ndos.ox.ac.uk
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Abstract |
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Objective. A genetic association with knee osteoarthritis (OA) of a single nucleotide polymorphism (SNP) in intron 1 of the LRCH1 gene was recently reported in a UK Caucasian case-control sample and confirmed in a Newfoundland Caucasian sample. Our objective was to assess whether the SNP was associated with OA in our large UK Caucasian sample.
Methods. The SNP was genotyped in 1521 cases that had undergone elective joint replacement of the hip (1098 cases), of the knee (340 cases) or of the hip and knee (83 cases) due to end-stage primary OA. The SNP was also genotyped in 736 controls of similar ages in the cases.
Results. There was no significant difference (all P-values >0.05) in genotype or allele frequencies between our cases and our controls. There was also no significant difference when the cases were stratified by sex, by joint replaced or by sex combined with joint replaced.
Conclusion. Our data on 2257 individuals implies that the LRCH1 intron 1 SNP is not a risk factor for OA aetiology of the knee or of the hip in our UK Caucasian sample.
KEY WORDS: Osteoarthritis, LRCH1, Genetic susceptibility, SNP
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Introduction |
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Through a gene-based association scan involving over 25 000 single nucleotide polymorphisms (SNPs), Spector et al. [1] reported an association with knee osteoarthritis (OA) of a C/T transition SNP (rs912428) located in intron 1 of the LRCH1 gene on chromosome 13q14. LRCH1 is a novel gene that codes for Leucine-rich repeats and calponin homology domain containing protein 1. Nothing is currently known about the normal biological function of this protein [1].
In the Spector et al. [1] study, possessing a copy of the T-allele of SNP rs912428 was a risk factor for knee OA in females (n = 335) with an odds ratio (OR) of 1.44 and P-value of 0.0078, and a risk factor for knee OA in males (n = 197) with an OR of 1.64 and P-value of 0.0036. Spector et al. [1] confirmed this result in Newfoundland OA cases with knee OA (males and females, n = 211) with an OR of 1.43 and P-value of 0.0234. However, Newfoundland cases, with hand OA (males and females, n = 99) or with hip OA (males and females, n = 68), showed no evidence of association (P-values >0.05) with SNP rs912428. This absence of association in hand or hip OA could be the result of two possibilities: (i) the small size of the hand and hip case samples used by Spector and colleagues [1] which make the detection of a genuine, but relatively weak effect, unlikely or (ii) the restriction to the knee of the LRCH1 OA susceptibility.
Our objective was to assess whether SNP rs912428 was associated with OA susceptibility in our large UK Caucasian case-control sample. We genotyped the SNP in 1521 cases that had undergone elective joint replacement of the hip, of the knee or of the hip and knee due to end-stage primary OA. Allele and genotype frequencies were then compared with 736 Caucasian controls who had no symptoms of OA and whose ages are similar to the cases.
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Patients and methods |
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Case-control sample
The cases (n = 1521; 845 females and 676 males) were ascertained through the Nuffield Orthopaedic Centre in Oxford. They had undergone total joint replacement of hip (n = 1098; 619 females and 479 males), of knee (n = 340; 182 females and 158 males), or of hip and knee (n = 83; 44 females and 39 males) for primary OA. The cases were ascertained using the criteria of signs and symptoms of OA sufficiently severe to require joint replacement surgery. All had pain with rest and night symptoms for more than 6 months duration. The radiological stage of the disease was a Kellgren and Lawrence (KL) grade of two or more in all cases with over 90% being grade three or four. Inflammatory arthritis (rheumatoid, polyarthritic or autoimmune disease) was excluded, as was post-traumatic or post-septic arthritis. No cases suggestive of a skeletal dysplasia or developmental dysplasia were included. The average age of the cases at replacement surgery was 65 yrs with an age range of 56–85 yrs. The controls comprised 736 individuals (379 females and 357 males) with no signs or symptoms of arthritis or joint disease (pain, swelling, tenderness or restriction of movement). The average age of the controls at recruitment was 69 yrs with an age range of 55–89 yrs. Due to ethical and financial constraints, the joints of the controls were not subjected to radiographic analysis. All cases and all controls were UK Caucasians.
Written consent was obtained from cases and from controls according to the declaration of Helsinki. The Oxfordshire Clinical Research Ethics Committee approved the design of the study.
Genotyping SNP rs912428
The SNP alters an AlwnI restriction enzyme site and was genotyped using a PCR-restriction enzyme analysis. The primers used are located in intron 1 of LRCH1 and have the sequence 5'-TCAACAGTGGCTATAGACCT-3' and 5'-AGGTCTGTGAGTTTGCGGCAT-3'. The 357 bp PCR product encompasses the polymorphic AlwnI site as well as an invariant AlwnI site, which acted as a positive control for enzyme digestion. Following digestion with AlwnI (New England Biolabs, Hitchin, UK), a T-allele generates two fragments of 271 and 86 bp, whilst a C-allele generates three fragments of 220, 86 and 51 bp. Digestion products were electrophoresed through 3% agarose and scored following ethidium bromide staining.
Statistical analysis
Genotype and allele distributions in cases and controls were compared using standard chi-square analysis-of-contingency tables. ORs were calculated with 95% confidence intervals (CIs). For stratification analysis, female cases were compared with female controls, and male cases were compared with male controls.
The minimum detectable ORs under the log additive model with power 
80% and significance level of 5% were calculated for each comparison using Quanto version 1.1 (http://hydra.usc.edu/gxe) [2, 3]. The T-allele frequency was set to 19% (the frequency for all 736 of our controls) and the population risk of OA was set to 5%.
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Results |
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Power of the study
To insure that we had adequate power to detect the OR values observed by Spector et al. [1], we calculated the minimum detectable ORs that could lead to an 80% probability of rejection of the null hypothesis of no association at a significance level of 0.05. The minimum detectable ORs are given in Table 1. These were calculated under the log additive model, which is equivalent to calculating the OR for the T-allele, as in Spector et al. [1]. The minimum detectable ORs were less than the ORs calculated by Spector et al. [1] for the comparable case-control comparisons except for females with knee OA. In this case, the minimum detectable OR was 1.54, whereas the value calculated by Spector and colleagues [1] was 1.47. Our sample size (female cases with knee OA = 182 and female controls = 379) has greater than 80% power to detect an OR of 1.47 when the significance level was raised to 0.10.
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Association analysis
Table 2 lists the genotype and allele frequencies of SNP rs912428 in our cases and controls. The data have also been stratified by sex. We compared the genotype frequencies between the cases and the controls, and the allele frequencies between the cases and the controls. None of the genotype or allele frequencies differed significantly (all P-values >0.05). The only stratum that approached significance (P-values <0.1) was males with hip OA, with a P-value of 0.07 for the allelic comparison. This was accounted for by an increased frequency of the T-allele in male hip cases (22.5%) vs the male controls (18.8%).
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The genotype and allele frequencies of SNP rs912428 in our study are comparable with those of the Spector et al. study [1]. For example, we observed a T-allele frequency of 19.0% in our controls, compared with T-allele frequencies of 14.0–18.4% in the Spector et al. [1] controls.
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionAcknowledgementsReferences |
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A large number of OA susceptibility loci have been reported and some have been confirmed in independent studies. The most compelling in this regard is the secreted frizzled-related protein 3 gene FRZB on chromosome 2q, which was originally shown to harbour OA susceptibility in a UK study [4] and which was subsequently confirmed in studies carried out in the Netherlands, Belgium and the USA [5–8]. Occasionally, for a complex disease, a very compelling find in one ethnic group is not found to be a risk factor in other ethnic groups [9]. For OA, a striking example of this is the asporin gene ASPN on chromosome 9q, which showed strong association in the Japanese OA patients [10] but which has not been confirmed in the European OA patients [11–13]. This implies the existence, at different frequencies between Asians and Europeans, of other polymorphic loci or of environmental factors that can influence the penetrance of the ASPN encoded OA susceptibility.
Our aim in this manuscript was to assess whether the LRCH1 SNP that had been previously reported to be a risk factor for knee OA in Caucasians was also a risk factor in our large UK Caucasian OA sample. Our study was adequately powered to detect an association comparable with that reported in the original report and by focusing on the same ethnic group as used in the original study, we avoided the potential confounding factor of trying to confirm a genetic association in a different ethnic group. Despite these precautions, we did not detect an association either when the cases were studied as a whole or when they were subjected to stratification by sex and by the joint replaced.
Our knee sample is of a size comparable with that studied in the original report (340 cases vs 335 cases in the Spector discovery sample, 443 cases in the Spector replication sample and 211 cases in the Spector Newfoundland sample). It is possible that our knee result is an example of an independent study observing a much weaker effect than that observed in the original report, which is not an uncommon observation for a complex trait risk allele [14]. Our hip sample is much larger than any of the case samples used by Spector et al. [1] and therefore had the greatest power to see an effect on OA susceptibility of SNP rs912428. However, no association was observed. As already noted, Spector et al. [1] also reported no association between SNP rs912428 and hip OA, although the case sample size that they studied was small (n = 68) and, therefore, lacked power. Based on our observation, it seems reasonable to conclude that SNP rs912428 is not a risk factor for OA of the hip. However, our study does not have the power to exclude a very weak effect of LRCH1 on OA susceptibility of the hip. Nor can we exclude an association with a different polymorphism within this gene.
Overall, our studies do not substantiate the previous findings of an association between the LRCH1 SNP rs912428 and OA. As more independent studies are reported, an accurate assessment of the effect of the LRCH1 SNP on OA susceptibility will emerge.
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Acknowledgements |
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Research into Ageing and the Arthritis Research Campaign supported this study. We thank Kim Clipsham and Bridget Watkins, who helped organize the collection of samples from individuals evaluated in this report.
The authors have declared no conflict of interest.
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References |
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