Heterotopic ossification (HO) is lamellar bone formation that occurs within tissues that do not normally have properties of ossification. The pathoaetiology of HO is poorly understood. We conducted a genome wide association study to better understand the genetic architecture of HO.

891 patients of European descent (410 HO cases) following THA for primary osteoarthritis were recruited from the UK. HO was assessed from plain AP radiographs of the pelvis. Genomic DNA was extracted, genotyped using the Illumina 610 beadchip and referenced using the 1000 Genome Project panel. HO susceptibility case-control analysis and an evaluation of disease severity in those with HO was undertaken using SNPTESTv2.3.0 on>10 million variants. We tested variants most strongly associated with HO in an independent UK THA replication cohort comprising 209 cases and 211 controls. The datasets were meta-analysed using PLINK.

In the discovery cohort 70 signals with an index variant at p<9×10–5 were suggestively associated with HO susceptibility. The strongest signal lay just downstream of the gene ARHGAP18 (rs59084763, effect allele frequency (EAF) 0.19, OR1.87 [1.48–2.38], p=2.48×10–8), the second strongest signal lay within the long non-coding (LNC) RNA gene CASC20 (rs11699612, EAF 0.25, OR1.73 [1.1.40–2.16, p=9.3×10–8). In the discovery cohort 73 signals with an index variant at p<9×10–5 were associated with HO severity. At replication, 12 of the leading 14 susceptibility signals showed a concordant direction of allelic effect and 5 replicated at nominal significance. Following meta-analysis, the lead replicating susceptibility signal was the CASC20 variant rs11699612 (p=2.71×10–11).

We identify consistent replicating association of variation within the LNC RNA CASC20 with HO susceptibility after THA. Although the function of CASC20 is currently unknown, possible mechanisms include transcriptional, post-transcriptional and epigenetic regulation of downstream target genes. The work presented here provides new avenues for the development of novel predictive and therapeutic approaches towards HO.

Introduction

Periprosthetic osteolysis resulting in aseptic loosening is a leading cause for total hip arthroplasty (THA) failure. Individuals vary in their susceptibility to osteolysis, and it is thought that heritable factors contribute to this variation. We conducted two genome-wide association studies to identify genetic risk loci associated with osteolysis and genetic risk loci associated with time to prosthesis failure due to osteolysis.

Systemic concentrations of metal ions (cobalt and chromium) are persistently elevated in patients with metal-on-metal hip resurfacing (MOMHR) compared to conventional total hip arthroplasty (THA). Several studies by us and others have described the detrimental effects of metal exposure on survival and function of various cell types in-vitro, but the mechanisms for these effects remain unclear. Epigenetic modifications following chronic metal exposure is a possible mechanism that could mediate these effects. Here we test the methylation status in genomic DNA from MOMHR (“cases”) and THA (“controls”) patient-groups, and its correlation with circulating metal levels.

The cohort consisted of 34 patients with a well-functioning MOMHR at a median follow-up of 9.75 years. These were individually matched for gender, age and time-since-surgery to a non-exposure group consisting of patients with THA. Genomic DNA was isolated from blood samples and cell composition estimated using the ‘estimateCellCounts’ function in ‘minfi R-package’. Methylation was assessed using the Illumina 450k BeadChip array analysing 426,225 probes. Logit model was fitted at each probe with case/control status as independent variable and covariates of gender, age, time-since-surgery, smoking, non-arthroplasty metal exposure, and cell composition. DNA methylation age was assessed using an online calculator (https://dnamage.genetics.ucla.edu/) and comparisons made between cases and controls, and correlated with circulating metal levels.

Cell distributions did not differ between the cases and controls (Wilcoxon test p<0.17) with no probe having an association at 5% FDR. Circulating metal levels and LVEDD also had no association with any probe at 5% FDR. There was no preferential age acceleration between cases and controls (Wilcox p<0.7), and it had no correlation with plasma-chromium or blood-cobalt levels (p<0.9).

In summary, large methylation changes following MOMHR seem to be absent, compared to THA. Future research with larger samples will be needed to clarify the presence and extent of small methylation changes.

Introduction

Although DDH is one of the most common skeletal dysplasias (incidence 1.5 cases per 1000 births), it remains slow and costly to recruit large-scale patient cohorts for powerful genetic association studies. In this work we have successfully used the NJR as a platform to generate a DDH biobank of 907 individuals, upon which we have conducted the first ever genome-wide association study (GWAS) for DDH.

Aims: Tumour necrosis factor-alpha is a proinflammatory cytokine that has been implicated in the inflammatory response to bacterial infection and wear debris particles around loosened total hip replacements (THR). Individual TNF responses to such stimuli may be dictated by genetic variation and we have studied the effect of single nucleotide polymorphisms (SNPs) within the TNF gene.

Methods: We performed a case control study of 9 SNPs (−1031, −863, −857, −376, −308, −238, +489, +851 and +1304) for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THR. Cases consisted of 44 patients with early aseptic loosening and 30 patients with microbiological evidence at surgery of deep infection. Controls consisted of 85 THRs that were clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening. DNA was extracted from venous blood and genotyped by Snapshot assay.

Results: Genotype and allele frequencies for all SNPs were in Hardy-Weinberg equilibrium between THR controls and a random sample of UK Caucasians. A significant association was found for the -863 SNP and aseptic loosening (p< 0.05; OR=2.36; 95% CI: 0.976 – 5.71). A trend towards association was found between the -863A SNP and deep infection (p=0.80; OR=2.42; CI: 0.800 – 7.34).

Conclusions: Genetic polymorphism of TNF-alpha may play a significant role in THR aseptic loosening and possibly in deep infection. SNP markers may serve as predictors of implant survival and response to therapy such as anti-TNF treatment.

Tumour necrosis factor-alpha is a proinflammatory cytokine that has been implicated in the propagation of inflammatory responses to bacterial infection and wear debris particles around loosened total hip replacements (THR). Individual TNF responses to such stimuli may be dictated by genetic variation. Single nucleotide polymorphisms (SNPs) at several loci within the TNF gene are associated with disease severity and susceptibility in a number of inflammatory conditions, but only a few SNPs have been screened in any one study.

14 SNPs have been identified within the TNF gene. Our unit has previously demonstrated that 5 SNPs are monomorphic in a sample group of UK Caucasians. We performed a case control study of the remaining 9 polymorphic positions (−1031, −863, −857, −376, −308, −238, +489, +851 and +1304) for possible association with deep sepsis or aseptic loosening.

All patients included in the study were Caucasian and had had a cemented Charnley THR and polyethylene cup. Cases consisted of 44 patients with early aseptic loosening (defined as that occurring within 6 years of implantation and findings at revision surgery or by the criteria of Hodgkinson et al for the acetabulum and Harris for the femoral stem) and 30 patients with microbiological evidence at surgery of deep infection. Controls consisted of 85 THRs that had remained clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening or ‘at risk’ signs as described by Wroblewski et al. DNA was extracted from venous blood and genotyped by Snapshot assay.

Genotype and allele frequencies for all SNPs were in Hardy-Weinberg equilibrium between THR controls and a random sample of UK Caucasians. The most significant associations were between the −238A (p< 0.05) and −863T (p< 0.05) alleles and aseptic loosening. A trend towards association was found between the −863A SNP and deep infection (p=0.80). The −238 A/G and −863 G/T genotypes were associated with deep infection (p< 0.05). No other significant associations were found.

Genetic polymorphism of TNF appears to play a significant role in THR aseptic loosening and possibly in deep infection. SNP markers may serve as predictors of implant survival and response to therapy such as anti-TNF treatment.