Purpose: There is a clinical unmet need for prognostic biomarkers of post-traumatic osteoarthritis which are peripherally accessible. We studied whether there was a detectable transcriptional response to knee joint injury in the cellular compartment of whole blood, and whether any significantly regulated transcripts were associated with patient-reported outcomes at the time of injury and 2 years after the injury.
Methods: Gene expression profiling (GEP, Illumina) was carried out on globin-depleted RNA extracted from whole blood (Paxgene tubes; Qiagen) from 22 participants in the Knee Injury Cohort @ Kennedy (KICK) sustaining structural knee injury in the past 20 days and 12 healthy age matched controls. The 45 highest differentially-regulated transcripts between the 2 groups were selected to populate microfluidic RT-PCR cards (Thermofisher Scientific). RT-PCR of the 45 transcripts was then carried out in a further group of 40 KICK participants’ baseline samples and 16 controls to validate the GEP findings, normalising to 3 housekeeping genes (GPDH, PPIA, YWHA2). Clinical data, including Knee injury and Osteoarthritis Outcome Score (KOOS) from which KOOS-4 composite measure was calculated, were collected at baseline and 2 years. Differences in transcript fold changes were tested by a linear parametric but also non-parametric method. Linear regression analysis of the association between transcript fold changes (normalised by log transformation where necessary) and the primary outcome KOOS-4 was carried out (StataIC 13). Adjustment was made for time from injury, age and gender.
Results: The mean ages of the control group and KICK participants for the GEP study were not significantly different (33±7.1, and 24±6.1 respectively). After globin depletion, RNA was still of high quality (RIN>7). ∼70 transcripts were significantly regulated in cases with acute knee injury compared with controls by GEP. Significant differences in many of the fold changes of these mRNAs were subsequently confirmed by RT-PCR; for example, IGF Binding Protein 2 was most downregulated (q=4.425x10-8) and Enolase-3 the most upregulated transcript compared with control samples (q=8.867x10-5). Fold changes of 4/45 transcripts were associated with KOOS-4 at baseline, of which 3 (ZAP70, AMT, CLEC5A) were independently associated with KOOS-4 in a multivariate model, unadjusted coeff. -8 (-15.5, -2.2); adjusted coeff. (for time from injury, age and gender) -10 (-16.7, -3.4); adj. R 0.30. Fold changes of 4/45 transcripts measured at baseline were associated with KOOS-4 at 2 years; only 1 of these was independently associated with KOOS-4 in a multivariate model; unadjusted coeffic.7.6 (0.45, 14.76); adjusted coeffic. 8.2 (0.5, 15.9); adj R 0.20.
Conclusions: Our findings support the approach of measuring the systemic response to joint injury by GEP, in that this response differs significantly from control. The whole blood transcriptional response varied substantially from the protein response measured by us previously in the joint. Many transcripts were associated with T cells (such as ZAP70), monocytes (such as CLEC5A) or other cells not typically studied in our field. This pilot study was carried out in relatively small numbers which risks type 1 or type 2 errors, but suggests that, as with protein biomarkers, some regulated transcripts are associated with patient-reported outcomes including pain and dysfunction at the time of injury (higher levels were associated with worse symptoms at the time of injury). Our preliminary finding of a transcript which is apparently associated with improved patient-reported outcomes at 2 years will now be tested in the full study population.
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