Using multivariable Mendelian randomization to estimate the causal effect of bone mineral density on osteoarthritis risk, independent of body mass index

OBJECTIVES: Observational analyses suggest that high bone mineral density (BMD) is a risk factor for osteoarthritis (OA); it is unclear whether this represents a causal effect or shared aetiology and whether these relationships are body mass index (BMI)-independent. We performed bidirectional Mendelian randomization (MR) to uncover the causal pathways between BMD, BMI and OA. METHODS: One-sample (1S)MR estimates were generated by two-stage least-squares regression. Unweighted allele scores instrumented each exposure. Two-sample (2S)MR estimates were generated using inverse-variance weighted random-effects meta-analysis. Multivariable MR (MVMR), including BMD and BMI instruments in the same model, determined the BMI-independent causal pathway from BMD to OA. Latent causal variable (LCV) analysis, using weight-adjusted femoral neck (FN)–BMD and hip/knee OA summary statistics, determined whether genetic correlation explained the causal effect of BMD on OA. RESULTS: 1SMR provided strong evidence for a causal effect of BMD estimated from heel ultrasound (eBMD) on hip and knee OA {odds ratio [OR](hip) = 1.28 [95% confidence interval (CI) = 1.05, 1.57], p = 0.02, OR(knee) = 1.40 [95% CI = 1.20, 1.63], p = 3 × 10(–5), OR per standard deviation [SD] increase}. 2SMR effect sizes were consistent in direction. Results suggested that the causal pathways between eBMD and OA were bidirectional (β(hip) = 1.10 [95% CI = 0.36, 1.84], p = 0.003, β(knee) = 4.16 [95% CI = 2.74, 5.57], p = 8 × 10(–9), β = SD increase per doubling in risk). MVMR identified a BMI-independent causal pathway between eBMD and hip/knee OA. LCV suggested that genetic correlation (i.e. shared genetic aetiology) did not fully explain the causal effects of BMD on hip/knee OA. CONCLUSIONS: These results provide evidence for a BMI-independent causal effect of eBMD on OA. Despite evidence of bidirectional effects, the effect of BMD on OA did not appear to be fully explained by shared genetic aetiology, suggesting a direct action of bone on joint deterioration

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases.

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52-kb intronic deletion 3'). Family members were assessed for HBM segregation with identified variants. Three-dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (∼prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non-LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5-HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST-LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z-scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z-scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (∼3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance.This study was supported by The Wellcome Trust and NIHR CRN (portfolio number 5163). CLG was funded by a Wellcome Trust Clinical Research Training Fellowship (080280/Z/06/Z), the EU 7th Framework Programme under grant agreement number 247642 (GEoCoDE), a British Geriatric Society travel grant, and is now funded by Arthritis Research UK (grant ref 20000). SH acknowledges Arthritis Research UK support (grant ref 19580). KESP acknowledges the support of Cambridge NIHR Biomedical Research Centre. KAW is supported by the core programme of the MRC Nutrition and Bone Health group at MRC Human Nutrition Research, funded by the UK Medical Research Council (Grant code U10590371). EM acknowledges support of the Sheffield Teaching Hospitals Foundation Trust Clinical Research Facility. The SGC is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada (Ontario Genomics Institute OGI- 055), GlaxoSmithKline, Janssen, Lilly Canada, Novartis Research Foundation, Ontario Ministry of Economic Development & Innovation, Pfizer, Takeda, and Wellcome Trust (092809/Z/10/Z).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/jbmr.270

A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre- and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre- and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer.This study was partly funded by a project grant from Cancer Research UK. R.C.F. is funded by an NIHR Professorship and receives core funding from the Medical Research Council and infrastructure support from the Biomedical Research Centre and the Experimental Cancer Medicine Centre. We acknowledge the support of The University of Cambridge, Cancer Research UK (C14303/A17197) and Hutchison Whampoa Limited

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Characterisation of Copy Number Changes in the Progression of Barrett’s Oesophagus

Introduction: The main risk factor for the development of oesophageal adenocarcinoma is Barrett’s oesophagus (BE). To diagnose those patients who will progress to cancer early to improve the dismal survival rate of oesophageal adenocarcinoma, patients with BE undergo regular endoscopic surveillance. The vast majority of patients, however, will never progress and are therefore monitored unnecessarily. Copy number changes have been shown to be important in the progression of BE to oesophageal adenocarcinoma (Li et al., 2014). Shallow whole genome sequencing (sWGS) has been established as a cost-effective method of investigating copy number changes in formalin fixed paraffin embedded (FFPE) tissue (Scheinin et al., 2014). We hypothesised that copy number alterations may be valuable markers in disease progression and aimed to characterise them in the progression of Barrett’s using sWGS in order to predict progression in patients from a point in time as close to baseline endoscopy as possible and to integrate p53 staining. Methods: To optimise sWGS we compared 50X WGS on frozen tissue with 0.1X WGS from FFPE tumour material from the same patient. To address poor cellularity in endoscopic biopsies, shallow WGS data from a 50% cellularity biopsy with a 90% frozen sample from a single patient were compared. Accounting for poor biopsy cellularity 0.4X coverage was used. We performed FFPE shallow WGS on 806 samples from an 89-patient cohort comprising a 1:1 ratio of patients who progressed to high grade dysplasia (HGD) and patients who never progressed. 1-31 samples per patient were collected over time and space throughout surveillance. Non-progressors had significantly longer follow-up (p-value = 0.0008). Data was processed based on published bioinformatic pipelines. Copy number analysis was carried out using a generalised linear model (GLM) in order to develop a predictive algorithm. Results: During optimisation, ˃85% of copy number changes were detected in both frozen and FFPE samples from spatially distinct regions of an individual tumour. We found 91% and 93% agreement in copy number calls using orthogonal platforms between 90% (frozen) and 50% (FFPE) cellularity samples from one tumour. In the 806 sample Barrett’s cohort, we observed larger copy number alterations in patients who progressed to cancer compared with non-progressors and significantly more CN alterations in progressor patients (p-value ˂ 0.001). More cancer-associated genes were affected in progressors and we observed significant heterogeneity between patients. There was also a greater level of complexity seen in the progressor patients when analysed using affinity propagation clustering. These data allowed us to develop a regression model to predict progression. Using the GLM model, we successfully classified samples as early as progressor or not with an AUC of 85.75% and a sensitivity and specificity of 84 and 79% respectively. At the patient level 94% progressor patients had at least one sample classified as at risk of progression and non-dysplastic progressor samples were classified as early as 13 years prior to HGD diagnosis. Depending on the classification threshold used, all samples over time and space were not classified as being at risk of progression in at least 60% patients who have not yet progressed to HGD/cancer. We observed 2 pathways to progression supporting previous observations. 90% of progressors had samples prior to their HGD or cancer diagnosis classified as being predisposed to progression suggestive of genetically unstable lesions from early on in surveillance that progressed to HGD over time. The remaining 10% appeared as non-progressors until their diagnosis of HGD. We investigated p53 expression in our patient cohort as the only biomarker to have successfully transitioned into the clinic for Barrett’s surveillance. Whilst we found our cohort to be representative in staining compared to other published cohorts, it did not contribute to the GLM and the copy number data out-performed the use of p53 IHC in the context of Barrett’s surveillance. Conclusions: We have optimised the use of shallow WGS in oesophageal adenocarcinoma and Barrett’s. Using these copy number data, we can confidently distinguish between patients who will progress to cancer and the majority of patients who will never progress. This approach has led to the development of a model for predicting progression in the clinical setting which is promising for further clinical validation.Funded by the MR

Genomic copy number predicts esophageal cancer years before transformation

Summary Recent studies show that aneuploidies and driver gene mutations precede cancer diagnosis by many years1–4. We assess whether these genomic signals can be used for early detection and pre-emptive cancer treatment using the neoplastic precursor lesion Barrett’s esophagus, as an exemplar5. Shallow whole genome sequencing of 777 biopsies, sampled from 88 patients in Barrett’s surveillance over a period of up to 15 years shows that genomic signals can distinguish progressive from stable disease even ten years prior to histopathological transformation. These findings are validated on two independent cohorts of 76 and 248 patients. These methods are low cost and applicable to standard clinical biopsy samples. Compared with current management guidelines based on histopathology and clinical presentation, genomic classification enables earlier treatment for high risk patients as well as reduction of unnecessary treatment and monitoring for patients who are unlikely to develop cancer.The laboratory of R.C.F. is funded by a Core Programme Grant from the Medical Research Council (RG84369). This work was also funded by a United European Gastroenterology Research Prize (RG76026)

Genomic copy number predicts esophageal cancer years before transformation

Summary Recent studies show that aneuploidies and driver gene mutations precede cancer diagnosis by many years1–4. We assess whether these genomic signals can be used for early detection and pre-emptive cancer treatment using the neoplastic precursor lesion Barrett’s esophagus, as an exemplar5. Shallow whole genome sequencing of 777 biopsies, sampled from 88 patients in Barrett’s surveillance over a period of up to 15 years shows that genomic signals can distinguish progressive from stable disease even ten years prior to histopathological transformation. These findings are validated on two independent cohorts of 76 and 248 patients. These methods are low cost and applicable to standard clinical biopsy samples. Compared with current management guidelines based on histopathology and clinical presentation, genomic classification enables earlier treatment for high risk patients as well as reduction of unnecessary treatment and monitoring for patients who are unlikely to develop cancer.The laboratory of R.C.F. is funded by a Core Programme Grant from the Medical Research Council (RG84369). This work was also funded by a United European Gastroenterology Research Prize (RG76026)

Safety and immunogenicity of heterologous prime-boost immunisation with Plasmodium falciparum malaria candidate vaccines, ChAd63 ME-TRAP and MVA ME-TRAP, in healthy Gambian and Kenyan adults.

BACKGROUND Heterologous prime boost immunization with chimpanzee adenovirus 63 (ChAd63) and Modified vaccinia Virus Ankara (MVA) vectored vaccines is a strategy recently shown to be capable of inducing strong cell mediated responses against several antigens from the malaria parasite. ChAd63-MVA expressing the Plasmodium falciparum pre-erythrocytic antigen ME-TRAP (multiple epitope string with thrombospondin-related adhesion protein) is a leading malaria vaccine candidate, capable of inducing sterile protection in malaria naïve adults following controlled human malaria infection (CHMI). METHODOLOGY We conducted two Phase Ib dose escalation clinical trials assessing the safety and immunogenicity of ChAd63-MVA ME-TRAP in 46 healthy malaria exposed adults in two African countries with similar malaria transmission patterns. RESULTS ChAd63-MVA ME-TRAP was shown to be safe and immunogenic, inducing high-level T cell responses (median >1300 SFU/million PBMC). CONCLUSIONS ChAd63-MVA ME-TRAP is a safe and highly immunogenic vaccine regimen in adults with prior exposure to malaria. Further clinical trials to assess safety and immunogenicity in children and infants and protective efficacy in the field are now warranted