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Background: Collection of real-world evidence (RWE) is important in achondroplasia. Development of a prospective, shared, international resource that follows the principles of findability, accessibility, interoperability, and reuse of digital assets, and that captures long-term, high-quality data, would improve understanding of the natural history of achondroplasia, quality of life, and related outcomes. Methods: The Europe, Middle East, and Africa (EMEA) Achondroplasia Steering Committee comprises a multidisciplinary team of 17 clinical experts and 3 advocacy organization representatives. The committee undertook an exercise to identify essential data elements for a standardized prospective registry to study the natural history of achondroplasia and related outcomes. Results: A range of RWE on achondroplasia is being collected at EMEA centres. Whereas commonalities exist, the data elements, methods used to collect and store them, and frequency of collection vary. The topics considered most important for collection were auxological measures, sleep studies, quality of life, and neurological manifestations. Data considered essential for a prospective registry were grouped into six categories: demographics; diagnosis and patient measurements; medical issues; investigations and surgical events; medications; and outcomes possibly associated with achondroplasia treatments. Conclusions: Long-term, high-quality data are needed for this rare, multifaceted condition. Establishing registries that collect predefined data elements across age spans will provide contemporaneous prospective and longitudinal information and will be useful to improve clinical decision-making and management. It should be feasible to collect a minimum dataset with the flexibility to include country-specific criteria and pool data across countries to examine clinical outcomes associated with achondroplasia and different therapeutic approaches.

Additional file 7: Baseline characteristics of the study population included in the exploratory analyses and full OLS regression models between independent variables (height z-score, absolute height [cm], age, gender and complications) and patient-reported outcomes (total scores).

Additional file 6: Growth charts (height and head circumference) of study population against published achondroplasia growth data, by time of measurement, age and sex.

Additional file 3: Table of descriptions, including ages assessed, by each patient-reported outcomes questionnaire.

Additional file 5: Methods for exploratory analyses between height/z-score and patient-reported outcomes.

Additional file 1: List of data categories collected from patient medical records.

Additional file 2: List of pre-defined complications of interest by relevant age population.

Additional file 4 : Imputation methods for missing data.

Additional file 1: Table S1. Exclusion and starting criteria of the MAA. Table S2. Overview of assessments in the MAA. Figure S1. Flow chart of patient disposition. Figure S2. Urinary keratan sulfate (uKS) over time by age at treatment initiation. Error bars are standard error. Figure S3. Weight over time by age at treatment initiation. Error bars are standard error. Figure S4. 6-minute walk test (6MWT) distance outcomes over time for patients from MOR-002 compared to natural history data from MOR-001 (MOR-001 data linearly extrapolated beyond year 2). Error bars are standard error. Figure S5. 6-minute walk test (6MWT) distance over time by age at treatment initiation compared to results from untreated patients from the MOR-001 natural history study (MOR-001 data linearly extrapolated beyond year 2). Error bars are standard error. Figure S6. Change in FVC (A) and FEV1 (B) over time by age group with comparison to MOR-001 natural history (MOR-001 data available for FVC only, linearly extrapolated beyond year 2). Error bars are standard error. Figure S7. Changes in pulmonary function from baseline to last follow-up (N = 40). Decline: ≥ 0.1 L decrease; Improvement: ≥ 0.1 L increase; Stability

This thesis comprises of three clinical studies looking at the role oxidative stress might play in making a plaque vulnerable, and thereby tries to identify new diagnostic markers and possible therapeutic targets. It explores whether gradients of markers of oxidative stress and vessel inflammation exist across coronary atherosclerotic plaque, and whether these relate to plaque characteristics, coronary remodelling and clinical presentation (stable/unstable). It also investigates the possible role of the cytoprotective enzyme heme oxygenase-l (HO-l) in coronary artery disease (CAD) and its clinical presentation. The research presented in this thesis also looks at whether acutely increasing iron would result in oxidative stress in vivo and produce endothelial dysfunction. Atherosclerosis is now recognized as a multifactorial process. It is well known that progression of atherosclerosis is neither linear nor predictable. We are now aware that noncritical lesions are the substrate for most fatal and non-fatal myocardial infarctions (MI). Available screening and diagnostic methods are insufficient to identify victims before the event occurs in most cases. While mortality from MI has improved for patients who present to the hospital over the last decade, we still are a long way away from reaching the preventative goal of identifying patients who are high risk of MI before their event. One of the key unanswered questions still remains the identification and treatment of a vulnerable patient before their sentinel event; hence it remains imperative that we continue to improve our understanding of the mechanism of atherothrombosis. The "oxidative-modification hypothesis" of atherosclerosis suggests that low-density lipoprotein cholesterol (LDL) accumulates in the subendothelial space of arteries, and through the action of proximate vascular cells, becomes oxidized; this can initiate the early events in atherosclerosis. Superoxide production is increased in a number of disease states, where it has been implicated in reduced endothelium-derived nitric oxide (EDNO) bioactivity, including hypercholesterolemia, hypertension, diabetes mellitus and atherosclerosis. This thesis in particular looks at the role of redox active iron and the antioxidant enzyme HO-l. Redox-active iron catalyses the production of ROS, causes lipid peroxidation, and has been implicated in the pathogenesis of atherosclerosis. Isoprostanes are prostaglandin isomers formed by free radical-catalysed oxidation of arachidonic acid esterified in membrane phospholipids, and are a marker of oxidative stress. We performed a prospective study on consecutive patients undergoing PCI at the Alfred Hospital cardiac catheterization laboratory to see if systemic and coronary trans-Iesional markers of iron stores would relate to coronary plaque characteristics and clinical presentation as stable versus unstable coronary syndromes; these data are presented in Chapter 3. This study confirmed that positive coronary remodeling at the site of maximum stenosis was more frequent in unstable patients. It showed that in both aortic and distal coronary blood samples at baseline, that ferritin consistently correlated with plaque area and was higher in patients with positive (versus negative) remodelling. Positive coronary remodelling has been associated with vulnerable plaques and coronary artery disease. Distal coronary ferritin levels post-PCI also correlated with plaque area, and were higher in patients with positive versus negative remodeling. There was a positive coronary trans-Iesional gradient of ferritin 3 minutes post PCI, and a corresponding positive trans-Iesional gradient of F2-isoprostanes at baseline. These findings did not vary according to clinical presentation, suggesting that markers of iron stores and oxidative stress are increased in coronary atherosclerotic plaque in vivo, and are subsequently released with plaque disruption. Increased intra-plaque redoxactive iron may affect plaque vulnerability directly by inducing oxidative stress. HO-l is an inducible isoform of a heme degradation enzyme that has been demonstrated to exert potent antioxidant and anti-inflammatory effects in the setting of atherosclerotic vascular disease, and in people with risk factors for atherosclerosis. It is emerging as an important cytoprotective, vasculoprotective and cardioprotective enzyme with pleiotropic potential. In light of current limited clinical data, we conducted a case-control study in consecutive patients presenting to the catheterization laboratory with de novo (i.e. newly diagnosed) CAD to systematically determine the relationship between HO-l promoter gene polymorphisms, HO-l plasma levels, and heme degradation products, along with markers of inflammation and iron parameters in patients with CAD. These data were compared against an aged and gender-matched healthy control group; these data are presented in Chapter 4. Specifically, we examined whether HO-I promoter gene polymorphisms andlor HO-l plasma levels could predict CAD clinical presentation in the presence of traditional strong CAD risk factors. This study showed that the HO-l promoter gene polymorphism SIS in this predominantly Caucasian population appeared protective against CAD. HO-l plasma levels (and its catalytic end products bilirubin) were raised in those with an unstable presentation of CAD. These data suggest that HO-l is an inducible enzyme that is up regulated in unstable CAD. In experimental models of atherosclerosis, vascular iron deposition is closely related to endothelial dysfunction, the progression of atherosclerosis, arterial thrombosis, and lowdensity lipoprotein oxidation. The endothelium plays a pivotal role in regulating key vascular processes that affect the progression of atherosclerosis such as regulating vascular tone, platelet activity, leukocyte adhesion, vascular smooth muscle cell proliferation, and vascular remodeling by the release of several paracrine factors, including nitric oxide. Endothelial dysfunction is not only an early marker for increased cardiovascular risk, it likely contributes to the pathogenesis of atherosclerosis. We previously demonstrated that iron chelation with desferrioxamine improved nitric-oxide mediated, endothelial-dependent vasodilatation in patients with CAD. Hence we hypothesized that increasing iron levels acutely would increase oxidative stress in vivo, and result in endothelial dysfunction in healthy humans and patients with CAD. To test this hypothesis we infused iron sucrose into the forearm of healthy volunteers and measured iron parameters, a marker of lipid oxidative stress, and endotheliumdependent and -independent vascular function. This study, presented in Chapter 5, demonstrates that acute iron infusion induces oxidative stress in vivo, without affecting nitric oxide-mediated vascular reactivity. Given previous evidence (including that from our own laboratory), this suggests that chronic iron overload is necessary to affect endothelial function, and by inference, atherosclerosis.