ObjectiveSystemic lupus erythematosus (SLE) features high frequency of cardiovascular disease (CVD) and fluctuating complement levels. The clinical trial Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) aimed to evaluate whether atorvastatin treatment reduced the progression of atherosclerosis in 221 patients with childhood-onset SLE (cSLE), using carotid intima media thickness (CIMT) as surrogates. We leveraged APPLE biorepository and trial data to investigate the relationship between complement and CVD in cSLE.MethodsGene copy numbers (GCNs) for total C4, C4A and C4B were measured by TaqMan-based real-time PCR and Southern blotting, and analysed with laboratory and clinical parameters through Student’s t-test and χ2 analyses. Effects of total C4, C4A and C4B GCNs on the response to placebo or atorvastatin treatment and progression of CIMT were examined by regression analyses.ResultsAt baseline, C4 protein levels strongly correlated with GCNs of total C4 (p=1.8×10−6). Each copy of C4 gene increased mean serum C4 by 3.28 mg/dL. Compared with those without hypertension (N=142), individuals with hypertension demonstrated significantly elevated serum levels for C4 and C3 at baseline and serially (C4: P=5.0×10−25; C3: P=5.84×10−20). Individuals with ≥2 C4B genes had 2.5 times the odds of having hypertension (p=0.016) and higher diastolic blood pressure (p=0.015) compared with those with C4B deficiency. At the study end, subjects with ≥2 C4B and atorvastatin treatment had significantly slower increase in CIMT compared with those treated with placebo (p=0.018).ConclusionscSLE with hypertension had elevated serum levels of C4 and C3 and higher GCN of C4B; cSLE with ≥2 C4B genes would benefit from statins therapy to prevent atherosclerosis.

BackgroundIdiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels and muscle fibres, chronic weakness and fatigue. While complement-mediated destruction of capillary endothelia is implicated in paediatric and adult dermatomyositis, the complex diversity of complement C4 in IIM pathology was unknown.MethodsWe elucidated the gene copy number (GCN) variations of total C4, C4A and C4B, long and short genes in 1644 Caucasian patients with IIM, plus 3526 matched healthy controls using real-time PCR or Southern blot analyses. Plasma complement levels were determined by single radial immunodiffusion.ResultsThe large study populations helped establish the distribution patterns of various C4 GCN groups. Low GCNs of C4T (C4T=2+3) and C4A deficiency (C4A=0+1) were strongly correlated with increased risk of IIM with OR equalled to 2.58 (2.28–2.91), p=5.0×10−53 for C4T, and 2.82 (2.48–3.21), p=7.0×10−57 for C4A deficiency. Contingency and regression analyses showed that among patients with C4A deficiency, the presence of HLA-DR3 became insignificant as a risk factor in IIM except for inclusion body myositis (IBM), by which 98.2% had HLA-DR3 with an OR of 11.02 (1.44–84.4). Intragroup analyses of patients with IIM for C4 protein levels and IIM-related autoantibodies showed that those with anti-Jo-1 or with anti-PM/Scl had significantly lower C4 plasma concentrations than those without these autoantibodies.ConclusionsC4A deficiency is relevant in dermatomyositis, HLA-DRB1*03 is important in IBM and both C4A deficiency and HLA-DRB1*03 contribute interactively to risk of polymyositis.

BackgroundHuman SLE is characterised by fluctuating serum levels of complement proteins. There are frequent copy number variations (CNVs) of complement C4A and C4B genes among different individuals. Previously, we demonstrated that C4A deficiency is a strong genetic risk factor for SLE.ObjectivesTo investigate how CNVs of C4 contribute to the great variability of C4 serum levels and how deficiencies of C4A or C4B modulate the clinical presentations, including organ damage, of SLE.MethodsOur study population included 499 patients from Hong Kong, who fulfilled ≥4 of the 2013 ACR/SLICC criteria for SLE. Among them 93% were women, the mean age of SLE onset was 32.8±13.0 years, and SLE duration was 14.4±7.6 years. Gene copy numbers (GCNs) of total C4 (C4T), C4A and C4B were determined by real-time PCRs. Serial serum levels over the past 5 years for C4 and C3 of each patient were retrieved through the laboratory data registry system. Serum C4 and C3 levels are shown as mg/100 ml (unit). Clinical manifestations and organ damage of SLE were correlated with CNVs of C4 genes and serum levels. Continuous data between groups were compared by t-tests and categorical data by χ2 analyses. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals for effects of C4 CNVs on cumulative clinical manifestations of SLE and accrued organ damage, adjusted for durations of disease.ResultsSerum levels for C4 varied from 1–84 units (Median: 17) and for C3 from 8–314 units (Median: 86). There was a very strong correlation between C4 and C3 protein levels (R=0.70, p=5.3×10–75). The GCN of C4T varied between 2 and 9 with a median of 4 copies (54%), followed by 2 and 3 copies (21%). Each additional gene copy correlated to an increase of 4 and 6 units for the mean and maximum serum C4 levels, respectively. A higher GCN of C4T (≥3 vs<3) was protective against the development of neuropsychiatric disorder over time [OR 0.45 (0.21–0.98), p=0.04]. A high GCN of C4L (≥3 vs<3), or the absence of C4S (GCN=0), was negatively associated with the occurrence of thrombocytopenia [OR 0.64 (0.42–0.97), p=0.04]. A high GCN of C4B was associated with damage to any organ [OR 1.76 (1.05–2.93), p=0.03], but a high GCN of C4A (≥3 vs<3) was associated with cardiovascular damage [OR 2.30 (1.06–5.00), p=0.04]. Among the SLE patients studied, 18.3% had persistently low levels of C4 (mean ≤10.0 units). These patients mostly had GCNs of C4T=2 or 3 [OR 4.02 (2.47–6.56), p=4.7×10–8], or C4B=0 or 1 [OR 3.06 (1.89–4.96), p=9.0×10–6]. Patients with persistently low C4 levels had increased prevalence of mucosal ulceration [OR 2.09 (1.15–3.78), p=0.02], lymphopenia [OR 1.76 (1.01–3.05), p=0.045] and gastrointestinal disorders [OR 2.52 (1.31–4.84), p=0.005].ConclusionsCNVs of C4 genes confer great variability of serum C4 levels among SLE patients. While C4A deficiency contributes to genetic predisposition of SLE, persistently low levels of serum C4 among patients were strongly correlated with low GCN of total C4 and C4B deficiency. Elucidating C4-CN...