Background/Purpose: C4 is an important component of the Complement system and plays an essential role in the activation cascade of the classical Complement pathway. Complete C4 deficiency is among the strongest genetic risk factors for systemic lupus erythematosus (SLE). There are two C4 circulating isoforms (C4A and C4B) encoded by C4A and C4B genes, respectively, that differ by only five nucleotides. C4A protein is involved in the clearance of immune complex and apoptotic debris while C4B protein is relevant in the opsonization of microbes. C4A and C4B genes are located at a gene cassette within the MHC class III region and depict gene copy-number variation (CNV). The number of C4A copies may be related to the susceptibility to SLE. This study aimed to investigate the impact of the C4A and C4B gene CNV on juvenile SLE.

Methods: We evaluated 90 children and 170 adults with SLE (meeting SLE ACR criteria) sequentially retrieved from the rheumatology outpatient clinic. Two hundred healthy individuals (HI) without evidence of autoimmune diseases were retrieved among blood bank donors. Peripheral blood leukocyte DNA was amplified by quantitative real-time PCR (qPCR) with primers for C4 gene and sequence specific TaqMan® probes for C4A (5’ FAM-ACCCCTGTCCAGTGTTAG-MGB 3’) and C4B (5′ FAM-ACCTCTCTCCAGTGATAC-MGB 3′). Gene copy number (GCN) was determined by the delta-delta cycle threshold (DDCT) method.

Results: Children with SLE had lower GCN of total C4 (mean total C4=3.1; 95% CI=2.8-3.4), C4A (mean C4A=1.7; 95% CI=1.5-1.9) and C4B (mean C4B=1.5; 95% CI=1.3-1.6) than HI (C4=4.3 with 95% CI=4.1-4.5, p<0.001; C4A=2.3 with 95% CI=2.2-2.5, p<0.001; C4B=2.0 with 95% CI=1.8-2.1; p<0.001). The frequency of SLE children with total C4 low GCN (<4 copies) was significantly higher than in HI (SLE=59% versus HI=28%; OR=3.68; 95% CI=2.19-6.20; p<0.001). The same was observed for C4A low GCN (<2 copies) (SLE=52% versus HI=18%; OR=4.98; 95% CI=2.88-8.62; p<0.001) and C4B low GCN (SLE=60% versus HI=31%; OR=3.26; CI=1.95-5.47; p<0.001). The association between adult SLE and low GCN for total C4 (OR=2.03; 95% CI=1.32-3.13; p=0.001), C4A (OR=2.36; 95% CI=1.46-3.81; p<0.001) and C4B (OR=1.13; CI=0.73-1.74; p=0.593) was less pronounced than observed in juvenile SLE. Moreover, there was an association between cardiovascular damage and low GCN for C4 and C4A. 81% of SLE children with cardiovascular damage had less than 4 copies of C4 against 51% of the children without cardiovascular manifestation (OR= 4.13; CI 95%= 1.02-16.68; p=0.047).  The same was observed for C4A (81% of those with cardiovascular damage had <2C4A against 44% of those with no heart involvement; OR=5.54; CI 95%= 1.37-22.32; p=0.016).

Conclusion: Low GCN for total C4, C4A and C4B is associated with an increased risk for developing systemic lupus erythematosus and this association is stronger in childhood than in adults. Furthermore, low GCN for C4 and C4A seems to be a risk factor of cardiovascular damage in juvenile SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/low-gene-copy-number-for-c4-c4a-and-c4b-is-a-strong-risk-factor-for-developing-systemic-lupus-erythematosus-in-childhood/