Background: Genetic predisposition is a key factor in the etiology of systemic sclerosis (SSc), especially within the major histocompatibility complex (MHC) region, where among others, the Complement component 4 (C4), encoded by C4A and C4B , plays a critical role in immune regulation and immune complex clearance, which are key processes in SSc. It has been described that variations in the copy number (CN) of C4 influence disease risk [1]. However, the relationship with SSc clinical and serological subtypes remains unexplored.

Objectives: This study aimed to analyze the association between C4 CN and SSc clinical and serological subtypes. Additionally, it sought to investigate the independence of HLA alleles in regard to C4 CN in the different analyses.

Methods: C4 , C4A and C4B CNs were imputed from genotypic data of a cohort of SSc patients (stratified into 3,853 anti-centromere positive (ACA+), 2,456 anti-topoisomerase I positive (ATA+), 6,286 limited cutaneous SSc (lcSSc) and 2,908 diffuse cutaneous SSc (dcSSc) patients) and 17,608 unaffected individuals. Logistic regression analyses were performed to evaluate associations between C4 genes CN with SSc serological and clinical subtypes. HLA alleles were imputed through the Michigan imputation server, and conditional analyses were conducted to assess independent HLA associations from C4 CN, using a genome-wide significance threshold of 5x10 ^-8 .

Results: Our results revealed that higher C4 CNs were associated with significant protection against SSc, particularly in ATA+ patients, with the effect predominantly driven by C4A (OR=0.63, p=3.15x10 ^-16 ). Notably, C4A CN protection remained significant when comparing ATA+ to ACA+ patients (OR=0.63, p=3.82x10 ^-11 ). Conversely, ACA+ patients showed a significant association with C4B CN (OR=0.84, p=1.23x10 ^-5 ), but not with C4A , pointing out the relevance of C4B in ACA+ patients and the differences between serological groups. These genetic associations align with clinical findings: dcSSc patients had a strong association with C4A (OR=0.62, p=3.47x10 ^-20 ), closer to ATA+ patients, while lcSSc patients showed a moderate effect with both C4A and C4B (OR=0.80 and OR=0.83, respectively), similar to ACA+ patients. Conditional analyses identified, for the first time, an HLA-A allele ( HLA-A*29:02 ) associated with ACA+ and lcSSc, and three distinct HLA-C alleles ( HLA-C*07:01 , HLA-C*04:01 , and HLA-C*16:01 ). HLA-C*07:01 was linked to dcSSc and ACA+, HLA-C*04:01 was associated with ACA+ and HLA-C*16:01 to ACA+ and lcSSc. A total of 35 HLA alleles were independent from C4 CN at least in one subtype, and previous associations with SSc subtypes were replicated. Notably, HLA-DQB1*03:03:02:01 was associated with an increased risk for ATA+ but offered protection for ACA+ patients.

Conclusion: This study highlights the distinct contribution of C4A and C4B CN to SSc subtypes susceptibility. Conditioning analysis underscores the influence of C4 CN in the HLA genetic associations, revealing novel alleles potentially involved in the disease. Ultimately, our findings reveal that increased C4 CNs, particularly C4A , have a protective effect in dcSSc, the more severe form of SSc, suggesting that C4 may prevent disease progression by promoting immune complex clearance.

REFERENCES: [1] Kerick M, Acosta-Herrera M, Simeón-Aznar CP, et al. (2022) Complement component C4 structural variation and quantitative traits contribute to sex-biased vulnerability in systemic sclerosis. NPJ Genom Med. 7(1):57.

Acknowledgements: We thank Sofia Vargas for her excellent technical assistance and all the patients and control donors for their essential collaboration. J.M.L. is recipient of a PFIS fellowship (FI23/00231) from Instituto de Salud Carlos III. This research is part of the doctoral degree awarded to J.M.L., within the Biomedicine program from the University of Granada.

Disclosure of Interests: Javier Martinez-Lopez: None declared , Carlos Rangel-Peláez: None declared , Inmaculada Rodriguez-Martin: None declared , Alfredo Guillen-Del-Castillo: None declared , Carmen P. Simeón-Aznar: None declared , José Luis Callejas: None declared , Oliver Distler 4P-Pharma, Abbvie, Acceleron, Acepodia Biotech, Aera, Alcimed, Altavant, Amgen, AnaMar, Anaveon AG, Argenx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Calluna (Arxx), Cantargia AB, Catalyze Capital, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, MSD Merck, Miltenyi Biotec, Mitsubishi Tanabe, Nkarta Inc., Novartis, Orion, Pilan, Prometheus, Quell, Redxpharma, Roivant, EMD Serono, Topadur and UCB, 4P-Pharma, Abbvie, Acceleron, Acepodia Biotech, Aera, Alcimed, Altavant, Amgen, AnaMar, Anaveon AG, Argenx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Calluna (Arxx), Cantargia AB, Catalyze Capital, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, MSD Merck, Miltenyi Biotec, Mitsubishi Tanabe, Nkarta Inc., Novartis, Orion, Pilan, Prometheus, Quell, Redxpharma, Roivant, EMD Serono, Topadur and UCB, BI, Kymera, Mitsubishi Tanabe, UCB, International SSc Group: None declared , Susanna M. Proudman: None declared , Mandana Nikpour: None declared , Australian Scleroderma Interest Group (ASIG): None declared , Nicolas Hunzelmann: None declared , Gianluca Moroncini: None declared , Jeska K. de Vries-Bouwstra: None declared , Ariane L. Herrick: None declared , Yannick Allanore: None declared , Lorenzo Beretta: None declared , Shervin Assassi: None declared , Christopher P. Denton: None declared , Maureen D. Mayes: None declared , Marialbert Acosta-Herrera: None declared , Javier Martin: None declared , Martin Kerick: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.