Supplementary Material for: Combined heterozygous genetic variations in complement C2 and C8B - an explanation for multi-dimensional immune imbalance?

The complement system plays a crucial role in host defence, homeostasis, tissue regeneration and bridges the innate and the adaptive immune system. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here we report on a patient carrying their combination in heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and ROS generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient’s plasma with FFP as complement source could fully restore full complement functionality. This study describes a combined heterozygous genetic variation in complement C2 and C8B which cannot explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

补体系统（complement system）在宿主防御、稳态维持、组织再生过程中发挥关键作用，同时作为桥梁连接固有免疫与适应性免疫系统。尽管补体C2（complement C2）的c.839_849+17del；p.(Met280Asnfs*5)变异与补体C8B（complement C8B）的c.1625C>T；p.(Thr542Ile)变异此前已被单独报道，但本研究报告了一例同时以杂合形式携带这两种变异的患者。该患者全身状态欠佳，罹患多种自身免疫性疾病。尽管未检测到自身免疫疾病特异性自身抗体，但遗传分析显示其补体C2与C8B基因存在异常。为此，我们从细胞与体液层面开展了针对固有免疫系统的全面研究，以明确其功能层面的影响。我们发现中性粒细胞与单核细胞的吞噬作用、活性氧（Reactive Oxygen Species, ROS）生成功能存在轻度受损，且C5a受体1（C5a receptor 1, C5aR1）的表达水平降低。全面的补体分析显示，补体旁路途径与经典途径的激活能力均出现下降。将纯化的补体C2与C8蛋白加入患者血清后，未能使功能异常恢复至正常水平；而添加C3则可改善溶血活性。在临床转化研究中，以新鲜冰冻血浆（Fresh Frozen Plasma, FFP）作为补体来源对患者血浆进行体外补充，可完全恢复补体的整体功能。本研究描述了补体C2与C8B的联合杂合遗传变异，该变异无法解释患者整体的功能异常，因此呼吁开展更多补体缺陷相关研究与对应治疗方案的开发。