Journal of Clinical Immunology (2020) 40:515–523 https://doi.org/10.1007/s10875-020-00766-x ORIGINAL ARTICLE Complement Activation in 22q11.2 Deletion Syndrome Dina Grinde1 & Torstein Øverland2 & Kari Lima2,3 & Camilla Schjalm4 & Tom Eirik Mollnes4,5,6 & Tore G. Abrahamsen7,8 Received: 9 July 2019 /Accepted: 19 February 2020 /Published online: 9 March 2020 # The Author(s) 2020 Abstract The 22q11.2 deletion syndrome (22q11.2 del), also known as DiGeorge syndrome, is a genetic disorder with an estimated incidence of 1:3000 to 1:6000 births. These patients may suffer from affection of many organ systems with cardiac malformations, immuno- deficiency, hypoparathyroidism, autoimmunity, palate anomalies, and psychiatric disorders being the most frequent. The importance of the complement system in 22q11.2 del has not been investigated. The objective of this study was to evaluate the complement system in relation to clinical and immunological parameters in patients. A national cohort of patients (n = 69) with a proven heterozygous deletion of chromosome 22q11.2 and a group of age and sex matched controls (n = 56) were studied. Functional capacity of the classical, lectin, and alternative pathways of the complement system as well as complement activation products C3bc and terminal complement complex (TCC) were accessed and correlated to clinical features. All patients in our study had normal complement activation in both classical and alternative pathways. The frequency of mannose-binding lectin deficiency was com- parable to the normal population. The patients had significantly raised plasma levels of C3bc and a slight, but not significant, increase in TCC compared with controls. This increase was associated with the presence of psychiatric disorders in patients. The present study shows no complement deficiencies in 22q11.2 deletion syndrome. On the contrary, there are signs of increased complement activation in these patients. Complement activation is particularly associated with the presence of psychiatric disorders. Keywords 22q11.2del . 22q11.2 deletion syndrome . DiGeorge syndrome . primary immunodeficiencies . complement Introduction In 22q11.2 del, most of the patients have a typical 3 megabase (Mb) deletion on the long arm of chromosome 22 The 22q11.2 deletion syndrome (22q11.2 del), also known as containing over 30 different genes. Approximately 8% of the DiGeorge or velocardiofacial syndrome, is the most common patients have a smaller, 1.5 Mb, deletion and 2% have atypical microdeletion syndrome in humans with an estimated preva- deletions. The inheritance of the syndrome is autosomal dom- lence from 1:3000 to 1: 6000 live births [1]. Due to clinical inant, but 90% of cases appear to be de novo [3, 4]. The phe- variability and heterogeneity, the occurrence of the disorder notype variation is large, and syndrome presentation includes may be underestimated [2]. The prevalence is also expected to immunodeficiency of variable severity, congenital heart de- rise due to improved patient survival [1]. fects, hypoparathyroidism, velopharyngeal insufficiency Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10875-020-00766-x) contains supplementary material, which is available to authorized users. * Dina Grinde 4 Department of Immunology, Oslo University Hospital and [email protected] University of Oslo, Oslo, Norway 5 Research Laboratory, Nordland Hospital, Bodø, and K.G. Jebsen 1 Department of Pediatric Research, Oslo University Hospital, TREC, University of Tromsø, Tromsø, Norway Oslo, Norway 6 Centre of Molecular Inflammation Research, Norwegian University 2 of Science and Technology, Trondheim, Norway Department of Pediatric Medicine, Oslo University Hospital, Oslo, Norway 7 Center for Rare Diseases, Division of Pediatric and Adolescent Medicine, Oslo University Hospital, Oslo, Norway 3 Department of Endocrinology, Akershus University Hospital, Lørenskog, Norway 8 Faculty of Medicine, University of Oslo, Oslo, Norway 516 J Clin Immunol (2020) 40:515–523 (VPI), hypothyroidism, autoimmunity, obesity, variable cogni- Hospital, during the time period 2011–2015. Patients with tive delays, behavioral abnormalities, and psychiatric illnesses atypical and known additional deletions were not included. [1, 4, 5]. Indeed, over 180 clinical signs and symptoms have The patients did not have any clinical apparent infection when been described [6]. However, there is no apparent correlation sampled. Fifty-six healthy individuals, consisting of health between the size of the deletion and the clinical phenotype. care workers and their families or friends, 26 males, median The complement system is an integral component of the age 10 years (IQR 5.3–19 years), were included as a control innate immunity and can be activated through the classical, group. They had no known infection, inflammation, allergic lectin, and alternative pathways. All three pathways are activat- disease, or other acute or chronic illness at the time of blood ed by different stimuli, and they converge on Complement 3 sampling. In some individuals, a restricted subset of tests was (C3), generating convertases that catalyze the conversion of C3 performed due to insufficient blood volume obtained. This is into its active fragments C3a and C3b [7]. This activation can indicated in the figure legends. be detected by different activation products of C3, including the The study was conducted according to the guidelines at our C3b, iC3b, and C3c products (C3bc) exposing the same hospital and was approved by the Regional Committee for neoepitope in all fragments [8, 9]. C3b in the alternative path- Research Ethics, reference number 2011/1741. Before inclu- way is the amplification step that leads to all downstream com- sion, written informed consent was obtained from the partic- plement events in the cascade, which ends with the formation ipants and/or their parents. of the terminal C5b-9 complement complex (TCC) [7]. C5b-9 can be inserted into a membrane as the membrane attack com- Blood Sampling plex and lyse bacteria and cells. It can be made in the fluid phase as soluble TCC and used as a marker indicating that Peripheral venous blood was drawn into tubes containing the terminal pathway has been activated to its very end [8]. EDTA. For plasma preparation, samples were stored on ice Complement activation is normally highly regulated. immediately after blood sampling and centrifuged at × 2000 However, a disturbed complement activation can contribute for 15 min at 4 °C. All samples were aliquoted, stored at − to the pathology of several diseases associated with 22q11.2 80 °C and thawed < 3 times. del. Thus, complement activation is seen in autoimmune and inflammatory diseases such as systemic lupus erytomatosus, Clinical Chemistry and Immunology antiphospholipid antibody syndrome, or ANCA-associated vasculities [7]. Activation of the complement system has also Routine analysis including leukocyte differential count, im- been linked to obesity [10]. Moreover, dysregulation of com- munoglobulins, hemoglobin, thrombocytes count, and C- plement has been demonstrated in both schizophrenia, bipolar reactive protein was performed at the Department of disorders, and autism spectrum disorders (ASD), as involve- Medical Biochemistry, Oslo University Hospital. ment of the complement system in both synapse elimination Lymphocyte subpopulation phenotyping was assayed at the and neuron migration in the developing brain has been recog- Department of Immunology, Oslo University Hospital. nized [11–17]. On the other hand, deficiency of complement components, including mannose-binding lectin (MBL), which Complement Analyses is a part of lectin pathway, might increase the overall suscep- tibility to infectious disease [18]. Assay of functional capacity of the classical, lectin, and alter- The aim of the present study was to investigate the role of native pathways in the complement system was assayed in the complement system in the pathology of 22q11.2 del by serum using Wielisa COMPL300 Total Complement studying the degree of complement activation in vivo and Functional Screen kit from Wieslab AB, Lund, Sweden [19]. measure the level of MBL. Classical pathway deficiency was defined as < 40% capacity. Lectin and alternative pathway deficiencies were defined as < 10% capacity. Materials and Methods Serum concentrations of MBL were quantified if lectin pathway deficiency was found using an MBL ELISA kit Patients (BIOPORTO Diagnostics A/S, Hellerup, Denmark) according to the manufactures instructions. Low MBL value is most Sixty-nine patients, 32 males, median age 9 years (IQR 3.5– frequently set at < 500 ng/mL, and complete deficiency < 15 years) from all over Norway with a proven heterozygous 100 ng/mL. deletion of chromosome 22 by fluorescent in situ hybridiza- In vivo complement activation was measured using activa- tion or multiplex ligation-dependent probe amplification, were tion products from C3 (C3bc) and the terminal pathway included in the study. They all attended the Pediatric (TCC) using ELISA assays described in detail previously Outpatient Clinic at Rikshospitalet, Oslo University [20]. The C3bc assay is based on a capture monoclonal J Clin Immunol (2020) 40:515–523 517 antibody (bH6) reacting with an epitope exposed in C3b, diagnosed with pernicious anemia and atrophic gastritis (pari- iC3b, and C3c. The TCC assay is based a capture monoclonal etal-cell and intrinsic factor autoantibodies positive). In addi- antibody (aE11) exposed