Dissecting the impact of complement component 4A in bipolar disorder.

The genetic overlap between schizophrenia (SZ) and bipolar disorder (BD) is substantial. Polygenic risk scores have been shown to dissect different symptom dimensions within and across these two disorders. Here, we focused on the most strongly associated SZ risk locus located in the extended MHC region, which is largely explained by copy numbers of the gene coding for complement component 4A (C4A). First, we utilized existing brain tissue collections (N = 1,202 samples) and observed no altered C4A expression in BD samples. The generated C4A seeded co-expression networks displayed no genetic enrichment for BD. To study if genetically predicted C4A expression discriminates between subphenotypes of BD, we applied C4A expression scores to symptom dimensions in a total of 4,739 BD cases with deep phenotypic data. We identified a significant association between C4A expression and psychotic mood episodes in BD type 1 (BDI). No significant association was observed between C4A expression and the occurrence of non-affective psychotic episodes in BDI, the psychosis dimensions in the total BD sample, or any other subphenotype of BD. Overall, these results points to a distinct role of C4A in BD that is restricted to vulnerability for developing psychotic symptoms during mood episodes in BDI.

Evaluation of C4 Gene Copy Number in Pediatric Acute Neuropsychiatric Syndrome.

Pediatric acute-onset neuropsychiatric syndrome (PANS) is an abrupt-onset neuropsychiatric disorder. PANS patients have an increased prevalence of comorbid autoimmune illness, most commonly arthritis. In addition, an estimated one-third of PANS patients present with low serum C4 protein, suggesting decreased production or increased consumption of C4 protein. To test the possibility that copy number (CN) variation contributes to risk of PANS illness, we compared mean total C4A and total C4B CN in ethnically matched subjects from PANS DNA samples and controls (192 cases and 182 controls). Longitudinal data from the Stanford PANS cohort (n = 121) were used to assess whether the time to juvenile idiopathic arthritis (JIA) or autoimmune disease (AI) onset was a function of total C4A or C4B CN. Lastly, we performed several hypothesis-generating analyses to explore the correlation between individual C4 gene variants, sex, specific genotypes, and age of PANS onset. Although the mean total C4A or C4B CN did not differ in PANS compared to controls, PANS patients with low C4B CN were at increased risk for subsequent JIA diagnosis (hazard ratio = 2.7, p value = 0.004). We also observed a possible increase in risk for AI in PANS patients and a possible correlation between lower C4B and PANS age of onset. An association between rheumatoid arthritis and low C4B CN has been reported previously. However, patients with PANS develop different types of JIA: enthesitis-related arthritis, spondyloarthritis, and psoriatic arthritis. This suggests that C4B plays a role that spans these arthritis types.

The complement system and human autoimmune diseases.

Genetic deficiencies of early components of the classical complement activation pathway (especially C1q, r, s, and C4) are the strongest monogenic causal factors for the prototypic autoimmune disease systemic lupus erythematosus (SLE), but their prevalence is extremely rare. In contrast, isotype genetic deficiency of C4A and acquired deficiency of C1q by autoantibodies are frequent among patients with SLE. Here we review the genetic basis of complement deficiencies in autoimmune disease, discuss the complex genetic diversity seen in complement C4 and its association with autoimmune disease, provide guidance as to when clinicians should suspect and test for complement deficiencies, and outline the current understanding of the mechanisms relating complement deficiencies to autoimmunity. We focus primarily on SLE, as the role of complement in SLE is well-established, but will also discuss other informative diseases such as inflammatory arthritis and myositis.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies.

BACKGROUND: Idiopathic 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: C4A 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.

Cerebrospinal fluid concentration of complement component 4A is increased in first episode schizophrenia.

Postsynaptic density is reduced in schizophrenia, and risk variants increasing complement component 4A (C4A) gene expression are linked to excessive synapse elimination. In two independent cohorts, we show that cerebrospinal fluid (CSF) C4A concentration is elevated in patients with first-episode psychosis (FEP) who develop schizophrenia (FEP-SCZ: median 0.41 fmol/ul [CI = 0.34-0.45], FEP-non-SCZ: median 0.29 fmol/ul [CI = 0.22-0.35], healthy controls: median 0.28 [CI = 0.24-0.33]). We show that the CSF elevation of C4A in FEP-SCZ exceeds what can be expected from genetic risk variance in the C4 locus, and in patient-derived cellular models we identify a mechanism dependent on the disease-associated cytokines interleukin (IL)-1beta and IL-6 to selectively increase neuronal C4A mRNA expression. In patient-derived CSF, we confirm that IL-1beta correlates with C4A controlled for genetically predicted C4A RNA expression (r = 0.39; CI: 0.01-0.68). These results suggest a role of C4A in early schizophrenia pathophysiology.

The plasma level of complement component 4A decreases with aripiprazole treatment in patients with early psychosis.

The complement component 4 (C4) gene has been reported to be significantly associated with schizophrenia, and C4A RNA expression was found to increase in postmortem brains of schizophrenia patients. This study aimed to examine the plasma levels of C4A and C4B proteins in patients with early psychosis and their changes following aripiprazole treatment. We recruited 45 patients, including 17 patients with ultra-high-risk and 28 patients with first-episode psychosis, and 45 age-matched and sex-matched controls. All patients received aripiprazole treatment for 4 weeks. Each patient received symptom evaluation before and after the treatment period. We measured the plasma levels of C4A and C4B in the pretreatment and posttreatment stages of patients and controls using an enzyme-linked immunosorbent assay. We found no significant differences in C4A and C4B levels between patients and controls, but the C4A level decreased significantly with aripiprazole treatment. Multivariate analysis showed that the decrease rate of C4A was significantly associated with the treatment response of the positive symptom dimension. In summary, we found that the plasma level of C4A decreased with aripiprazole treatment, and the decrease rate was associated with the treatment response of the positive dimension in patients with early psychosis. This mechanism deserves further clarification.

Deregulation of complement components C4A and CSMD1 peripheral expression in first-episode psychosis and links to cognitive ability.

Up-regulation of the complement component 4A (C4A) in the brain has been associated with excessive synaptic pruning and increased schizophrenia (SZ) susceptibility. Over-expression of C4A has been observed in SZ postmortem brain tissue, and the gene encoding for a protein inhibitor of C4A activity, CUB and Sushi multiple domains 1 (CSMD1) gene, has been implicated in SZ risk and cognitive ability. Herein, we examined C4A and CSMD1 mRNA expression in peripheral blood from antipsychotic-naive individuals with first-episode psychosis (FEP; n = 73) and mentally healthy volunteers (n = 48). Imputed C4 locus structural alleles and C4A serum protein levels were investigated. Associations with symptom severity and cognitive domains performance were explored. A significant decrease in CSMD1 expression levels was noted among FEP patients compared to healthy volunteers, further indicating a positive correlation between C4A and CSMD1 mRNA levels in healthy volunteers but not in FEP cases. In addition, C4 copy number variants previously associated with SZ risk correlated with higher C4A mRNA levels in FEP cases, which confirms the regulatory effect of C4 structural variants on gene expression. Evidence also emerged for markedly elevated C4A serum concentrations in FEP cases. Within the FEP patient group, higher C4A mRNA levels correlated with more severe general psychopathology symptoms and lower CSMD1 mRNA levels predicted worse working memory performance. Overall, these findings suggest C4A complement pathway perturbations in individuals with FEP and corroborate the involvement of CSMD1 in prefrontal-mediated cognitive functioning.

Human Complement C4B Allotypes and Deficiencies in Selected Cases With Autoimmune Diseases.

Human complement C4 is one of the most diverse but heritable effectors for humoral immunity. To help understand the roles of C4 in the defense and pathogenesis of autoimmune and inflammatory diseases, we determined the bases of polymorphisms including the frequent genetic deficiency of C4A and/or C4B isotypes. We demonstrated the diversities of C4A and C4B proteins and their gene copy number variations (CNVs) in healthy subjects and patients with autoimmune disease, such as type 1 diabetes, systemic lupus erythematosus (SLE) and encephalitis. We identified subjects with (a) the fastest migrating C4B allotype, B7, or (b) a deficiency of C4B protein caused by genetic mutation in addition to gene copy-number variation. Those variants and mutants were characterized, sequenced and specific techniques for detection developed. Novel findings were made in four case series. First, the amino acid sequence determinant for C4B7 was likely the R729Q variation at the anaphylatoxin-like region. Second, in healthy White subject MS630, a C-nucleotide deletion at codon-755 led to frameshift mutations in his single C4B gene, which was a private mutation. Third, in European family E94 with multiplex lupus-related mortality and low serum C4 levels, the culprit was a recurrent haplotype with HLA-A30, B18 and DR7 that segregated with two defective C4B genes and identical mutations at the donor splice site of intron-28. Fourth, in East-Asian subject E133P with anti-NMDA receptor encephalitis, the C4B gene had a mutation that changed tryptophan-660 to a stop-codon (W660x), which was present in a haplotype with HLA-DRB1*04:06 and B*15:27. The W660x mutation is recurrent among East-Asians with a frequency of 1.5% but not detectable among patients with SLE. A meticulous annotation of C4 sequences revealed clusters of variations proximal to sites for protein processing, activation and inactivation, and binding of interacting molecules.

Increased activation product of complement 4 protein in plasma of individuals with schizophrenia.

Structural variation in the complement 4 gene (C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood-brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC (mean = 654 ± 16 ng/mL, 557 ± 94 respectively, p = 0.01). The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.

Variants at the MHC Region Associate With Susceptibility to Clostridioides difficile Infection: A Genome-Wide Association Study Using Comprehensive Electronic Health Records.

Background: Clostridioides difficile is a major cause of healthcare-associated and community-acquired diarrhea. Host genetic susceptibility to Clostridioides difficile infection has not been studied on a large-scale. Methods: A total of 1,160 Clostridioides difficile infection cases and 15,304 controls were identified by applying the eMERGE Clostridioides difficile infection algorithm to electronic health record data. A genome-wide association study was performed using a linear mixed model, adjusted for significant covariates in the full dataset and the antibiotic subgroup. Colocalization and MetaXcan were performed to identify potential target genes in Clostridioides difficile infection - relevant tissue types. Results: No significant genome-wide association was found in the meta-analyses of the full Clostridioides difficile infection dataset. One genome-wide significant variant, rs114751021, was identified (OR = 2.42; 95%CI = 1.84-3.11; p=4.50 x 10-8) at the major histocompatibility complex region associated with Clostridioides difficile infection in the antibiotic group. Colocalization and MetaXcan identified MICA, C4A/C4B, and NOTCH4 as potential target genes. Down-regulation of MICA, upregulation of C4A and NOTCH4 was associated with a higher risk for Clostridioides difficile infection. Conclusions: Leveraging the EHR and genetic data, genome-wide association, and fine-mapping techniques, this study identified variants and genes associated with Clostridioides difficile infection, provided insights into host immune mechanisms, and described the potential for novel treatment strategies for Clostridioides difficile infection. Future replication and functional validation are needed.

Complement C4 associations with altered microbial biomarkers exemplify gene-by-environment interactions in schizophrenia.

Schizophrenia is a complex brain disorder with genetic and environmental factors contributing to its etiology. Complement C4 genes are schizophrenia susceptibility loci and are activated in response to infections and gut microbiome imbalances. We hypothesize that C4 genetic susceptibility predisposes individuals to neuropathological effects from pathogen exposures or a microbiome in dysbiosis. In 214 individuals with schizophrenia and 123 non-psychiatric controls, we examined C4 gene copy number and haplotype groups for associations with schizophrenia and microbial plasma biomarkers. C4A copy number and haplotypes containing HERV-K insertions (C4A-long; C4AL-C4AL) conferred elevated odds ratios for schizophrenia diagnoses (OR 1.58-2.56, p < 0.0001), while C4B-short (C4BS) haplogroups conferred decreased odds (OR 0.43, p < 0.0001). Haplogroup-microbe combinations showed extensive associations with schizophrenia including C4AL with Candida albicans IgG (OR 2.16, p < 0.0005), C4AL-C4BL with cytomegalovirus (CMV) IgG (OR 1.79, p < 0.008), C4BS with lipopolysaccharide-binding protein (LBP) (OR 1.18, p < 0.0001), and C4AL-C4AL with Toxoplasma gondii IgG (OR = 17.67, p < 0.0001). In controls, only one haplogroup-microbe combination was significant: C4BS with CMV IgG (OR 0.52, p < 0.02). In schizophrenia only, LBP and CMV IgG levels were inversely correlated with C4A and C4S copy numbers, respectively (R2 = 0.13-0.16, p < 0.0001). C4 haplogroups were associated with altered scores of cognitive functioning in both cases and controls and with psychiatric symptom scores in schizophrenia. Our findings link complement C4 genes with a susceptibility to infections and a dysbiotic microbiome in schizophrenia. These results support immune system mechanisms by which gene-environmental interactions may be operative in schizophrenia.

Impact of Homozygous C4A Deficiency on Clinical Presentation of Systemic Lupus Erythematosus.

OBJECTIVE: To investigate the association of C4A null allele (C4AQ0) with systemic lupus erythematosus (SLE) and determine the clinical presentation of SLE in relation to C4A null allele. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Pathology (AFIP), Rawalpindi, Immunology Department, from December 2018 to December 2019. METHODOLOGY: Patients referred to AFIP, who fulfilled American College of Rheumatology (ACR) criteria of 1997 for diagnosis of SLE were included in the study. Approval from the Institutional Ethical Review Board was taken. C4A and C4B null alleles were determined in 66 SLE patients and 40 age- and gender-matched healthy controls by polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP). Various clinical features and laboratory findings in the SLE patients were analysed in relation with C4A null allele. RESULTS: The mean age of the study population was 30.56 ±10.08 years. C4A null allele was detected in 7 (10.6%) patients; whereas, C4B null allele was detected in only two (3%) patients. SLE patients with C4A null allele had increased incidence of arthritis (100%) and renal damage (85.7%); compared to those with normal C4A allele, 57.6% and 32%, respectively. Fisher's Exact test revealed strong association of C4A null allele with arthritis and renal damage, (p = 0.039 and 0.01, respectively). CONCLUSION: Homozygous absence of C4A alleles was encountered in 10.6% of Pakistani patients of SLE and is closely related with clinical features of arthritis and renal damage. Knowledge of C4A null allele in SLE patients at diagnosis can predict disease course. Key Words: SLE, C4A null alleles, C4AQ0, Homozygous C4A deficiency.

The complement system in schizophrenia: where are we now and what's next?

The complement system is a set of immune proteins involved in first-line defense against pathogens and removal of waste materials. Recent evidence has implicated the complement cascade in diseases involving the central nervous system, including schizophrenia. Here, we provide an up-to-date narrative review and critique of the literature on the relationship between schizophrenia and complement gene polymorphisms, gene expression, protein concentration, and pathway activity. A literature search identified 23 new studies since the first review on this topic in 2008. Overall complement pathway activity appears to be elevated in schizophrenia. Recent studies have identified complement component 4 (C4) and CUB and Sushi Multiple Domains 1 (CSMD1) as potential genetic markers of schizophrenia. In particular, there is some evidence of higher rates of C4B/C4S deficiency, reduced peripheral C4B concentration, and elevated brain C4A mRNA expression in schizophrenia patients compared to controls. To better elucidate the additive effects of multiple complement genotypes, we also conducted gene- and gene-set analysis through MAGMA which supported the role of Human Leukocyte Antigen class (HLA) III genes and, to a lesser extent, CSMD1 in schizophrenia; however, the HLA-schizophrenia association was likely driven by the C4 gene. Lastly, we identified several limitations of the literature on the complement system and schizophrenia, including: small sample sizes, inconsistent methodologies, limited measurements of neural concentrations of complement proteins, little exploration of the link between complement and schizophrenia phenotype, and lack of studies exploring schizophrenia treatment response. Overall, recent findings highlight complement components-in particular, C4 and CSMD1-as potential novel drug targets in schizophrenia. Given the growing availability of complement-targeted therapies, future clinical studies evaluating their efficacy in schizophrenia hold the potential to accelerate treatment advances.

Relationships of complement components C3 and C4 and their genetics to cardiometabolic risk in healthy, non-Hispanic white adolescents.

BACKGROUND: Complement promotes inflammatory and immune responses and may affect cardiometabolic risk. This study was designed to investigate the effect of complement components C3 and C4 on cardiometabolic risk in healthy non-Hispanic white adolescents. METHODS: Body mass index (BMI), BMI percentile, waist circumference, and percent body fat were assessed in 75 adolescents. Arterial stiffness was assessed using arterial tomography and endothelial function using reactive hyperemia. Fasting lipids, inflammatory markers, and complement levels were measured and oral glucose tolerance test was performed. A single C3 polymorphism and C4 gene copy number variations were assessed. RESULTS: C3 plasma levels increased with measures of obesity. Endothelial function worsened with increased C3 and C4 levels. Triglycerides and low-density lipoprotein increased and high-density lipoprotein (HDL) and insulin sensitivity decreased with increasing C3 levels, but the relationships were lost when body habitus was included in the model. C4 negatively related to HDL and positively to inflammatory markers. Subjects with at least one C3F allele had increased BMI and fat mass index. HDL was significantly related to C4L, C4S, C4A, and C4B gene copy number variation. CONCLUSIONS: C3 levels increase with increasing body mass and increased C4 levels and copy number are associated with increased cardiometabolic risk in healthy adolescents.

Impact of C4, C4A and C4B gene copy number variation in the susceptibility, phenotype and progression of systemic lupus erythematosus.

BACKGROUND: Complement component 4 (C4) gene copy number (GCN) affects the susceptibility to systemic lupus erythematosus (SLE) in different populations, however the possible phenotype significance remains to be determined. This study aimed to associate C4A, C4B and total C4 GCN and SLE, focusing on the clinical phenotype and disease progression. METHODS: C4, C4A and C4B GCN were determined by real-time PCR in 427 SLE patients and 301 healthy controls, which underwent a detailed clinical evaluation according to a pre-established protocol. RESULTS: The risk of developing SLE was 2.62 times higher in subjects with low total C4 GCN (< 4 copies, OR = 2.62, CI = 1.77 to 3.87, p < 0.001) and 3.59 times higher in subjects with low C4A GCN (< 2 copies; OR = 3.59, CI = 2.15 to 5.99, p < 0.001) compared to those subjects with normal or high GCN of total C4 (≥4) and C4A (≥2), respectively. An increased risk was also observed regarding low C4B GCN, albeit to a lesser degree (OR = 1.46, CI = 1.03 to 2.08, p = 0.03). Furthermore, subjects with low C4A GCN had higher permanent disease damage as assessed by the Systemic Lupus International Collaborating Clinics - Damage Index (SLICC-DI; median = 1.5, 95% CI = 1.2-1.9) than patients with normal or high copy number of C4A (median = 1.0, 95% CI = 0.8-1.1; p = 0.004). There was a negative association between low C4A GCN and serositis (p = 0.02) as well as between low C4B GCN and arthritis (p = 0.02). CONCLUSIONS: This study confirms the association between low C4 GCN and SLE susceptibility, and originally demonstrates an association between low C4A GCN and disease severity.

A gene co-expression network-based analysis of multiple brain tissues reveals novel genes and molecular pathways underlying major depression.

Major depression is a common and severe psychiatric disorder with a highly polygenic genetic architecture. Genome-wide association studies have successfully identified multiple independent genetic loci that harbour variants associated with major depression, but the exact causal genes and biological mechanisms are largely unknown. Tissue-specific network approaches may identify molecular mechanisms underlying major depression and provide a biological substrate for integrative analyses. We provide a framework for the identification of individual risk genes and gene co-expression networks using genome-wide association summary statistics and gene expression information across multiple human brain tissues and whole blood. We developed a novel gene-based method called eMAGMA that leverages tissue-specific eQTL information to identify 99 biologically plausible risk genes associated with major depression, of which 58 are novel. Among these novel associations is Complement Factor 4A (C4A), recently implicated in schizophrenia through its role in synaptic pruning during postnatal development. Major depression risk genes were enriched in gene co-expression modules in multiple brain tissues and the implicated gene modules contained genes involved in synaptic signalling, neuronal development, and cell transport pathways. Modules enriched with major depression signals were strongly preserved across brain tissues, but were weakly preserved in whole blood, highlighting the importance of using disease-relevant tissues in genetic studies of psychiatric traits. We identified tissue-specific genes and gene co-expression networks associated with major depression. Our novel analytical framework can be used to gain fundamental insights into the functioning of the nervous system in major depression and other brain-related traits.

Identification of the primate-specific gene BTN3A2 as an additional schizophrenia risk gene in the MHC loci.

BACKGROUND: Schizophrenia is a complex mental disorder resulting in poor life quality and high social and economic burden. Despite the fact that genome-wide association studies (GWASs) have successfully identified a number of risk loci for schizophrenia, identifying the causal genes at the risk loci and elucidating their roles in disease pathogenesis remain major challenges. METHODS: The summary data-based Mendelian randomization analysis (SMR) was used to integrate a large-scale GWAS of schizophrenia with brain expression quantitative trait loci (eQTL) data and brain methylation expression quantitative trait loci (meQTL) data, to identify novel risk gene(s) for schizophrenia. We then analyzed the mRNA expression and methylation statuses of the gene hit BTN3A2 during the early brain development. Electrophysiological analyses of CA1 pyramidal neurons were performed to evaluate the excitatory and inhibitory synaptic activity after overexpression of BTN3A2 in rat hippocampal slices. Cell surface binding assay was used to test the interaction of BTN3A2 and neurexins. FINDINGS: We identified BTN3A2 as a potential risk gene for schizophrenia. The mRNA expression and methylation data showed that BTN3A2 expression in human brain is highest post-natally. Further electrophysiological analyses of rat hippocampal slices showed that BTN3A2 overexpression specifically suppressed the excitatory synaptic activity onto CA1 pyramidal neurons, most likely through its interaction with the presynaptic adhesion molecule neurexins. INTERPRETATION: Increased expression of BTN3A2 might confer risk for schizophrenia by altering excitatory synaptic function. Our result constitutes a paradigm for distilling risk gene using an integrative analysis and functional characterization in the post-GWAS era. FUND: This study was supported by the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB02020003 to Y-GY), the National Natural Science Foundation of China (31730037 to Y-GY), and the Bureau of Frontier Sciences and Education, Chinese Academy of Sciences (QYZDJ-SSW-SMC005 to Y-GY).

Insights on the relationship between complement component C4 serum concentrations and C4 gene copy numbers in a Western Australian systemic lupus erythematosus cohort.

The relationship between serum concentration of complement C4 ([C4]) and C4 gene copy number (GCN) was investigated in 56 systemic lupus erythematosus (SLE) patients and 33 age and sex-matched controls in a Western Australian population. C4A and C4B gene copy numbers (C4A & B GCN) together with the presence or absence of the ≈6.4-kb human endogenous retroviral element type K (hereafter HERV-K) in intron 9 were estimated by two TaqMan™ real-time PCR (RT-PCR) assays that measured total C4 and HERV-K GCNs, respectively. There was good correlation between the two methods; however, the HERV-K GCN method showed a positive bias (≈6%) relative to the C4A & B total GCN. Despite individual variation, excellent correlation between total C4 GCN and mean [C4] per GCN was observed for both the SLE and control cohorts ( R2 = 88% and R2 = 99%, respectively). It was noted that serum [C4] was significantly lower in the SLE patients than the controls ( p = 0.006) despite there being no difference between C4A and C4B GCN in both cohorts. The data therefore confirm previous reports that the C4A genes are preferentially associated with the presence of the HERV-K insertion relative to C4B genes and does not support the hypothesis that low [C4] in SLE is explained by low C4A GCNs. There was no evidence also that the presence of the HERV-K insertion in C4 genes influenced [C4]. This study supports the view that low [C4] in SLE patients is due to consumption rather than deficient synthesis related to lower C4A & B GCN.

Neuropil contraction in relation to Complement C4 gene copy numbers in independent cohorts of adolescent-onset and young adult-onset schizophrenia patients-a pilot study.

A recent report suggested Complement 4 (C4A) gene copy numbers (GCN) as risk factors for schizophrenia. Rodent model showed association of C4 with synaptic pruning suggesting its pathophysiological significance (Sekar, A. et al. (2016)). We, therefore, predicted that C4A GCN would be positively correlated with neuropil contraction in the human brain among schizophrenia patients showing more prominent correlations in ventral regions among young adults and dorsal regions among adolescents since neuromaturation progresses dorsoventrally. Whole-brain, multi-voxel, in vivo phosphorus magnetic resonance spectroscopy (31P MRS) assessed neuropil changes by estimating levels of membrane phospholipid (MPL) precursors and catabolites. Increased MPL catabolites and/or decreased MPL precursors indexed neuropil contraction. Digital droplet PCR-based assay was used to estimate C4A and C4B GCN. We evaluated two independent cohorts (young adult-onset early-course schizophrenia (YASZ = 15) and adolescent-onset schizophrenia (AOSZ = 12) patients), and controls matched for each group, n = 22 and 15, respectively. Separate forward stepwise linear regression models with Akaike information Criterion were built for MPL catabolites and precursors. YASZ cohort: Consistent with the rodent model (Sekar, A. et al. 2016)), C4A GCN positively correlated with neuropil contraction (increased pruning/decreased formation) in the inferior frontal cortex and inferior parietal lobule. AOSZ cohort: C4A GCN positively correlated with neuropil contraction in the dorsolateral prefrontal cortex and thalamus. Exploratory analysis of C4B GCN showed positive correlation with neuropil contraction in the cerebellum and superior temporal gyrus among YASZ while AOSZ showed neuropil contraction in the prefrontal and subcortical structures. Thus, C4A and C4B GCN are associated with neuropil contraction in regions often associated with schizophrenia, and may be neuromaturationally dependent.

Clinical features of patients with homozygous complement C4A or C4B deficiency.

INTRODUCTION: Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. MATERIAL AND METHODS: Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. RESULTS: Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%CI = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%CI = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%CI = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%CI = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%CI = 1.22-4.88, p = 0.010). CONCLUSION: This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.