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Altered expression of the complement component gene is a known risk factor for schizophrenia. Further, predicted brain expression has also been associated with memory function highlighting that altered C4A expression in the brain may be relevant for cognitive and behavioral traits. We obtained genetic information and performance measures on seven cognitive tasks for up to 329 773 individuals from the UK Biobank, as well as brain imaging data for a subset of 33 003 participants. Direct genotypes for variants ( = 3213) within the major histocompatibility complex region were used to impute C4 structural variation, from which predicted expression of the and genes in human brain tissue were predicted. We investigated if predicted brain or expression were associated with cognitive performance and brain imaging measures using linear regression analyses. We identified significant negative associations between predicted expression and performance on select cognitive tests, and significant associations with MRI-based cortical thickness and surface area in select regions. Finally, we observed significant inconsistent partial mediation of the effects of predicted expression on cognitive performance, by specific brain structure measures. These results demonstrate that the risk locus is associated with the central endophenotypes of cognitive performance and brain morphology, even when considered independently of other genetic risk factors and in individuals without mental or neurological disorders.

Low copy numbers (CNs) of C4 genes are associated with systemic autoimmune disorders and affects autoantibody diversity and disease subgroups. The primary objective of this study was to characterize diversity of complement (C4) and C4-Human Endogenous Retrovirus (HERV) gene copy numbers in SLE. We also sought to assess the association of C4 and C4-HERV CNs with serum complement levels, autoantibodies, disease phenotypes and activity. Finally, we checked the association of C4 and HERV CNs with specific HLA alleles. Genomic DNA from 70 SLE and 90 healthy controls of south Indian Tamil origin were included. Demographic, clinical and serological data was collected in a predetermined proforma. CNs of C4A and C4B genes and the frequency of insertion of 6.4kb HERV within C4 gene (C4AL, C4BL) was determined using droplet digital polymerase chain reaction (ddPCR). A four digit high resolution HLA genotyping was done using next generation sequencing. In our cohort, the total C4 gene copies ranged from 2 to 6. Compared to controls, presence of two or less copies of C4A gene was associated with SLE risk (p = 0.005; OR = 2.79; 95% CI = 1.29–6.22). Higher frequency of HERV insertion in C4A than in C4B increases such risk (p = 0.000; OR = 12.67; 95% CI = 2.80-115.3). AL-AL-AL-BS genotype was significantly higher in controls than SLE (9%vs1%, p = 0.04; OR = 0.15, 95% CI = 0.00-0.16). Distribution of HLA alleles was not different in SLE compared to controls as well as in SLE subjects with ≤ 2 copies and > 2 copies of C4A, but HLA allele distribution was diverse in subjects with C4B ≤ 2 copies and > 2 copies. Finally, there was no correlation between the C4 and the C4-HERV diversity and complement levels, autoantibodies, disease phenotypes and activity. In conclusion, our data show that, low C4A copy number and higher insertion of HERV-K in C4A increases the risk for SLE. C4 and C4-HERV CNs did not correlate with serum complements, autoantibodies, disease phenotypes and activity in SLE. Further validation in a larger homogenous SLE cohort is needed.

BackgroundSystemic Lupus Erythematosus (SLE) is characterized by low levels of serum C3 and C4. Low complements in SLE may either be due to increased consumption or decreased synthesis, the exact mechanisms of which is unknown. C4 gene has copy number variations and exists as two functionally distinct but genetically homologous isoforms, C4A (0-8 copies) and C4B (0-3 copies). The genes are either long (L) or short (S) based on a 6.4kb Human Endogenous Retrovirus K (HERV-K) insertion. Low copy numbers of complement C4 genes in systemic autoimmune disorders and its link with autoantibodies, disease subgroups have been reported.ObjectivesTo characterize C4 and C4-HERV genetic diversity in SLE and its relationship with serum complement levels, autoantibodies, disease activity, clinical subgroups and HLA.MethodsGenomic DNA from 70 SLE and 90 controls were used to characterize the copy numbers of C4A and C4B genes and the frequency of HERV insertion (C4AL, C4BL) using droplet digital polymerase chain reaction. High resolution typing of 8 HLA genes was done using next generation sequencing. Chi-square test was used to compare the frequencies of C4, C4-HERV structural elements and HLA between groups. Correlation with complement levels, number of autoantibodies present, SLEDAI and clinical subgroups was done using Spearman’s nonparametric test.ResultsIn this cohort, total C4 gene copies ranged from 2-6 with two copies of C4A and C4B genes being frequent; there were no C4 null alleles, and a negative correlation was noted between C4A and C4B copy numbers (r=-0.3713; p<0.001; Figure 1). Compared to controls, presence of two or less copies of C4A gene was associated with SLE risk while C4B frequency was comparable between the groups. Significantly increased frequency of HERV insertion in C4A than in C4B gene was observed. Of the 34 different C4-HERV genotypes observed, 7 were at a frequency greater than 5%. Though the frequency of AL-AL-BL-BS genotype was overrepresented in SLE, the difference was not statistically significant. The AL-AL-AL-BS genotype was significantly higher in controls than SLE (9% vs 1%, p=0.04; Table 1). HLA-DPA1*02 and DPB1*13 were more frequent in subjects with C4B >2 copies than in those with C4B ≤2 copies (33% vs 61%; Pc=0.04 & 5% vs 28%; Pc=0.04). There was no correlation between the C4 as well as C4-HERV gene copy numbers and serum levels of complement, autoantibodies, SLEDAI and SLE clinical subgroups (Figure 1)ConclusionOur data show that, although the C4A and C4B gene diversity is different between cases and controls, it does not correlate with serum complement levels, autoantibodies, disease activity and clinical subgroups in SLE. Low C4A copy number and increased insertion of HERV in C4A may be a risk for SLE. Our findings in this small cohort need further validation in a larger homogenous SLE cohort.Table 1.C4 Genetic diversity in SLEC4A copy numbersSLE; n=70%HC; n=90%POR95% CI≤25680%5359%0.0052.791.29 to 6.22>21420%3741%0.0050.360.16 to 0.77C4B copy numbers≤25680%8190%NS0.440.16 to 1.20>21420%910%NS2.250.84 to 6.30SLE C4-HERVC4A; n=70%C4B; n=70%POR95% CI0 HERV insertion2319270.0000.080.01-0.361-3 HERV insertion689751730.00012.672.81-115.30C4 genotypesSLE; n=70%HC; n=90%P valueOR95% CIAL-AL-BL-BS1420%1213%NSAL-AL-AL-BS11%89%0.040.150.00 to 1.16AL-AL-AS-BL-BS811%89%NSAL-AL-AS-BL-BL11%78%NSAL-AL-BL46%78%NSAL-AL-BL-BL710%67%NSAL-AL-BS-BS710%67%NSAL-AL-BS57%44%NSC4, Complement C4; HERV, Human Endogenous Retro Virus; SLE, Systemic Lupus Erythematosus; HC, Healthy Controls; P<0.05 considered significantFigure 1.Association of C4 gene copy numbers with serum complements, autoantibodies & SLEDAIAcknowledgementsThis study was funded by “Centre Franco-Indien pour la Promotion de la Recherche Avancée (5103-1-Tamouza/Negi)” and CMM was a post-doctoral fellow of CEFIPRA 5103-1Disclosure of InterestsNone Declared.

Complement system plays an important role in the immune defense against pathogens; however, recent studies demonstrated an important role of complement subunits C1q, C4, and C3 in normal functions of the central nervous system (CNS) such as non-functional synapse elimination (synapse pruning), and during various neurologic pathologies. Humans have two forms of C4 protein encoded by C4A and C4B genes that share 99.5% homology, while mice have only one C4B gene that is functionally active in the complement cascade. Overexpression of the human C4A gene was shown to contribute to the development of schizophrenia by mediating extensive synapse pruning through the activation C1q-C4-C3 pathway, while C4B deficiency or low levels of C4B expression were shown to relate to the development of schizophrenia and autism spectrum disorders possibly via other mechanisms not related to synapse elimination. To investigate the potential role of C4B in neuronal functions not related to synapse pruning, we compared wildtype (WT) mice with C3- and C4B- deficient animals for their susceptibility to pentylenetetrazole (PTZ)- induced epileptic seizures. We found that C4B (but not C3)–deficient mice were highly susceptible to convulsant and subconvulsant doses of PTZ when compared to WT controls. Further gene expression analysis revealed that in contrast to WT or C3-deficient animals, C4B-deficient mice failed to upregulate expressions of multiple immediate early genes (IEGs) Egrs1-4, c-Fos, c-Jus, FosB, Npas4, and Nur77 during epileptic seizures. Moreover, C4B-deficient mice had low levels of baseline expression of Egr1 on mRNA and protein levels, which was correlated with the cognitive problems of these animals. C4-deficient animals also failed to upregulate several genes downstream of IEGs such as BDNF and pro-inflammatory cytokines IL-1β, IL-6, and TNF. Taken together, our study demonstrates a new role of C4B in the regulation of expression of IEGs and their downstream targets during CNS insults such as epileptic seizures.

The Major Histocompatibility Complex (MHC, 6p21) codes for traditional HLA and other host response related genes. The polymorphic HLA-DRB1 gene in MHC Class II has been associated with several complex diseases. In this study we focus on MHC haplotype structures in the Finnish population. We explore the variability of extended HLA-DRB1 haplotypes in relation to the other traditional HLA genes and a selected group of MHC class III genes. A total of 150 healthy Finnish individuals were included in the study. Subjects were genotyped for HLA alleles (HLA-A, -B, -DRB1, -DQB1, and -DPB1). The polymorphism of TNF, LTA, C4, BTNL2 and HLA-DRA genes was studied with 74 SNPs (single nucleotide polymorphism). The C4A and C4B gene copy numbers and a 2-bp silencing insertion at exon 29 in C4A gene were analysed with quantitative genomic realtime-PCR. The allele frequencies for each locus were calculated and haplotypes were constructed using both the traditional HLA alleles and SNP blocks. The most frequent Finnish A∼B∼DR -haplotype, uncommon in elsewhere in Europe, was A*03∼B*35∼DRB1*01∶01. The second most common haplotype was a common European ancestral haplotype AH 8.1 (A*01∼B*08∼DRB1*03∶01). Extended haplotypes containing HLA-B, TNF block, C4 and HLA-DPB1 strongly increased the number of HLA-DRB1 haplotypes showing variability in the extended HLA-DRB1 haplotype structures. On the contrary, BTNL2 block and HLA-DQB1 were more conserved showing linkage with the HLA-DRB1 alleles. We show that the use of HLA-DRB1 haplotypes rather than single HLA-DRB1 alleles is advantageous when studying the polymorphisms and LD patters of the MHC region. For disease association studies the HLA-DRB1 haplotypes with various MHC markers allows us to cluster haplotypes with functionally important gene variants such as C4 deficiency and cytokines TNF and LTA, and provides hypotheses for further assessment. Our study corroborates the importance of studying population-specific MHC haplotypes.

The major histocompatibility complex (MHC) on chromosome 6p21 is a key contributor to the genetic basis of systemic lupus erythematosus (SLE). Although SLE affects African Americans disproportionately compared to European Americans, there has been no comprehensive analysis of the MHC region in relationship to SLE in African Americans. We conducted a screening of the MHC region for 1,536 single nucleotide polymorphisms (SNPs) and the deletion of the C4A gene in a SLE case–control study (380 cases, 765 age-matched controls) nested within the prospective Black Women’s Health Study. We also genotyped 1,509 ancestral informative markers throughout the genome to estimate European ancestry to control for population stratification due to population admixture. The most strongly associated SNP with SLE was the rs9271366 (odds ratio, OR = 1.70, p  = 5.6 × 10 −5 ) near the HLA - DRB1 gene. Conditional haplotype analysis revealed three other SNPs, rs204890 (OR = 1.86, p  = 1.2 × 10 −4 ), rs2071349 (OR = 1.53, p  = 1.0 × 10 −3 ), and rs2844580 (OR = 1.43, p  = 1.3 × 10 −3 ), to be associated with SLE independent of the rs9271366 SNP. In univariate analysis, the OR for the C4A deletion was 1.38, p  = 0.075, but after simultaneous adjustment for the other four SNPs the odds ratio was 1.01, p  = 0.98. A genotype score combining the four newly identified SNPs showed an additive risk according to the number of high-risk alleles (OR = 1.67 per high-risk allele, p  < 0.0001). Our strongest signal, the rs9271366 SNP, was also associated with higher risk of SLE in a previous Chinese genome-wide association study (GWAS). In addition, two SNPs found in a GWAS of European ancestry women were confirmed in our study, indicating that African Americans share some genetic risk factors for SLE with European and Chinese subjects. In summary, we found four independent signals in the MHC region associated with risk of SLE in African American women.

Genetic variants resulting in non-expression of complement C4A and C4B genes are common in healthy European populations and have shown association with a number of diseases, most notably the autoimmune disease, systemic lupus erythematosus. The most frequent cause of a C4 \"null\" allele, following that of C4 gene copy number variation (CNV), is a non-sense mutation arising from a 2 bp CT insertion into codon 1232 of exon 29. Previous attempts to accurately genotype this polymorphism have not been amenable to high-throughput typing, and have been confounded by failure to account for CNV at this locus, as well as by inability to distinguish between paralogs. We have developed a novel, high-throughput, paralog-specific assay to detect the presence and copy number of this polymorphism. We have genotyped healthy cohorts from the United Kingdom (UK) and Spain. Overall, 30/719 (4.17%) individuals from the UK cohort and 8/449 (1.78%) individuals from the Spanish cohort harboured the CT insertion in a C4A gene. A single Spanish individual possessed a C4B CT insertion. There is weak correlation between the C4 CT insertion and flanking MHC polymorphism. Therefore it is important to note that, as with C4 gene CNV, disease-association due to this variant will be missed by current SNP-based genome-wide association strategies.

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.

Polymorphisms in genes in the human leukocyte antigen (HLA) class II region comprise the most important inherited risk factors for many autoimmune diseases, including type 1 diabetes (T1D) and celiac disease (CD): both diseases are positively associated with the HLA-DR3 haplotype ( DRB1*03:01-DQA1*05:01-DQB1*02:01 ). Studies of two different populations have recently documented that T1D susceptibility in HLA-DR3 homozygous individuals is stratified by a haplotype consisting of three single nucleotide polymorphisms (‘tri-SNP’) in intron 1 of the HLA-DRA gene. In this study, we use a large cohort from the longitudinal ‘The Environmental Determinants of Diabetes in the Young’ (TEDDY) study to further refine the tri-SNP association with T1D and with autoantibody-defined T1D endotypes. We found that the tri-SNP association is primarily in subjects whose first-appearing T1D autoantibody is to insulin. In addition, we discovered that the tri-SNP is also associated with CD, and that the particular tri-SNP haplotype (‘101’) that is negatively associated with T1D risk is positively associated with risk for CD. The opposite effect of the tri-SNP haplotype on two DR3-associated diseases can enhance and refine current models of disease prediction based on genetic risk. Finally, we investigated possible functional differences between the individuals carrying high and low-risk tri-SNP haplotypes and found that differences in complement system genes C4A and C4B may underlie the observed divergence in disease risk.

Complement C4, a key molecule in the complement system that is one of chief constituents of innate immunity for immediate recognition and elimination of invading microbes, plays an essential role for the functions of both classical (CP) and lectin (LP) complement pathways. Complement C4 is the most polymorphic protein in complement system. A plethora of research data demonstrated that individuals with C4 deficiency are prone to microbial infections and autoimmune disorders. In this review, we will discuss the diversity of complement C4 proteins and its genetic structures. In addition, the current development of the regulation of complement C4 activation and its activation derivatives will be reviewed. Moreover, the review will provide the updates on the molecule interactions of complement C4 under the circumstances of bacterial and viral infections, as well as autoimmune diseases. Lastly, more evidence will be presented to support the paradigm that links microbial infections and autoimmune disorders under the condition of the deficiency of complement C4. We provide such an updated overview that would shed light on current research of complement C4. The newly identified targets of molecular interaction will not only lead to novel hypotheses on the study of complement C4 but also assist to propose new strategies for targeting microbial infections, as well as autoimmune disorders.