Compelling Evidence Linking CD40 Gene With Graves’ Disease in the Chinese Han Population

Mutations in CD40 have been widely reported to be risk factors for Graves’ disease (GD). The gene, along with its cognate ligand CD40L, may regulate pro-inflammatory and immune responses. Rs1883832, located at the -1 position of the Kozak sequence, is the most well-studied single nucleotide polymorphism (SNP) of CD40, and has been confirmed to predispose those with the alteration to GD, regardless of ethnicity. Our genome-wide association study (GWAS) indicated that several SNPs, including rs1883832 located within the vicinity of CD40 were associated with GD in the Han Chinese population. Aiming at identifying the most consequential SNP and its underlying pathogenic mechanism, we performed a two-stage refined study on 8,171 patients with GD and 7,906 controls, and found rs1883832 was the most significantly GD-associated SNP in the CD40 gene region (PCombined = 9.17×10-11, OR = 1.18). Through searching the cis-expression quantitative trait locus database and using quantitative RT-PCR, we further discovered that the rs1883832 genotype can influence CD40 gene transcription. Furthermore, we demonstrated that rs1883832 is a susceptibility locus for pTRAb+ GD patients. In conclusion, the current study provides robust evidence that rs1883832 can regulate CD40 gene expression and affect serum TRAb levels, which ultimately contributes to the development of GD.

Precision Medicine in Graves’ Disease: CD40 Gene Variants Predict Clinical Response to an Anti-CD40 Monoclonal Antibody

BackgroundCD40, a key co-stimulatory molecule expressed on antigen-presenting cells, is genetically associated with a number of autoimmune diseases including Graves’ disease (GD). Therefore, recent therapies targeting CD40 have been developed, including the anti-CD40 monoclonal antibody Iscalimab. In a recent pilot study, Iscalimab was shown to induce clinical remission in ~ 50% of GD patients, but the reason why only 50% of GD patients responded is not known. The aim of our study was to test the hypothesis that specific CD40 single nucleotide polymorphism (SNP) genotypes and haplotypes are associated with clinical response of GD patients to Iscalimab.MethodsWe extracted genomic DNA from the whole blood of 13 GD patients treated with Iscalimab, and genotyped seven CD40 single nucleotide polymorphisms (SNPs) associated with autoimmunity. Additionally, we analyzed CD40 mRNA expression levels in whole blood. The patients’ CD40 SNP genotypes and mRNA levels were tested for association with clinical response to Iscalimab.ResultsThree common haplotypes, designated haplotypes A, B, and C, were identified. Haplotypes B and C were associated with higher CD40 mRNA levels and clinical response to Iscalimab (i.e., patients achieving euthyroidism without need for additional medications), while haplotype A was associated with decreased CD40 mRNA levels and no response to Iscalimab.ConclusionOur data suggest that genetic polymorphisms in the CD40 gene drive its expression levels and response to Iscalimab. Polymorphisms associated with higher CD40 levels are also associated with clinical response to CD40-targeted therapies. These results set the stage to implementing precision medicine in the therapeutic approach to GD.

Association of CD40 Gene Polymorphisms With Systemic Lupus Erythematosus and Rheumatoid Arthritis in a Chinese Han Population

Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are complex autoimmune diseases. CD40 participates in inflammatory response, and promotes fibroblast proliferation, leading to occurrence and progression of SLE, RA. This study explores CD40 gene polymorphisms in SLE and RA patients from a Chinese Han population. Two hundred SLE patients, 340 RA patients, and 900 healthy controls were enrolled. Genomic DNA was extracted from peripheral blood, and six polymorphisms of CD40 gene (rs3765456, rs1569723, rs73115010, rs13040307, rs1883832, and rs4810485) were detected by KASP method. Frequencies of rs1569723 genotypes AA, AC, AA+AC were significantly higher in RA patients as compared to those in healthy controls (P = 0.049, P = 0.024, P = 0.022). Frequencies of genotypes CT, CC+CT of rs1883832, and GT, GG+GT of rs4810485 were significantly higher in RA patients as compared to those in healthy controls (P = 0.012, P = 0.018, P = 0.009, P = 0.015). RA patients carrying rs13040307 C allele and rs73115010 T allele showed increased number of swollen joints. Moreover, frequency of allele T of rs13040307 was lower in SLE patients with positive anti-dsDNA and hematuria as compared to that in patients without these parameters (P = 0.038, P = 0.045). There were increased frequencies of genotype TT, allele T for rs13040307 and lower frequencies of genotype TT, allele T for rs73115010 in lupus patients with myositis (all P<0.05). Interestingly, frequencies of rs1569723 A allele, rs4810485 T allele were higher in SLE patients with myositis, and frequencies of rs3765456 A allele, rs1883832 T allele were lower in SLE patients with myositis (All P<0.05). In conclusion, CD40 gene polymorphisms may associate with susceptibility to SLE and RA.

Some Common SNPs of the T-Cell Homeostasis-Related Genes Are Associated with Multiple Sclerosis, but Not with the Clinical Manifestations of the Disease, in the Polish Population

Purpose. Multiple sclerosis (MS) is an autoimmune disease, and genetic factors play an important role in its pathogenesis and progression. The aim of our study was to evaluate the frequencies of alleles and genetic variants of the T-cell homeostasis-related genes, in subjects with MS, as well as to investigate the association with MS clinical manifestations and disability. Methods. 94 subjects with MS and 160 healthy individuals have been genotyped for seven common single-nucleotide variants in IL-2RA, CTLA4, CD40, and PADI4 genes. The ages of onset, duration of the disease, and clinical condition of the MS subjects were analysed. We used the Chi2 test confirmed with Fisher’s exact test for statistical analysis. Results. The frequency of allele T and CT/TT genotypes (rs7093069) in the IL2RA gene, as well as the T allele and CT/TT genotypes in rs12722598, were significantly higher in the control group. The significant differences between studied groups we also found for the G allele and GG/GA genotypes of rs3087243 in CTLA4 gene, which were more common among the control group. The heterozygous genotype TC (rs1883832) of CD40 gene was more common in the control subjects, and the frequency of the alleles and genotypes in the rs1748033 of the PADI4 gene did not differ between the studied groups. Between the studied genotypes, we did not observe any significant differences in the age of onset and duration of disease, including sex stratification. Conclusion. Our results highlight the protective role of some of the T-cell homeostasis-related genetic variants in MS development, but not in its clinical manifestation.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice

Hemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α–interferon-γ–driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

Association of antibodies to benzo[a]pyrene, estradiol and progesterone with gene polymorphisms of cytokines in postmenopausal women

Previous studies reported some associations between IgA and IgG antibodies specific to benzo[a] pyrene (Bp), estradiol (Es) and progesterone (Pg), and breast cancer (BC) in postmenopausal women. Likewise, the individual ratios of these antibodies (IgA-Bp/IgA-Pg, IgG-Bp/IgG-Pg, IgG-Es/IgG-Pg, IgG-Es/IgG-Pg) were associated with BC. It was suggested that development of antibodies to chemical carcinogens and steroid hormones was determined by functional polymorphisms of cytokine genes. The purpose of this study was to identify the suggested associations of antibodies to Bp, Es, Pg, and their individual ratios to the following gene polymorphisms: IL1RN (rs4251961), IL1B (rs16944), IL6 (rs1800795, rs1800796, rs1554606), IL8 (rs4073), TNFA (rs1800629) and CD40 (rs6074022) detected in postmenopausal healthy women and BC patients.The serum IgA and IgG antibodies specific to Bp, Es and Pg were studied in 470 healthy women and 995 BC patients by non-competitive solid phase immunoassay. The conjugates of Bp, Es, Pg with bovine serum albumin were used as adsorbed antigen. The goat antibodies against human IgA or IgG conjugated with horseradish peroxidase were used for the detection of bound hapten-specific antibodies. Cytokine gene polymorphisms were analyzed by the real-time PCR.Associations between the studied antibodies and their ratios with the gene polymorphisms in IL1RN (rs4251961), IL6 (rs1800795), TNFA (rs1800629) and CD40 (rs6074022) were found in healthy women. Higher individual ratios of IgA-Bp/IgA-Pg (p = 0.0001), IgG-Bp/IgG-Pg (p < 0.0001), IgG-Es/IgG-Pg (p = 0.0003) were associated with the allele C gene IL1RN. The higher IgG-Es levels were more common in the persons with allele G gene IL6 (p = 0.007), and with C allele of CD40 gene (p = 0.005). The high IgA-Pg levels were associated with A allele gene of TNFA (p = 0.008). Associations of antibodies were found only with genes polymorphisms in CD40 (rs6074022) in BC patients. Higher IgG-Es levels were more common in persons with allele T gene CD40 (p = 0.007).In conclusion, we revealed the participation of cytokines in immune regulation of antibody genesis for environmental chemical carcinogens and endogenous steroid hormones in healthy women and BC patients. The future investigations of antibodies specific to Bp, Es and Pg combined with the analysis genes polymorphisms in cytokines will be useful for detection of the individual hormone-dependent cancer risks in humans.

Functional association of a CD40 gene single-nucleotide polymorphism with the pathogenesis of coronary heart disease

Aims Endothelial dysfunction is a major contributor to the pathogenesis of atherosclerosis. CD40–CD40 ligand interactions confer a pro-inflammatory phenotype to endothelial cells (ECs). Recently, a thymine to cytosine transition (−1T&gt;C) in the Kozak sequence of the CD40 gene (rs1883832) has been associated with coronary heart disease (CHD) in an Asian population. As there are no reports yet regarding its role in other ethnic groups, this study determines if the −1T&gt;C single-nucleotide polymorphism (SNP) could be a risk factor for CHD in Caucasians by performing an association study and elucidates its functional consequence in cultured ECs. Methods and results Molecular and biochemical techniques, cell adhesion assays were used for genotype-stratified human EC characterization. SNP distribution in Caucasians was examined in a hospital-based case–control CHD study and serum levels of soluble CD40 (sCD40) were quantified by ELISA. The SNP in the CD40 gene affected baseline CD40 protein abundance on ECs. There was a genotype-dependent difference in CD40-mediated expression of pro-inflammatory genes. Monocyte adhesion was highest on the surface of cells homozygous for the C allele. Homozygosity for the C allele was associated with significant 2.32-fold higher odds of developing CHD as compared to TT genotype carriers. sCD40 plasma levels were genotype-dependently elevated in CHD patients, indicating a possible prognostic value. Conclusion The C allele of the CD40 SNP provokes a pro-inflammatory EC phenotype, compensated by an enhanced CD40 shedding to neutralize excess CD40 ligand. Homozygosity for the C allele is the cause for a genetic susceptibility to atherosclerosis and its sequelae.