Risk factors for autoimmune-mediated congenital heart block

Placental transfer of maternal Ro/SSA and La/SSB autoantibodies during pregnancy is associated with conduction disturbances and inflammation in the developing fetal heart, termed autoimmune-mediated congenital heart block (CHB). Maternal Ro/SSA and La/SSB autoantibodies are the main risk factors associated with the fetal cardiac manifestations to date, however, the low recurrence rate despite persisting autoantibodies in subsequent pregnancies indicates that additional factors determine fetal susceptibility. The complex interactions between fetal genetic variants and factors that influence the intrauterine environment are thought to trigger or prevent the onset of CHB in Ro/SSA and/or La/SSB exposed pregnancies. The identification of such variants and factors was the main aim of this thesis. Genome-wide SNP association studies in families with at least one child affected by CHB and an anti-Ro52/SSA positive mother identified distinct cellular pathways associated with CHB. Exploration of potential candidate genes in the CHB-associated regions identified auxilin as a novel fetal susceptibility gene affecting cardiac excitation-contraction coupling. Discovery of additional CHB-associated variants affecting genes involved in vesicular or transmembrane transport and cardiac function further supported the idea that genetic variants in pathways connected to cardiac conduction and contractility may influence fetal susceptibility to disease. Furthermore, CHB-associated variants affecting genes with function assigned to immune responses emerged from our association studies and are likely to contribute to the inflammatory and tissue destructive processes connected with CHB. Ro/SSA autoantibodies are associated with interferon activation, and we found that cardiomyocyte expression of CHB-associated genes is affected by interferon-alpha stimulation. PBMCs from neonates with CHB and exposed to Ro/SSA autoantibodies in utero also displayed differential expression of several CHB-associated genes. Interestingly, expression of auxilin was altered in cardiomyocytes and PBMCs, validating the relevance of this particular gene and its pathway in CHB pathogenesis. We further identified and confirmed distinct class I and II HLA allele associations with CHB implementing potential impact for disease. Among the factors that may influence the intrauterine environment, we found that seasonal timing of pregnancy, infections, outdoor activity and psychological stress associated with the risk for CHB in Ro/SSA positive pregnancies. Finally, we also investigated potential cross-targets for the maternal anti-Ro52/p200 antibodies, and fetal intrauterine exposure to these maternal autoantibody specificities may further influence clinical outcomes of CHB. In summary, our data expands the current understanding of CHB pathogenesis, and suggests that the overall fetal susceptibility to CHB and degree of disease severity depends on a combination of genetic risk variants, their overall functional consequences, and their interactions with intrauterine factors in addition to the effect of fetal exposure to maternal Ro/SSA autoantibodies

Auxilin is a novel susceptibility gene for congenital heart block which directly impacts fetal heart function

Objective: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%–16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function. Methods: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography. Results: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals. Conclusions: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB.publishedVersio

H1N1 vaccination in Sjögren’s syndrome triggers polyclonal B cell activation and promotes autoantibody production

Objectives: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren’s syndrome (pSS) to an H1N1 influenza vaccine. Methods: Patients with Sjögren’s syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. Results: Surprisingly, treatment-naïve patients with Sjögren’s syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. Conclusions: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren’s syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients

European families reveal MHC class I and II associations with autoimmune-mediated congenital heart block

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