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.

Natural killer cells and type II interferon in Ro/SSA and La/SSB autoantibody-exposed newborns at risk of congenital heart block.

OBJECTIVE: Congenital heart block (CHB) with immune cell infiltration develops in the fetus after exposure to maternal Ro/La autoantibodies. CHB-related serology has been extensively studied, but reports on immune-cell profiles of anti-Ro/La-exposed neonates are lacking. In the current study, we characterised circulating immune-cell populations in anti-Ro/La+mothers and newborns, and explored potential downstream effects of skewed neonatal cell populations. METHODS: In total, blood from mothers (n=43) and neonates (n=66) was sampled at birth from anti-Ro/La+ (n=36) and control (n=30) pregnancies with or without rheumatic disease and CHB. Flow cytometry, microarrays and ELISA were used for characterising cells and plasma. RESULTS: Similar to non-pregnant systemic lupus erythematosus and Sjögren-patients, anti-Ro/La+mothers had altered B-cell subset frequencies, relative T-cell lymphopenia and lower natural killer (NK)-cell frequencies. Surprisingly, their anti-Ro/La exposed neonates presented higher frequencies of CD56dimCD16hi NK cells (p<0.01), but no other cell frequency differences compared with controls. Type I and II interferon (IFN) gene-signatures were revealed in neonates of anti-Ro/La+ pregnancy, and exposure of fetal cardiomyocytes to type I IFN induced upregulation of several NK-cell chemoattractants and activating ligands. Intracellular flow cytometry revealed IFNγ production by NK cells, CD8+ and CD4+ T cells in anti-Ro/La exposed neonates. IFNγ was also detectable in their plasma. CONCLUSION: Our study demonstrates an increased frequency of NK cells in anti-Ro/La exposed neonates, footprints of type I and II IFN and an upregulation of ligands activating NK cells in fetal cardiac cells after type I IFN exposure. These novel observations demonstrate innate immune activation in neonates of anti-Ro/La+pregnancy, which could contribute to the risk of CHB.

Detection of IgG anti-Domain I beta2 Glycoprotein I antibodies by chemiluminescence immunoassay in primary antiphospholipid syndrome.

BACKGROUND: IgG anti-Domain I (anti-DI) ß2 Glycoprotein I (ß2GPI) antibodies are associated to thrombotic risk in antiphospholipid syndrome (APS), but their detection is technically difficult. In this study, a chemiluminescent immunoassay (CLIA) was used to evaluate the clinical significance of IgG anti-DI in a large cohort of patients with primary APS (PAPS). METHODS: The study population included 88 patients with PAPS, 63 ELISA-negative subjects and 166 controls. IgG anti-DI, IgG anticardiolipin (aCL) and IgG anti-ß2GPI antibodies were assayed using CLIA (HemosIL AcuStar®). RESULTS: The sensitivity and specificity of IgG anti-DI antibodies were comparable to those of IgG aCL and IgG anti-ß2GPI antibodies. There was a significant agreement, association and titre correlation between IgG anti-DI and IgG aCL as well as IgG anti-ß2GPI antibodies (p<0.001 for all). IgG anti-DI antibody showed lesser prevalence and mean titres in the pregnancy morbidity than in thrombotic and PAPS patients with both involvements (p<0.001). Regarding the conventional aPL antibody profiles, the triple positivity group had higher prevalence and mean titres than single and double positivity ones (p<0.001). CONCLUSIONS: This study provides further evidence that anti-DI antibodies can be considered a promising biomarker for risk assessment particularly in patients having vascular thrombosis and triple conventional aPL positivity.

A contribution to detection of anticardiolipin and anti-ß2glycoprotein I antibodies: Comparison between a home-made ELISA and a fluorescence enzyme immunoassay.

BACKGROUND: Currently, ELISA for detection of anticardiolipin (aCL) and anti-ß2glycoprotein I (anti-ß2GPI) antibodies is not standardized. Recently, few studies have compared the performance of ELISA with that of fluorescence enzyme immunoassay (FEIA), but they have produced debatable results. The aim of this investigation was to compare ELISA with FEIA results in detecting aCL and anti-ß2GPI antibodies. METHODS: The study cohort included 94 primary antiphospholipid syndrome (PAPS) patients, 65 subjects with the clinical criteria for PAPS classification but ELISA negative for the laboratory criteria and 165 control subjects. Serum IgG/IgM aCL/anti-ß2GPI antibodies were determined using FEIA-EliA™ and a home-made ELISA. RESULTS: The sensitivities of the two methods were similar with the exception of IgM aCL which was found to be significantly higher in the PAPS patients using the ELISA method, even if IgM aCL was detected at a low level by both techniques. The two assays had a comparable specificity, a high/significant agreement and a significant correlation between the antibody levels. FEIA testing uncovered no significant prevalence of any antiphospholipid (aPL) antibody in the ELISA negative patients. CONCLUSION: Our results suggest that FEIA is comparable to a home-made ELISA.

The clinical performance of a chemiluminescent immunoassay in detecting anti-cardiolipin and anti-ß2 glycoprotein I antibodies. A comparison with a homemade ELISA method.

BACKGROUND: Fully automated chemiluminescence immunoassays (CLIAs) are emerging technologies for the detection of anti-cardiolipin (aCL) and anti-ß2 glycoprotein I (anti-ß2GPI) antibodies for anti-phospholipid syndrome (APS) classification, which is commonly based on an enzyme-linked immunosorbent assay (ELISA) test result. CLIA and a homemade ELISA were used in this study to detect these antibodies, and their performances were compared. METHODS: Sera were collected from 104 patients with primary APS, 88 seronegative subjects who met the clinical but not the laboratory criteria for APS, and 150 control subjects. IgG/IgM aCL and IgG/IgM anti-ß2GPI antibodies were determined in the sera using a CLIA (HemosIL AcuStar®) and a homemade ELISA. RESULTS: CLIA had a significantly lower comparative sensitivity for IgM aCL and IgG/IgM IgG anti-ß2GPI antibodies; its comparative specificity was higher with respect to ELISA for IgM aCL and IgM anti-ß2GPI antibodies. The two techniques showed a high, significant agreement (p<0.001) and a significant titer correlation (p<0.001). CLIA also detected IgG/IgM aCL and IgG anti-ß2GPI antibodies in the seronegative patients. There was a significantly higher prevalence of IgG aCL and IgG anti-ß2GPI antibodies (p<0.001 and p=0.01, respectively) in those patients with respect to that in the control population. CONCLUSIONS: Despite a lower comparative sensitivity, CLIA showed a higher comparative specificity for some aPL and a good level of agreement and correlation with a homemade ELISA. CLIA also detected some aCL and anti-ß2GPI antibodies in the seronegative patients not usually identified by homemade ELISA.

Relationship between antiphosphatidylserine/prothrombin and conventional antiphospholipid antibodies in primary antiphospholipid syndrome.

BACKGROUND: Antiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are emerging as an important marker for antiphospholipid syndrome (APS). We aimed to compare their performance with that of conventional antiphospholipid antibodies (aPL) such as lupus anticoagulant (LA), anticardiolipin (aCL), and anti-ß2-glycoprotein I (anti-ß2GPI) in APS and to assess their frequency in APS-negative (APS-ne) patients. METHODS: We considered 160 APS patients and 128 APS-ne patients with clinical criteria for APS but tested negative for conventional aPL. Immunoglobulin (Ig)G/IgM aPS/PT, IgG/IgM aCL, and IgG/IgM anti-ß2GPI were detected using ELISA assay and LA with a series of coagulation tests. RESULTS: IgG aPS/PT were significantly associated with IgG aCL, IgG anti-ß2GPI, and LA (p<0.0001 for all). IgM aPS/PT were significantly associated only with LA (p<0.0001) instead. There was a significant correlation between IgG aPS/PT and both IgG aCL and IgG anti-ß2GPI levels (ρ=0.42 and ρ=0.40, respectively). Both IgG aPS/PT and IgM aPS/PT positivity significantly correlated with LA (ρ=0.44 and ρ=0.5, respectively). IgG and IgM aPS/PT were significantly more frequent in triple than in double and in single positivity (p<0.0001). According to multivariate analysis, IgG and/or IgM aPS/PT were independent risk factors for LA. APS/PT antibodies were found in 9.4% of the APS-ne patients vs. 2% of healthy control (p=0.043); those antibodies were significantly more frequent in the thrombosis with respect to the pregnancy morbidity subset (p=0.01). CONCLUSIONS: Our data attribute a clinical relevance to both IgG and IgM aPS/PT antibodies. In particular, the significant prevalence of aPS/PT in APS-ne patients suggests including them as additional laboratory criterion for APS.

IgA anticardiolipin and IgA anti-ß2 glycoprotein I antibody positivity determined by fluorescence enzyme immunoassay in primary antiphospholipid syndrome.

BACKGROUND: Primary antiphospholipid syndrome (PAPS) is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity as well as blood antiphospholipid (aPL) antibodies such as anticardiolipin (aCL), anti-ß2 glycoprotein I (anti-ß2GPI) antibodies of the IgG/IgM isotype and lupus anticoagulant (LA). The clinical significance of aCL and anti-ß2GPI antibodies of the IgA isotype in PAPS is still a controversial issue. METHODS: Sera and plasma were collected from 84 PAPS patients (54 with thrombosis and/or pregnancy morbidity and 30 with pregnancy morbidity alone), 66 seronegative patients (subjects with clinical manifestations of PAPS although with negative results on conventional antiphospholipid antibody testing), and 78 healthy blood donors. IgA aCL and IgA anti-ß2GPI were determined using fluorescence enzyme immunoassay (FEIA), (EliATM, Phadia AB, Uppsala, Sweden). For comparison purposes, the sera were also tested for IgG/IgM aCL/anti-ß2GPI antibodies using the same immunoassay method. LA was assayed following internationally accepted guidelines. RESULTS: Present respectively in 19% and 50% of the PAPS patients studied, IgA aCL and IgA anti-ß2GPI antibody frequencies were both statistically significant (p=0.001 and p<0.001, respectively). The mean titers of both IgA aCL and IgA anti-ß2GPI antibodies were higher in the thrombotic patients, but only the latter were significantly associated with thrombosis. Isolated IgA anti-ß2GPI antibody positivity was significantly prevalent (p=0.04) in seven (10.6%) of the seronegative patients. CONCLUSIONS: Positivity to IgA anti-ß2GPI antibody detected using FEIA was found to be clinically relevant in PAPS patients. Moreover the prevalence of isolated IgA anti-ß2GPI antibody positivity was significant in the seronegative patients.