Cross-reactivity of anti-Ro52 antibodies and genetic susceptibility in congenital heart block

Introduction. Congenital heart block (CHB) is a passively acquired autoimmune disease associated with transplacental transfer of maternal anti-Ro/SSA and/or anti-La/SSB antibodies mediating inflammation and subsequent fibrosis of the atrio-ventricular node in the fetal heart. Despite the long-established association of maternal antibodies in directly inducing CHB both with in vitro and in vivo studies, the pathogenic mechanisms involved remain unclear and identification of cross-reactive targets of anti-Ro52 antibodies in the fetal heart is matter of debate. CHB occurs in 1–2% of anti-Ro/SSA antibody-positive pregnancies and has a recurrence rate of 12–20% in a subsequent pregnancy, suggesting that additional factors, such as genetic and environmental components may determine the outcome in terms of CHB development in autoantibody exposed fetuses. Objectives. This thesis is divided into two parts and investigates the pathogenic mechanism and the genetic association with CHB. The first study aims to identify cross-reactive targets of anti-Ro52/p200 antibodies by screening a library of peptides covering the whole human proteome and using monoclonal antibodies specific for the Ro52/p200 region and to confirm the reactivity at peptide level and on the whole antigen level. The second study aims to identify fetal susceptibility genes among the Human Leucocyte Antigen (HLA) locus in anti-Ro/SSA autoantibody-mediated CHB from DNA samples collected from a multi-centric European cohort of families in which children affected by CHB were born. Part I. A whole proteome microarray revealed 17 peptides to be significantly cross-reactive with a Ro52/p200 monoclonal antibody (Ab31) and two linear motifs (“YSDF” and “YSNF) were shared among these sequences. Among the targets, cross-reactivity was studied in detail for TG and GAK, for which reactivity was showed both at the peptide and whole protein level with anti-p200 monoclonal antibodies and with sera from mothers whose children have CHB, suggesting that reactivity to these protein could represent a risk factor for development of CHB in the fetuses in anti-Ro/SSA-positive pregnancies. Part II. DNA from 636 individuals of 173 European families in which children affected from CHB were born (119 Swedish, 38 Finnish, 2 Norwegian and 14 Italian families) was genotyped and imputation of HLA class I and HLA class II loci was performed. From the analysis, HLA-Cw*06, -DRB1*13, -DQA1*01 and -DQB1*06 emerged as a protective alleles associated with CHB development, while HLA-DQA1*04 transmissions were associated with susceptibility. Furthermore, haplotype analysis revealed that the DRB1-DQA1-DQB1 13-01:03-06:03 haplotype significantly associated with protection from CHB development, while DRB1-DQA1-DQB1 08-04:01-04:02 was significantly associated with CHB susceptibility. A parent-of-origin effect was seen for the following alleles: a lower maternal transmission to affected children was associated with Cw*06, while lower paternal transmission was observed for DQB1*06 and DRB1*07. With these findings, we propose that HLA typing in anti-Ro52-positive pregnancies might be a useful tool to assess the risk of CHB in the fetuses

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

Cross-reactivity of anti-Ro52 antibodies and genetic susceptibility in congenital heart block

Introduction. Congenital heart block (CHB) is a passively acquired autoimmune disease associated with transplacental transfer of maternal anti-Ro/SSA and/or anti-La/SSB antibodies mediating inflammation and subsequent fibrosis of the atrio-ventricular node in the fetal heart. Despite the long-established association of maternal antibodies in directly inducing CHB both with in vitro and in vivo studies, the pathogenic mechanisms involved remain unclear and identification of cross-reactive targets of anti-Ro52 antibodies in the fetal heart is matter of debate. CHB occurs in 1–2% of anti-Ro/SSA antibody-positive pregnancies and has a recurrence rate of 12–20% in a subsequent pregnancy, suggesting that additional factors, such as genetic and environmental components may determine the outcome in terms of CHB development in autoantibody exposed fetuses. Objectives. This thesis is divided into two parts and investigates the pathogenic mechanism and the genetic association with CHB. The first study aims to identify cross-reactive targets of anti-Ro52/p200 antibodies by screening a library of peptides covering the whole human proteome and using monoclonal antibodies specific for the Ro52/p200 region and to confirm the reactivity at peptide level and on the whole antigen level. The second study aims to identify fetal susceptibility genes among the Human Leucocyte Antigen (HLA) locus in anti-Ro/SSA autoantibody-mediated CHB from DNA samples collected from a multi-centric European cohort of families in which children affected by CHB were born. Part I. A whole proteome microarray revealed 17 peptides to be significantly cross-reactive with a Ro52/p200 monoclonal antibody (Ab31) and two linear motifs (“YSDF” and “YSNF) were shared among these sequences. Among the targets, cross-reactivity was studied in detail for TG and GAK, for which reactivity was showed both at the peptide and whole protein level with anti-p200 monoclonal antibodies and with sera from mothers whose children have CHB, suggesting that reactivity to these protein could represent a risk factor for development of CHB in the fetuses in anti-Ro/SSA-positive pregnancies. Part II. DNA from 636 individuals of 173 European families in which children affected from CHB were born (119 Swedish, 38 Finnish, 2 Norwegian and 14 Italian families) was genotyped and imputation of HLA class I and HLA class II loci was performed. From the analysis, HLA-Cw*06, -DRB1*13, -DQA1*01 and -DQB1*06 emerged as a protective alleles associated with CHB development, while HLA-DQA1*04 transmissions were associated with susceptibility. Furthermore, haplotype analysis revealed that the DRB1-DQA1-DQB1 13-01:03-06:03 haplotype significantly associated with protection from CHB development, while DRB1-DQA1-DQB1 08-04:01-04:02 was significantly associated with CHB susceptibility. A parent-of-origin effect was seen for the following alleles: a lower maternal transmission to affected children was associated with Cw*06, while lower paternal transmission was observed for DQB1*06 and DRB1*07. With these findings, we propose that HLA typing in anti-Ro52-positive pregnancies might be a useful tool to assess the risk of CHB in the fetuses.Introduzione. Il blocco avtrio-ventricolare (BAV) congenito è una malattia autoimmune passiva associata al trasferimento transplacentare di anticorpi materni anti-Ro/SSA e/o anti-La/SSB, causando infiammazione e fibrosi del nodo atrio-ventricolare con successivo blocco cardiaco nel feto. Nonostante l’associazione diretta degli anticorpi materni nell'indurre il BAV sia da lungo nota, i meccanismi patogenetici coinvolti rimangono poco chiari e l'identificazione di bersagli di cross-reattività degli anticorpi anti-Ro52 nel cuore fetale è oggetto di dibattito. In aggiunta, Il BAV si verifica nel 1-2% delle gravidanze in cui sono presenti anticorpi anti-Ro/SSA e ha un tasso di ricorrenza del 12-20% nelle una successive gravidanze, suggerendo che fattori aggiuntivi, quali genetici e ambientali sono coinvolti nello sviluppo del BAV. Obiettivi. La prima parte della tesi riguarda uno studio che ha avuto lo scopo di identificare i bersagli di cross-reattività degli anticorpi anti-Ro52/p200 attraverso uno screening di una libreria peptidica coprente l'intero proteoma umano e l'utilizzo di anticorpi monoclonali specifici per la regione p200 della proteina Ro52. Ulteriore obiettivo è quello di confermare la reattività a livello di peptide e a quello di intero antigene. Il secondo studio ha lo scopo di studiare l’associazione genetica del locus “Human Leucocyte Antigen” (HLA) con il BAV in una coorte di famiglie europee in cui sono nati bambini con BAV. Parte I. Lo screening su base proteomica ha individuato 17 peptidi significativamente cross-reattivi con un anticorpo monoclonale specifico per la regione p200 (Ab31). Tra le 17 sequenze sono stati trovati due motivi lineari condivisi ("YSDF" e "YSNF). Tra i target positivamente legati dall’anticorpo Ab31, la cross-reattività è stata ulteriormente studiata per TG e GAK, per i quali la reattività è stata dimostrata sia a livello peptidico, che e a livello di proteina intera, usando anticorpi monoclonali anti-p200 e sieri di madri i cui figli hanno il BAV. Reattività verso queste proteine potrebbe rappresentare un fattore di rischio maggiore per lo sviluppo del BAV nei feti in gravidanze positive per gli anticorpi anti-Ro/SSA. Parte II. Campioni di DNA provenienti da 636 individui da 173 famiglie europee in cui sono nati bambini affetti da BAV (119 svedesi, 38 finlandesi, 2 norvegese e 14 italiane) è stato genotipizzato ed è stata eseguita l'imputazione dei loci HLA di classe I ed HLA di classe II. Dall'analisi è emerso che gli alleli HLA-Cw*06, -DRB1*13, -DQA1*01 e -DQB1*06 conferiscono protezione dallo sviluppo BAV, mentre la trasmissione ai feti di HLA-DQA1*04 conferisce suscettibilità alla malattia. Inoltre, l'analisi ha rivelato che l’aplotipo DRB1-DQA1-DQB1 13-01:03-06:03 è significativamente associato con la protezione dallo sviluppo BAV, mentre DRB1-DQA1-DQB1 08-04:01-04:02 è significativamente associato con suscettibilità al BAV. Infine, è’ stato osservato anche un effetto di origine parentale per questi alleli: -Cw*06 è associato una minore trasmissione materna ai bambini affetti, mentre DQB1*06 e DRB1*07 sono associati ad una inferiore trasmissione paterna. Con questi risultati, proponiamo che la genotipizzazione di questi alleli in gravidanze anti-Ro52-positive potrebbe essere un utile strumento per valutare il rischio di sviluppo del BAV nei feti

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

Abstract 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-\u3b22-glycoprotein I (anti-\u3b22GPI) 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-\u3b22GPI 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-\u3b22GPI, 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-\u3b22GPI levels (\u3c1=0.42 and \u3c1=0.40, respectively). Both IgG aPS/PT and IgM aPS/PT positivity significantly correlated with LA (\u3c1=0.44 and \u3c1=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

The clinical performance of a chemiluminescent immunoassay in detecting anti-cardiolipin and anti-\u3b2<inf>2</inf> 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-\u3b22 glycoprotein I (anti-\u3b22GPI) 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-\u3b22GPI antibodies were determined in the sera using a CLIA (HemosIL AcuStar\uae) and a homemade ELISA. RESULTS: CLIA had a significantly lower comparative sensitivity for IgM aCL and IgG/IgM IgG anti-\u3b22GPI antibodies; its comparative specificity was higher with respect to ELISA for IgM aCL and IgM anti-\u3b22GPI 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-\u3b22GPI antibodies in the seronegative patients. There was a significantly higher prevalence of IgG aCL and IgG anti-\u3b22GPI 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-\u3b22GPI antibodies in the seronegative patients not usually identified by homemade ELISA

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

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