Faculty Bibliography

Purpose: Maternal anti-Ro antibodies are highly associated with heart block and/or cardiomyopathy (cardiac neonatal lupus (NL) in an offspring. Monozygotic twin studies and the 10 fold increased recurrence rate of cardiac NL in a subsequent pregnancy implicate a strong genetic influence on risk of disease. Here, we report the first genome-wide association study of cardiac NL. Methods: Caucasian children (n=117) with cardiac NL were identified from an extensive collection of DNA in the U.S. Research Registry for Neonatal Lupus (RRNL). Two criteria were required: 1) cardiac NL defined as heart block (1st, 2nd,3rd, degree) documented by electrocardiogram (if 1st degree), echocardiogram, history of pacemaker, or statement in the medical record; and/or presence of cardiac injury which included autopsy evidence of a mononuclear infiltrate in the endocardium, myocardium and pericardium and/or endocardial fibroelastosis on echocardiogram always associated with cardiac dysfunction 2) maternal antibodies to 52kD SSA/Ro, 60kD SSA/Ro, or 48kD SSB/La. In 96%, 2nd or 3rd degree block was present. Cardiac NL subjects were genotyped using the Illumina 370K SNP platform and merged with 3351 Illumina out-of-study controls from SLEGEN (Harley 2008). Standard quality control and admixture adjusted tests of association were computed. Results: The HLA region (6p21) showed strong evidence of association. Two HLA SNPs in high linkage disequilibrium were rs3135353 (p_dom= 3.99E-08; OR = 2.87; 2.8 kb from HLA-DRB5) and rs3129963 (p_dom= 8.96E-07; OR = 2.57; 75 kb from HLA-DRA). Outside the HLA region, rs1810636 near 20p13 showed significant association to cardiac NL (p_rec= 1.53E-12, OR = 4.07). In proximity to this region are the DNA sequences of five noncoding RNAs that associate with the spliceosome. An additional suggestive association at 5q11.2 includes rs 2432143 (p_dom=5.87E-05, OR=2.28) within the integrin, alpha 1, a receptor involved in cell-cell adhesion, which may play a role in inflammation and fibrosis. Importantly, none of the fifteen prominent non-HLA polymorphisms reported in SLE association studies were associated with cardiac NL at less than 1E-5. Conclusion: These analyses are the first genome-wide association study for cardiac NL and identify several strong statistical associations. These associations, including the HLA region, corroborate the genetic influences on cardiac NL and may provide a basis for exuberant fibrosis of the conduction system

Purpose: Patients (pts) with SLE develop widespread vascular inflammation and life threatening thrombotic events which are not completely explained by typical antiphospholipid antibodies. Since EPCR is a critical regulator at a crossroads of inflammation and thrombosis, two SNPs were evaluated in 199 active SLE pts to determine if genotype has an impact on circulating sEPCR, autoantibodies to EPCR, and thrombotic risk. Methods: Genotyping was performed by RTPCR with TaqMan primers for rs6936 A-G (serine to glycine) and rs7014 G-C (3' UTR). Circulating sEPCR was quantified by the ASSERACHROM sEPCR ELISA (Stago). Antibodies to EPCR were measured by a novel ELISA, calibrated against sera from 60 healthy individuals. Medical history and disease activity scores were obtained at the time of sampling. Results: Mean pt age was 39.1, 89.9% female, 62.3% Caucasian, 25.6% African descent and 12.1% Asian. There was no demographic bias in rs6936 genotypes, but most African descent pts were homozygous for G at rs7014 (40/51 vs 46/148 in others, p <0.001). Lupus activity (by BILAG) was the same between groups with overall mean (SD) score of 6.9 (4.3), suggesting moderate vascular inflammation throughout the population. In pts with the genotype rs6936 AA, median sEPCR was 145 ng/ml vs 317 ng/ml in non-AA (p <0.001). There was also a difference between genotypes at rs7014: 130 ng/ml in CC pts vs 164 in non-CC (p=0.005). Median sEPCR of 37 pts who carried both of the low sEPCR genotypes was 130 ng/ml vs 317 ng/ml in the rest of the population (p<0.001). Low C4 complement was unevenly distributed in rs7014 genotypes, found in 25% of GG, 41.6% of GC and 33.3% of CC, (p=0.041, GG vs GC). More women with rs6936 AA had a history of pregnancy loss and/or pre-eclampsia (23.6% vs 8% of those with G alleles in that gene, p=0.054) and the same for rs7014 (37.8% CC vs 17% with G alleles, p=0.011). Overall pts with a history of pregnancy complications, arterial and/or venous thromboses were also increased in rs6396 AA, 32.9% vs 15% in non-AA (p=0.06) and rs7014 CC, 37.8% vs 26.7% in non-CC (p=ns). Combining genotypes in assessing thrombotic risk, AA/CC women had a high rate of pregnancy complications (14/35 or 40% vs 3/35 or 8.57% of non-AA/non-CC (p=0.004). Pts with any type of thrombotic event included 14/37 (37.8%) of AA/CC vs 6/38 (15.7%) of non-AA or CC (p=0.039). 38 pts had autoantibodies to EPCR, more common in rs6936 AA than in pts with the G allele (32/162 vs 6/38 (p=ns) and more common in rs7014 pts with G allele than CC (34/163 vs 4/37) (p=ns). There were no pts with both 6936 AA and a G allele in 7014 even though each genotype represents about 19% of the population. Conclusion: These data suggest that genetic variants influence sEPCR levels in active lupus and may increase complement consumption, autoantibody formation and thrombotic complications in these patients

The association of isolated congenital heart block (CHB) with maternal autoantibodies to SSA/Ro and SSB/La ribonucleoproteins is approaching the predictable, even in mothers who are completely asymptomatic. Indeed, this model of passively acquired autoimmunity offers an exceptional opportunity to examine the effector arm of immunity and define the pathogenicity of an autoantibody in mediating tissue injury. The study of CHB exemplifies not only translational research, which inherently draws upon clinical observations and explores them in the laboratory, but 'integrational' research which attempts to fit critical clinical and basic observations together, even those seemingly at odds. The spectrum of conduction abnormalities includes second and third-degree block, but injury can extend to the myocardium and endocardium, in rare cases without AV nodal dysfunction. The rarity of disease continues to drive the search for factors (fetal and environmental) that might amplify the effects of the maternal autoantibodies. The identification of exaggerated apoptosis, macrophage/myfibroblast crosstalk, TGF beta expression, and extensive fibrosis in the conducting system and in some cases surrounding myocardium in fetuses dying with CHB, provide in vivo support for several parallel lines of in vitro investigation. Specifically, the consideration of exaggerated apoptosis as the initial link between maternal antibody and tissue injury led to the observation that cardiocytes are capable of phagocytosing autologous apoptotic cardiocytes and anti-Ro/La antibodies inhibit this function. Recognizing that this perturbation of physiologic efferocytosis might divert uptake to professional Fc gamma R-bearing phagocytes fits well with experiments demonstrating macrophage secretion of pro-inflammatory and fibrosing cytokines when coincubated with apoptotic cardiocytes bound by Ro/La antibodies. While CHB is rare, its study should set precedent for defining the role of autoantibodies in driving end organ disease

OBJECTIVE: Identification of isolated congenital heart block (CHB) predicts, with near certainty, the presence of maternal anti-SSA/Ro antibodies; however, the 2% incidence of CHB in first offspring of anti-SSA/Ro+ mothers, 20% recurrence in subsequent pregnancies, and discordance in identical twins suggest that an environmental factor amplifies the effect of the antibody. Accordingly, this study was carried out to explore the hypothesis that hypoxia potentiates a profibrosing phenotype of the fetal cardiac fibroblast. METHODS: Evidence of an effect of hypoxia was sought by immunohistologic evaluation of CHB-affected fetal heart tissue and by determination of erythropoietin levels in cord blood. The in vitro effect of hypoxia on gene expression and phenotype in fibroblasts derived from fetal hearts and lungs was investigated by Affymetrix arrays, quantitative polymerase chain reaction (PCR), immunofluorescence, and immunoblotting. RESULTS: In vivo hypoxic exposure was supported by the prominent intracellular fibroblast expression of hypoxia-inducible factor 1alpha in conduction tissue from 2 fetuses in whom CHB led to death. The possibility that hypoxia was sustained was suggested by significantly elevated erythropoietin levels in cord blood from CHB-affected, as compared with unaffected, anti-SSA/Ro-exposed neonates. In vitro exposure of cardiac fibroblasts to hypoxia resulted in transdifferentiation to myofibroblasts (a scarring phenotype), as demonstrated on immunoblots and immunofluorescence by increased expression of smooth muscle actin (SMA), an effect not seen in lung fibroblasts. Hypoxia-exposed cardiac fibroblasts expressed adrenomedullin at 4-fold increased levels, as determined by Affymetrix array, quantitative PCR, and immunofluorescence, thus focusing attention on cAMP as a modulator of fibrosis. MDL12,330A, an adenylate cyclase inhibitor that lowers the levels of cAMP, increased expression of fibrosis-related proteins (mammalian target of rapamycin, SMA, plasminogen activator inhibitor type 1, and type I collagen), while the cAMP activator forskolin attenuated transforming growth factor beta-elicited fibrosing end points in the cardiac fibroblasts. CONCLUSION: These findings provide evidence that hypoxia may amplify the injurious effects of anti-SSA/Ro antibodies. Modulation of cAMP may be a key component in the scarring phenotype. Further assessment of the susceptibility of cardiac fibroblasts to cAMP modulation offers a new research direction in CHB

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjogren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB