Autoantibodies to the Sodium/Potassium Pump Alpha-1 Subunit AT1A1 Identify At-Risk Pregnancies That Will Develop Fetal/Congenital Heart Block: Evidence and Translational Plan

Abstract

Objectives Autoimmune congenital heart block (CHB) has been associated with maternal autoimmune disease for a century, particularly in lupus and Sjögren’s disease pregnancies. Although 80+% of affected pregnancies are anti-Ro positive, CHB develops in only 1 in 50 such pregnancies, indicating that anti-Ro alone is insufficient. We aimed to identify more predictive maternal autoantibody targets. We hypothesized that the autoantibodies target would be a fetal cardiac protein. Methods We assessed sera from pregnancies with CHB outcome compared with anti-Ro-positive pregnancies without CHB. We used solubilized proteins from fetal heart tissue, and from developmentally immature stem cell-derived cardiomyocytes, to characterize the targets of maternal serum antibodies from affected pregnancies, compared to those from unaffected pregnancies. Targets were identified on 2D gels of cardiac proteins exposed to maternal sera and confirmed using single-lane westerns.[1,2] A targeted 4-peptide ELISA was developed based on the identified cardiac protein epitopes and is being assessed. Additional cohorts from Canada, Germany, Italy, Spain and U.S. are being assessed to refine and validate our assays. (Table 1) In collaboration with EuroImmun AG, we are developing a standardized research assay that can be translated to a laboratory-developed test for clinical care. TABLE 1 Results Pregnancies with prior CHB were assessed separately (Table 1, Columns 11 to 13). For no prior CHB, sera from 7 affected and 5 unaffected pregnancies identified expanding autoantibody target cardiac proteins (beyond Ro and La) throughout CHB pregnancies, which were absent in unaffected pregnancies. (7/7 vs 0/5; p=0.0013, Fisher Exact Test [FET]) The earliest target was AT1A1 (sodium/potassium pump alpha 1 subunit), and western blots identified these autoantibodies. (7/7 vs 0/5; p=0.0013, FET) A Padua cohort identified the same autoantibody fingerprint, and same AT1A1 reactivity by both western and ELISA. (20/20 vs 0/25; p<0.00001, FET). A Toronto (validation) cohort had equivalent results for 2/3 methods. (16/16 vs 0/24; p<0.00001, FET). In pregnancies with a prior CHB child, the antibody pattern and anti-AT1A1 ELISA were occasionally positive despite no CHB resulting in a reduced positive predictive value (0.429). Negative predictive value remained at 1.0. Ro-negative autoimmune CHB[3] provided equivalent results. Six additional cohorts are under evaluation. Conclusion We have identified AT1A1 as a robust auto-antibody target associated with autoimmune CHB outcome. This marker demonstrates strong discriminatory performance across multiple cohorts. Ongoing multicenter validation and assay development with commercial partner EuroImmun AG will support translation of these findings into a clinical diagnostic test aimed at improving CHB risk prediction. References [1.] Benjamin S. Lancet Rheumatol 2025;7:e554-64. [2.] Benjamin S. [Abstract]. Arthritis Rheumatol 2022;74 Suppl 9. https://acrabstracts.org/abstract/fetal-cardiac-targets-identify-the-autoantibodies-associated-with-congenital-heart-block/ . [3.] Brucato A. J Rheumatol 2009;36:1744-8.