BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis.

Background: Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative coccobacillus recognized as a pathogen in periodontitis and infective endocarditis. By producing a toxin (leukotoxin A, LtxA) that triggers global hypercitrullination in neutrophils, Aa has been recently linked to rheumatoid arthritis (RA) pathogenesis. Although mechanistic and clinical association studies implicate Aa infection in the initiation of autoimmunity in RA, direct evidence in humans is lacking.Case:We describe a 59-year-old man with anti-citrullinated protein antibody (ACPA)-positive RA who presented for evaluation of refractory disease. He was found to have Aa endocarditis. Following antibiotic treatment, joint symptoms resolved and ACPAs normalized. Given the implications for RA immunopathogenesis, we further investigated the bacterial, genetic and immune factors that may have contributed to the patient's clinical and autoimmune phenotypes.Methods:DNA was extracted from serum and used to amplify the Aa leukotoxin (ltx) promoter region by PCR, which was further analyzed by Sanger sequencing. High-resolution identification of HLA alleles was performed by sequenced based typing (SBT). TNF-α, IFN-γ, GM-CSF, IL-1β, IL-6, IL-8, IL-17A, IL-18, IL-21, and IL-22 were quantified in serum by a multiplex immunoassay. IgG and IgA antibodies to Aa LtxA were assayed by ELISA.Results:Aa genotyping confirmed infection with a highly leukotoxic strain carrying a 530-bp ltx promoter deletion, shown to result in 10- to 20-fold higher bacterial expression of LtxA. Immuno-phenotyping showed high anti-LtxA antibodies, elevated cytokines implicated in RA pathogenesis (Th1/Th17), and specific host susceptibility conferred by three HLA alleles strongly linked to ACPAs and RA (DRB1*04:04, DRB1*15:01, and DPB1*04:01). One year after eradication of Aa, the patient remained free of arthritis and anti-CCP antibodies.Conclusion: In the context of genetic risk for RA, systemic subacute infection with a leukotoxic strain of Aa can drive ACPA production and a clinical phenotype similar to RA.