Risk factors for ANA positivity in healthy persons

Abstract

Introduction: The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.

Methods: Biological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.

Results: Overall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.

Conclusions: Risks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.

Figure 1

Serum ANA levels measured by ELISA in individuals enrolled in DRADR. Results are shown for all individuals, regardless of diagnosis (top panel), for healthy control (HC) subjects only (middle panel) and for SLE patients only (bottom panel). For each set of results, mean and SEM values are shown on the left and individual values are plotted on the right; male and female groups are compared using t-tests. ELISA values greater than 20 units are considered positive.

Figure 2

Serum antibody levels measured by ELISA in RA patients and community-derived control subjects. ANA levels in RA patients from DRADR (L panel) were significantly higher in females than in males. By contrast, CCP antibodies measured in individuals from the Dallas Heart Study (middle panel) or in DRADR RA patients (right panel) did not show significant male-female differences.

Figure 3

Heat map with clustering of IgM autoantibodies HC subjects and SLE patients. Clusters of study subjects are highlighted to show groups of SLE patients (blue boxes) and ANA high HC (yellow ovals).

Figure 4

Heat map with clustering of IgG autoantibodies in HC subjects and SLE patients. Clusters of study subjects show groups of SLE patients (blue boxes) and ANA high HC (yellow ovals).

Figure 5

IgG autoantibodies showing significant stepwise elevations in the three study groups. P-values calculated using Kruskall-Wallis test.

Figure 6

IgG autoantibodies with elevated levels in high ANA HCs but not in SLE patients. IgM reactivity for these same three autoantigens had similar patterns (not shown).

Figure 7

Expression of Type I IFN signature genes in the three study groups. A stepwise pattern of increase is observed for IFITM3, MX1 and IFIT3. The genes IFI44L and IFI27 show increases that are relatively specific for SLE. Other IFN genes without elevation in the high ANA HC group shown with black symbols are: IFI6, HES4, ISG15, OAS1, IFIT3, HERC5, EIF2AK2.