Why Would Ana Come Positive Over and Over Again

Arthritis Res Ther. 2011; 13(2): R38.

Take a chance factors for ANA positivity in good for you persons

Quan-Zhen Li

^oneDepartment of Immunology, Academy of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9093, United states

David R Karp

ⁱⁱDepartment of Medicine, The Partitioning of Rheumatic Diseases, The Simmons Arthritis Eye, Academy of Texas Southwestern Medical Heart at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-8884, Usa

Jiexia Quan

²Department of Medicine, The Sectionalization of Rheumatic Diseases, The Simmons Arthritis Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-8884, USA

Valerie K Branch

ⁱⁱDepartment of Medicine, The Division of Rheumatic Diseases, The Simmons Arthritis Center, University of Texas Southwestern Medical Centre at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-8884, United states

Jinchun Zhou

¹Department of Immunology, University of Texas Southwestern Medical Eye at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9093, Usa

Yun Lian

¹Department of Immunology, Academy of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9093, USA

Benjamin F Chong

³Section of Dermatology, University of Texas Southwestern Medical Centre at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9069, USA

Edward K Wakeland

¹Section of Immunology, Academy of Texas Southwestern Medical Heart at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9093, USA

Nancy J Olsen

²Department of Medicine, The Partition of Rheumatic Diseases, The Simmons Arthritis Eye, University of Texas Southwestern Medical Eye at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-8884, United states of america

^fourElectric current address: Sectionalisation of Rheumatology, Penn State Hershey Medical Eye, 500 University Drive, Hershey PA 17033, USA

Received 2010 Aug 24; Revised 2010 December 23; Accustomed 2011 Mar two.

Supplementary Materials

Additional file ane Components of the Autoantigen Array. This tabular array lists the components of the array along with sources of the autoantigens.

GUID: E8E4D243-5E5B-4871-813A-67DFB5DB5932

Abstract

Introduction

The finding of antinuclear antibody (ANA) positivity in a salubrious individual is usually of unknown significance and in nigh cases is benign. However, a subset of such individuals is at run a 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 marking 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 Affliction Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an assortment with more than 100 specificities. Whole claret 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 every bit in good for you controls (HC). Age was non significantly associated with ANA levels and the elevated ANA values could not be explained past higher IgG levels. Some other autoantibody, anti- cyclic citrullinated peptide (CCP), did not testify gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in loftier ANA HCs. Some of these autoantibodies were directed to antigens in peel and others were related to autoimmune weather 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 pregnant ANA positivity. Genes upregulated in high ANA HCs included a celiac affliction autoantigen and some components of the Blazon I interferon (IFN) gene signature.

Conclusions

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

Introduction

Antinuclear antibodies (ANAs) are measurable in approximately 25% of the population, and the prevalence of significantly elevated levels may exist 2.5% [ane]. Findings from numerous studies show remarkable consistency beyond ethnically and racially various report populations despite the use of many different methods for ANA measurement. The persistence of this type of autoreactivity in the human population suggests that antinuclear antibodies may be an important component of the normal allowed response. Most individuals with a positive ANA do non accept an autoimmune affliction and most as well are unlikely to develop one. This is consistent with the fact that the prevalence of all autoimmune disorders is 5 to 7% [2]. Furthermore, the disease that is most closely linked to ANA positivity, systemic lupus erythematosus (SLE), is relatively rare, affecting no more than one to 1.5 per 1,000 persons (0.i to 0.15%) in the United States [3]. Still, since ANA positivity is for all practical purposes a requirement for SLE diagnosis, it must also be assumed that individuals who are in preclinical disease stages are represented in the ANA positive healthy population. Although many consultations for ANA positivity seen in rheumatology practice are not associated with any identifiable pathology, it is also true that if early detection of SLE is to go feasible, focus on the ANA positive population will be necessary.

Nosotros have considered the possibility that other claret markers could be used to differentiate benign ANA positivity from that which carries a loftier risk of autoimmune disease. These markers may include other autoantibodies, since it is well-known that autoantibody positivity increases in quantity and complexity in years preceding a diagnosis of SLE [4]. Gene dysregulation in peripheral claret cells has been closely associated with SLE diagnosis and illness status, so changes in cistron expression may also signal a condition with enhanced gamble.

To accost these questions, we studied salubrious individuals and patients with autoimmune diseases who had been enrolled in the Dallas Regional Autoimmune Affliction Registry (DRADR). A subgroup of healthy controls that were found to have high ANA levels was examined in greater particular using autoantigen and cistron expression arrays. The findings suggest the feasibility of identifying risk markers for development of SLE in the setting of ANA positivity, using both demographic features and profiling of autoantibodies and expressed genes in peripheral blood.

Materials and methods

Patients and salubrious controls

Samples for study were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Individuals are classified according to clinical diagnosis or healthy status at the time of enrollment into the registry. Disease features and the presence of SLE criteria were adamant by patient interview and medical record review [five]. Claret samples were obtained for banking of serum aliquots and whole blood samples were stored in PaxGene tubes for later isolation of RNA. All samples were maintained at -80°C until use. The overall report group included one,159 individuals from DRADR: 401 healthy controls (HC) who were negative for current or by autoimmune disease, 116 start-degree relatives (FDR), 294 patients with SLE, 151 patients with less than 4 SLE criteria and considered as having incomplete lupus (ILE), 154 with rheumatoid arthritis (RA) and 43 with other miscellaneous atmospheric condition including scleroderma, Sjogren's syndrome, ankylosing spondylitis and vasculitis. More detailed analyses were carried out on a subset of HC individuals with high ANA values (n = 18) and these were compared to gender- and historic period-matched HC with negative ANA values (n = xvi) and to SLE patients with high ANA levels of >100 Due east.U. (n = 14). In i experiment, plasma samples from the Dallas Middle Study population, which has been described previously [1], were employed. All subjects gave written informed consent for entry into the Dallas Regional Autoimmune Illness Registry. Research carried out under the auspices of this registry has been approved past the UT Southwestern Institutional Review Board.

Antibiotic assays

Serum ANA levels were measured by ELISA (Inova, San Diego, CA, United states) using the manufacturer's suggested cut-off of >xx units to define positive results. Other ELISA kits were used to measure CCP antibodies (Inova), thyroglobulin autoantibodies (Genway Biotech Inc., San Diego, CA, USA) and total IgG (Bethyl Laboratories Inc., Montgomery, TX, United states). The extractable nuclear antibiotic (ENA) panel for 8 additional specificities was a Luminex-based multiplex assay, and positive results were as defined by the manufacturer (Inova). Serum reactivity to a panel of approximately 101 autoantigens and six controls was measured on a slide-based array that has been described previously (Boosted file 1) [6]. Serum samples (1 μL, diluted 1:100) were added to each assortment in duplicate and autoantibodies were detected with Cy3-labelled anti-human being IgG and Cy-5 labeled anti-human IgM simultaneously. Images were generated for analysis and mean fluorescence intensities (MFI) were adamant as previously described. Rut maps were generated using Cluster and Treeview software (Michael Eisen, Berkeley CA, USA) [7]. On the estrus map, intensities higher than the row mean are colored red, those below the mean are greenish and cells with signals close to the mean are black. Gray was used to denote missing data.

Gene expression assay

Total RNA was prepared from 2.5 to five.0 ml of blood collected in Paxgene tubes that had been stored at -80°C. Purity and concentration of the isolated RNA was determined using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, U.s.). We used 250 ng of full RNA to generate biotinylated cRNA using a TotalPrep RNA Amplification kit (Ambion, Austin, TX, USA). The samples were applied to Ilumina HumanWG-6 v3.0 Expression Bead Fries (Illlumina Inc., San Diego, CA, United states) following the manufacturer's directions. The resulting assortment data were analyzed using Ilumina GenomeStudio software (version 3) and statistical analyses were carried out using Partek Genomic Suite (version 6; Partek Inc., Gladstone, MI, USA). Heat maps were generated using the Cluster and Treeview programs [7].

Statistical analyses

Data are presented as mean values and standard errors of the mean. Continuous variables in experiments with three or more groups of samples were analyzed using a one-way ANOVA with Tukey'due south multiple comparison test for postal service test analysis of pairs of samples or the Kruskal-Wallis test for data that did not fit a normal distribution. Analyses of experimental information with two comparison groups used an unpaired 2-tailed Pupil's t-examination, with Welch's correction for groups with unequal variances or Mann-Whitney U for data that did not fit a normal distribution. Pearson's correlation coefficient was used to compare continuous variables. Discontinuous variables were compared using Fisher'southward Exact Test. Graph pad PRISM software (version 5.0a; GraphPad Software, La Jolla, CA, USA) was used for information analysis and graphics. P-values <0.05 were considered significant.

Results

ANA and autoantibodies

Using the defined cutting-off of >20 ELISA units (EU), 615 individuals out of the 1,159 tested were ANA positive. For the subset of 401 HC, the average ANA was xix.5 EU and 101 individuals were in the positive range. This rate of 25% HC positivity is very close to what nosotros have reported previously [1]. Good for you FDRs had a slightly college overall boilerplate ANA (24.4 EU) and a prevalence of ANA positivity of 34%. These values were not significantly different than in the HCs (P >0.07 for both). For the overall group, ANA values were significantly higher in females than in males (Figure ​ ane) and no males had values >240 European union while iii.2% of females had values in this range (P = 0.0030). When the assay was limited to HC, females again showed significantly college ANA levels than males (21.4 vs. 15.6 European union; P = 0.033) and ANA positivity was as well more than prevalent in HC females (29%) than in HC males (17%; P = 0.014). This corresponds to a relative run a risk of ANA positivity in healthy females vs. males of 1.21 (95% confidence interval = 1.059 to 1.390). The highest ANA levels were seen almost exclusively in non-Hispanic females (data not shown), although the overall mean values for Hispanic and non-Hispanic females were not significantly dissimilar (P = 0.7). African-American (AA) HC (n = 32) had a college mean ANA value than non-AAs (27.47 ± five.viii EU vs. 18.1 ± 5.8 EU), but the difference did not reach statistical significance (P = 0.064). Within each gender, AA individuals also had college values than non-AA individuals (females: thirty.38 ± 8.12 Eu vs. twenty.59 ± 1.80 Eu; males 21.09 ± five.55 Eu vs. xv.17 ± i.31 EU); these differences were besides not significant (P >0.1 for each). However, the results are highly suggestive of higher ANA values in AA HC, and the differences might achieve significance in a larger sample size. Patients with SLE did not evidence gender differences in overall ANA values, although the very highest ANA values were again seen exclusively in females (Effigy ​ ane).

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 just (middle panel) and for SLE patients just (lesser 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 twenty units are considered positive.

We compared these findings to another autoimmune disease, RA, which is associated with the CCP autoantibody. While RA patients showed significantly higher ANA levels in females than in males (Figure ​ 2), antibodies to CCP did not show gender differences in either the control population represented by the Dallas Center Written report or in RA patients from DRADR (Effigy ​ 2). These findings suggest that female gender is a general risk factor for ANA but is non necessarily a correlate with other autoimmune disease-specific antibodies.

Serum antibiotic levels measured by ELISA in RA patients and community-derived control subjects. ANA levels in RA patients from DRADR (L console) were significantly higher in females than in males. By contrast, CCP antibodies measured in individuals from the Dallas Eye Written report (eye console) or in DRADR RA patients (correct panel) did not show pregnant male-female differences.

Age was non related to ANA positivity in HCs; high values were scattered throughout the age spectrum (Rⁱⁱ = 0.01). The high ANA levels in HC also were not explained by overall increases in full IgG equally the ii measures were not significantly correlated (R² = 0.11; P = 0.two).

To further understand high ANA levels in HC, a subgroup analysis was done using HC subjects with ANA values greater than 40 EU as the index group. This value is approximately ane standard deviation greater than the overall mean ANA for HC. A total of 18 of the 401 HC, or almost 4%, fit this definition of high ANA. Two comparator groups, HC with negative ANA (ANA values <12 European union) and SLE patients with high ANA (>100 EU) were generally matched for demographic features including gender, race and ethnicity (Table ​ 1). Autoantibodies on the ENA panel were generally not elevated in the high ANA HC group. Merely 3 of the 18 individuals showed positive ENA results, and in all three the lone positive specificity was anti-chromatin.

Tabular array 1

Demographic features of report groups

Group	Age (yrs)	ANA (EU)	Female (%)	AA (%)	Hispanic (%)
HC High ANA (n = 18)	33.eight ± iii.4*	78.4 ± 10.0*	83	22	6
HC Low ANA (north = sixteen)	43.1 ± 3.vii	7.0 ± 0.6	75	25	0
SLE (n = 14)	38.0 ± 4.0	182.4 ± 15.4	93	43	7
P**	0.2	0.001#	0.4	0.iv	0.6

*Values represent mean and standard error of the mean. **P-values were calculated by ane-way ANOVA or Fisher's exact test. ^# Mean ANA in SLE was significantly higher than in the High ANA HC group (P <0.05). ANA, antinuclear antibodies; SLE, systemic lupus erythematosus.

The three study groups were and then compared using the autoantigen assortment for both IgG and IgM autoantibodies (Figures ​ three and ​ four). Two major IgM clusters showed a trend for the SLE patients to cluster together, but this was not statistically significant (P = 0.08); the high ANA HC individuals had a similar tendency to be in the not-SLE cluster (P = 0.06; Figure ​ 3). For IgG, two chief IgG clusters were identified, and all of the SLE patients were in one of these clusters (P = 0.009; Figure ​ four).

Rut 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).

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 (yellowish ovals).

Antibodies detected on the arrays were then examined for specificities appearing in HC that were lupus-like. This was divers every bit overall hateful values for a given specificity showing no deviation between the loftier ANA HC and SLE groups (P >0.ane) while at the same time having lower values in the depression ANA HC group compared to SLE (P <0.one). The antibodies in these groups were examined to choose those with stepwise increases in the groups (low ANA HC <High ANA HC <SLE) and to exclude whatever with distributions that were highly skewed by one or two individuals. This analysis yielded fourteen IgGs; 9 of these with the most pregnant P-values (Kruskal-Wallis test) are shown (Effigy ​ five). Four of the nine specificities are directed confronting autoantigens establish in peel tissues (DSG4, MMP1, recombinant human collagen, integrinα6β4) [eight-10]. Others are associated with autoimmune kidney disease (GBM), thyroid disorders (thyroglobulin), scleroderma (PM/Scl100), and inflammatory arthritis (proteoglycan) [11]. A search for autoantibodies that might exist associated with a lowered take a chance of lupus was carried out by choosing specificities that were elevated in high ANA HC just were not loftier in SLE patients. The ratio of the mean values for the high ANA HC group to the corresponding SLE group mean was calculated, and iii specificities had IgG ratios greater than 4.0: Jurkat T cells, gliadin and SK-N-SH (Figure ​ 6). In each of these, a subset of the loftier ANA HC individuals showed potent reactivity while SLE patients did not have high values. For gliadin, an autoantigen associated with celiac disease, and Jurkat, a measure of anti-T cell and thymocyte antibodies, the distributions were significantly different for the 3 groups (P = 0.042 and 0.033, respectively). Although the antineuronal antibodies measured past SKNSH reactivity did not show significant differences across groups by ANOVA, a dichotomized analysis showed values greater than 400 MFI units were present only in the loftier ANA HC grouping (P = 0.0242). The 4 highest values for each of these different autoantibodies represented the same individuals.

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

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

An independent ELISA assay was carried out for thyroglobulin (TG) autoantibodies to compare with the findings on the assortment. Anti-TG levels of the IgG class measured by the two techniques were significantly correlated (P = 0.007) and values were significantly college in the HC high ANA group (ane.xviii ± 0.23 EU) than the HC depression ANA group (0.lx ± 0.07 European union; P = 0.030), consistent with the array results.

Gene expression profiles

Mean gene expression values were compared betwixt the two HC groups defined past ANA condition, and this assay identified 95 dysregulated genes at a significance level of P <0.01. Somewhat surprisingly, the vast majority of these genes (90 out of 95) were upregulated in the ANA-negative group. Of the five genes that were upregulated in the high ANA HC grouping, the highest (2-fold difference) was TGM2, which encodes the celiac affliction autoantigen transglutaminase 2.

To detect specificities that might exist informative of ANA and diagnosis status, a 2d analysis was carried out by commencement comparing the SLE and high ANA HC groups to determine gene specificities that were significantly different at a significance level of P <0.001. This list was then sorted based on the values for the calculated ratio SLE/ANA-High HC. The most highly expressed specificity in this list was IFI27, which was 65-fold higher in SLE than in ANA High HC. Overall, 69 genes in this listing had greater than ii-fold differences betwixt SLE and ANA High HC. These 69 genes were then resorted based on differences in the ratio of ANA High HC/ANA Low HC. The highest ratio in this fix was 2-fold and out of the top 10 specificities, 7 were in the Type I interferon signature (IFITM3, MX1, IFI6, IFI44L, ISG15, OAS1, IFIT3) and one encodes a protein that is activated by dsRNA as might exist nowadays in a viral infection (EIF2AK2). The specificities that most clearly showed stepwise increases in the three groups going from Low ANA HC to High ANA HC to SLE were IFITM3 and MX-i (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 superlative in the high ANA HC grouping shown with black symbols are: IFI6, HES4, ISG15, OAS1, IFIT3, HERC5, EIF2AK2.

Discussion

Detecting autoimmune disease in early or preclinical stages is clinically important considering the institution of treatment prior to the onset of organ damage has a greater chance to improve or fifty-fifty cure the affliction [12]. Yet, early and reliable diagnosis of lupus is a claiming, in large part due to the functioning profiles of available diagnostic tests. The optimal exam would be sensitive enough to detect all individuals who take a affliction while at the same fourth dimension delivering sufficient specificity to have reasonable predictive probability that the illness is likely. The classic screening exam for SLE is the presence in serum of antinuclear antibodies (ANAs) measured by immunofluorescence staining of a cellular substrate [13]. ANA positivity is for all applied purposes required to make a diagnosis of lupus since more than than 99% of patients with SLE have significant levels of this autoantibody detected at some time during the grade of disease. However, since the prevalence of SLE is low, most individuals presenting to a medico with ANA positivity do not in fact have lupus and are non at high risk for developing this illness. But there are few available quantitative and objective measures to establish prognosis for an individual with a positive ANA. This contrasts to the tools that are available for determining the risk of cardiovascular disease, where multifaceted profiles including elements of personal and family history, social habits, body measurements and lab tests tin can generate a reasonable and very personalized risk prediction for an individual patient [14].

In practice, physicians actually practise employ some profiling to assess the hazard associated with ANA positivity. Young women are more likely to develop lupus than old men, for example, so ANA positivity in the one-time is of greater concern. The present study confirms our previous observation that female gender is a take a chance gene for significant ANA positivity [1]. This result is too consistent with other findings in healthy control populations including a study of 500 normal individuals in Brazil showing that ANA positivity was near twice equally prevalent in females as in males [15]. Similar findings were reported in a rural Canadian population, with the gender difference being greatest at higher ANA levels, equally was also noted in the present study [16]. The enhanced female risk contour does not appear to extend to the anti-CCP antibiotic that is associated with RA, some other female-predominant autoimmune disorder. Reasons for the association of female gender with strong ANA positivity remain obscure.

Age was not correlated with ANA levels in HCs, which seems to contradict the generally-accustomed hypothesis that immunosenescence is associated with increased autoantibody product due to decreased self-regulatory mechanisms. The present findings are consistent, however, with other reports [15] and advise that in a cross-exclusive analysis such every bit this at that place are many reasons for ANA positivity. The younger individuals may in fact have abnormal immune regulation that predisposes to SLE-like disease while older persons may develop autoreactivity as office of crumbling immune responses that do not lead to development of pathology.

Other clues are available to propose approaches to stratifying risk in the ANA-positive population. One is the well-recognized presence of other autoantibodies that accrue prior to SLE diagnosis [4]. The prevalence of any i of these is depression, even so. For instance, ribosomal P autoantibodies are highly specific for SLE simply are present in less than one-third of patients [17]. This finding predicts that if autoantibodies are included in risk profiling, multiplexed assays will be required. I such approach that is available clinically, the ENA console, was not useful in the loftier ANA HC individuals in the present study considering very few positives were found. This result is consistent with our previous feel indicating that but the high ANA positives are probable to have ENA specificities [one] and even then, the prevalence of other autoantibodies in HCs is very low. The culling approach which has been practical in the present written report is to profoundly expand the repertoire of autoantibodies that can exist probed by use of an autoantigen assortment, which in other studies has been shown to provide novel insights into expressed autoantibody repertoires [eighteen]. The array information in the nowadays written report revealed increased autoreactivity in a grouping of loftier ANA HCs. One of the elevated autoantibodies was thyroglobulin, and since autoimmune thyroid disease is probably near 10-fold more than mutual than SLE [19], this outcome suggests that a significant proportion of the ANA positivity seen by rheumatologists is related to thyroid autoimmunity. Longitudinal studies propose that thyroid autoreactivity, particularly in women, may be predictive of thyroid dysfunction [xx]. Autoantibodies to cartilage proteoglycan can be measured in several systemic and joint-specific rheumatic diseases including Sjogren's Syndrome, rheumatoid arthritis, lupus and ankylosing spondylitis [21], suggesting that undetected or preclinical joint inflammation may contribute to ANA positivity. The relative increase in skin autoreactivity in the loftier ANA HC group might be related to a relative enrichment for peel antigens on the arrays, then this finding should be interpreted with circumspection. However, it does raise the interesting question of whether some early on autoimmune events might accept identify in cutaneous structures. Transient ANA positivity has been observed in patients with polymorphous lite eruption [22] and exposure to dominicus in susceptible individuals can trigger major organ-damaging lupus [23]. The interface dermatitis that characterizes the pathology in SLE as well as in other skin disorders may precede a diagnosis of lupus [24] and is associated with activation of the Type I interferon gene signature [25]. The triggering of this set of genes in skin, however, is not limited to inflammatory disease but tin likewise occur as part of the immune response to viruses, raising the question of whether cutaneous reactions to environmental agents in susceptible hosts might generalize to a systemic response.

The autoantibody arrays show that although the autoreactivity spectrum in SLE is broad, not all specificities are elevated. Upregulated autoantibodies to gliadin, and to T lymphocytes and neuroblastoma cells were nowadays only in the ANA high HC group, and not in the SLE patients. This result confirms in part our previous report that gliadin autoreactivity is associated with incomplete forms of lupus that are associated with myopathies [26]. Whether these autoantibodies are actively protective and lower the risk of lupus or alternatively are predictive of other autoimmune diseases developing in these individuals volition require further longitudinal investigation.

Upregulated genes observed in loftier ANA HC individuals include some in the Blazon I IFN signature that are associated with SLE [27,28]. While some of these genes, notably IFI27, were only elevated in SLE, others such as MX-1 showed an intermediate level in the High ANA HC grouping. In addition to being the about highly-upregulated of the IFN genes in our sample, other data suggest that IFI27 is relatively more than specific for lupus than at least some of the other IFN-inducible genes. A recent study demonstrated that IFI27 is more probable to be upregulated in lupus than in some other autoimmune condition, idiopathic thrombocytopenic purpura [29]. The specific functions of many of the proteins associated with IFN-related genes are obscure, just MX-ane is closely associated with the response to the influenza virus, then upregulation of this gene in normal individuals might reflect the ubiquitous exposure to this pathogen.

The present data propose possible components of a lupus risk profile. As in the cardiovascular risk profiling equations, gender volition be a cistron but in lupus the risk volition exist associated with females rather than males. The age category will be inverted from that of cardiovascular disease, with greater weight given to younger ages. The lupus gamble is correlated with ANA levels, not simply positivity, with values in the upper quartile having a two- to three-fold elevation of hazard [ane], and so this will be an of import component. Other autoantibodies that may add to risk include those that are clinically well-known like anti-dsDNA and anti-Sm, likewise as other novel specificities including the pare determinants identified in the present report. On the other hand, antineuronal, anti-thyroid or gliadin autoantibodies might steer attention abroad from SLE towards other autoimmune disorders. Elevated expression of genes related to the Type I IFN signature is likely to add points to the take a chance equation.

This report has several limitations. One is the lack of data regarding use of medications, especially hormones, by the HC. Whether administration of estrogen in the form of oral contraceptives or postmenopausal replacement therapy might induce high ANA levels in a healthy individual cannot be ascertained from our data. Another limitation is the cross-sectional design which does non permit insights into changes that evolve over time. And it would be of interest to make up one's mind reactivity to foreign antigens such every bit infectious agents to further translate the significance of the autoreactive responses. Ultimately, validation of run a risk profiles will require longitudinal studies.

Finally, since SLE is a relatively rare disorder, the probability of finding a new onset patient is low even later ANA positivity has been identified. I approach to increasing the likelihood of useful results would be to follow individuals who already have some of the identified adventure profile components. For example, studies could be carried out in individuals who have been sent for ANA testing for any reason. The very fact that the private sought medical attention and that an ANA was ordered is probable to increase the pre-test probability of disease. Such an approach has shown that in the population of individuals sent for rheumatoid cistron testing, the pre-test probability for RA is 17% [30].

Conclusions

This study shows that information technology may be possible to identify the pocket-size per centum of ANA positive individuals who are at gamble for development of SLE. Characterization of protein and gene expression profiles accompanying ANA positivity has potential to enable more than precise adventure quantification and ultimately to identify pre-clinical stages of this disease that would allow early and definitive treatment.

Abbreviations

ANA: antinuclear antibody; CCP: circadian citrullinated peptide; DRADR: Dallas Regional Autoimmune Disease Registry; ENA: extractable nuclear antibody; Eu: ELISA units; FDR: first degree relative; HC: healthy controls; IFN: interferon; ILE: incomplete lupus; MFI: mean fluorescence intensity; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; TG: thyroglobulin

Competing interests

NJO has equity involvement in ArthroChip LLC and has received inquiry grants from Medimmune, Novo Nordisk, Genentech/Roche and Homo Genome Sciences. DRK has received a research grant from Human being Genome Sciences. All other authors declare that they have no competing interests.

Authors' contributions

QZL supervised the protein and factor expression assortment assays and analyses. DRK organized the patient registry. JQ organized and performed laboratory assays. JZ performed the protein array assays, while YL performed data analyses for poly peptide and gene expression assortment assays. VKB coordinated patient recruitment for the registry. BFC contributed to design and validation of the autoantibody assortment. EKW conceived of the study design and NJO assembled the patient cohorts and supervised clinical phenotyping. QZL, DK, EKW and NJO wrote the manuscript with input and consensus from all authors. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1:

Components of the Autoantigen Assortment. This table lists the components of the array along with sources of the autoantigens.

Acknowledgements

This work was supported past NIH/NIAMS P50AR0455503. We capeesh the assist of Azza Mutwally, Alice Cox, Sarmistha Sen, Justin Ribault and Pratik Doshi for enrolling and evaluating the individuals enrolled in DRADR. Michelle Christadoss and Ferdicia Carr-Johnson processed the samples and carried out the laboratory assessments. Jessica Mayeux assisted with data assay. Heidi Jacobe MD shared with us a fix of peel-specific autoantigens for inclusion in the poly peptide arrays, and we appreciate her input. Thanks likewise to Chandra Mohan MBBS, PhD, for helpful discussions and support.

The data presented in this publication have been deposited in the NCBI Factor Expression Coach with accretion number GSE 24706 and may exist accessed online [31].

References

• Wandstrat A, Carr-Johnson F, Branch Five, Gray H, Fairhurst A, Reimold A, Karp D, Wakeland E, Olsen Northward. Autoantibody profiling to place individuals at chance for systemic lupus erythematosus. J Autoimmun. 2006;27:153–160. doi: ten.1016/j.jaut.2006.09.001. [PubMed] [CrossRef] [Google Scholar]
• Davidson A, Diamond B. Autoimmune diseases. North Engl J Med. 2001;345:340–350. doi: 10.1056/NEJM200108023450506. [PubMed] [CrossRef] [Google Scholar]
• Chakravarty Due east, Bush T, Manzi S, Clarke A, Ward Chiliad. Prevalence of adult systemic lupus erythematosus in California and Pennsylvania in 2000: estimates obtained using hospitalization information. Arthritis Rheum. 2007;56:2092–2094. doi: 10.1002/art.22641. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Arbuckle M, McClain M, Rubertone M, Scofield R, Dennis G, James J, Harley J. Evolution of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. 2003;349:1526–1533. doi: 10.1056/NEJMoa021933. [PubMed] [CrossRef] [Google Scholar]
• Tan Due east, Cohen A, Fries J, Masi A, McShane D, Rothfield N, Schaller J, Talal N, Winchester R. The 1982 revised criteria for the nomenclature of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271–1277. doi: 10.1002/art.1780251101. [PubMed] [CrossRef] [Google Scholar]
• Li Q, Zhou J, Wandstrat A, Carr-Johnson F, Branch V, Karp D, Mohan C, Wakeland E, Olsen North. Protein array autoantibody profiles for insights into systemic lupus erythematosus and incomplete lupus syndromes. Clin Exp Immunol. 2007;147:60–70. [PMC free article] [PubMed] [Google Scholar]
• Open Source Clustering Software. http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/
• Müller R, Heber B, Hashimoto T, Messer Thousand, Müllegger R, Niedermeier A, Hertl Thou. Autoantibodies against desmocollins in European patients with pemphigus. Clin Exp Dermatol. 2009;34:898–903. [PubMed] [Google Scholar]
• Bare Chiliad, Gisondi P, Mimouni D, Peserico A, Piaserico Due south, Shoenfeld Y, Reunala T, Zambruno 1000, Di Zenzo Chiliad, Girolomoni G. New insights into the autoantibody-mediated mechanisms of autoimmune bullous diseases and urticaria. Clin Exp Rheumatol. 2006;24:S20–25. [PubMed] [Google Scholar]
• Tomimura South, Ogawa F, Iwata Y, Komura K, Hara T, Muroi E, Takenaka Thousand, Shimizu Yard, Hasegawa G, Fujimoto K, Sato Due south. Autoantibodies against matrix metalloproteinase-1 in patients with localized scleroderma. J Dermatol Sci. 2008;52:47–54. doi: 10.1016/j.jdermsci.2008.04.013. [PubMed] [CrossRef] [Google Scholar]
• Polgár A, Falus A, Koó East, Ujfalussy I, Seszták M, Szuts I, Konrád Thousand, Hodinka L, Bene East, Mészáros 1000, Ortutay Z, Farkas E, Paksy A, Buzás EI. Elevated levels of synovial fluid antibodies reactive with the small proteoglycans biglycan and decorin in patients with rheumatoid arthritis or other joint diseases. Rheumatology (Oxford) 2003;42:522–527. [PubMed] [Google Scholar]
• Sisó A, Ramos-Casals Grand, Bové A, Brito-Zerón P, Soria N, Muñoz S, Testi A, Plaza J, Sentís J, Coca A. Previous antimalarial therapy in patients diagnosed with lupus nephritis: influence on outcomes and survival. Lupus. 2008;17:281–288. [PubMed] [Google Scholar]
• Meroni P, Schur P. ANA screening: an old examination with new recommendations. Ann Rheum Dis. 2010;69:1420–1422. doi: 10.1136/ard.2009.127100. [PubMed] [CrossRef] [Google Scholar]
• Sheridan Southward, Pignone Thou, Mulrow C. Framingham-based tools to calculate the global risk of coronary middle disease: a systematic review of tools for clinicians. J Gen Intern Med. 2003;eighteen:1039–1052. doi: ten.1111/j.1525-1497.2003.30107.x. [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
• Fernandez Due south, Lobo A, Oliveira Z, Fukumori L, Périgo A, Rivitti East. Prevalence of antinuclear autoantibodies in the serum of normal blood donors. Rev Hosp Clin Fac Med Sao Paulo. 2003;58:315–319. [PubMed] [Google Scholar]
• Semchuk 1000, Rosenberg A, McDuffie H, Cessna A, Pahwa P, Irvine D. Antinuclear antibodies and bromoxynil exposure in a rural sample. J Toxicol Environ Health A. 2007;seventy:638–657. doi: 10.1080/15287390600974593. [PubMed] [CrossRef] [Google Scholar]
• Heinlen L, Ritterhouse 50, McClain Chiliad, Keith M, Neas B, Harley J, James J. Ribosomal P autoantibodies are present earlier SLE onset and are directed confronting non-C-terminal peptides. J Mol Med. 2010;88:719–727. doi: x.1007/s00109-010-0618-1. [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
• Silverman M, Srikrishnan R, Germar 1000, Goodyear C, Andrews One thousand, Ginzler E, Tsao B. Genetic imprinting of autoantibody repertoires in systemic lupus erythematosus patients. Clin Exp Immunol. 2008;153:102–116. doi: 10.1111/j.1365-2249.2008.03680.x. [PMC costless article] [PubMed] [CrossRef] [Google Scholar]
• McGrogan A, Seaman H, Wright J, de Vries C. The incidence of autoimmune thyroid affliction: a systematic review of the literature. Clin Endocrinol (Oxf) 2008;69:687–696. doi: 10.1111/j.1365-2265.2008.03338.10. [PubMed] [CrossRef] [Google Scholar]
• Walsh J, Bremner A, Feddema P, Leedman P, Brown S, O'Leary P. Thyrotropin and thyroid antibodies every bit predictors of hypothyroidism: a thirteen-year, longitudinal study of a customs-based cohort using electric current immunoassay techniques. J Clin Endocrinol Metab. 2010;95:1095–1104. doi: 10.1210/jc.2009-1977. [PubMed] [CrossRef] [Google Scholar]
• Vynios DH, Tsagaraki I, Grigoreas GH, Samiotaki Chiliad, Panayotou G, Kyriakopoulou D, Georgiou P, Korbakis D, Panayotou A, Nanouri Thou, Assouti M, Andonopoulos AP. Autoantibodies against aggrecan in systemic rheumatic diseases. Biochimie. 2006;88:767–773. doi: 10.1016/j.biochi.2006.01.004. [PubMed] [CrossRef] [Google Scholar]
• Tzaneva S, Volc-Platzer B, Kittler H, Hönigsmann H, Tanew A. Antinuclear antibodies in patients with polymorphic light eruption: a long-term follow-upwards study. Br J Dermatol. 2008;158:1050–1054. doi: 10.1111/j.1365-2133.2008.08500.x. [PubMed] [CrossRef] [Google Scholar]
• Schmidt E, Tony H, Bröcker E, Kneitz C. Sun-induced life-threatening lupus nephritis. Ann North Y Acad Sci. 2007;1108:35–forty. doi: ten.1196/annals.1422.004. [PubMed] [CrossRef] [Google Scholar]
• Paradela S, Lorenzo J, Martínez-Gómez W, Yebra-Pimentel T, Valbuena L, Fonseca East. Interface dermatitis in skin lesions of Kikuchi-Fujimoto'south disease: a histopathological marker of evolution into systemic lupus erythematosus? Lupus. 2008;17:1127–1135. doi: ten.1177/0961203308092161. [PubMed] [CrossRef] [Google Scholar]
• Wenzel J, Tüting T. An IFN-associated cytotoxic cellular immune response confronting viral, self-, or tumor antigens is a mutual pathogenetic feature in "interface dermatitis". J Invest Dermatol. 2008;128:2392–2402. doi: 10.1038/jid.2008.96. [PubMed] [CrossRef] [Google Scholar]
• Olsen N, Prather H, Li Q, Burns D. Autoantibody profiles in two patients with non-autoimmune muscle illness implicate a part for gliadin autoreactivity. Neuromuscul Disord. 2010;xx:188–191. doi: 10.1016/j.nmd.2009.12.008. [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
• Bennett L, Palucka A, Arce E, Cantrell V, Borvak J, Banchereau J, Pascual V. Interferon and granulopoiesis signatures in systemic lupus erythematosus claret. J Exp Med. 2003;197:711–723. doi: 10.1084/jem.20021553. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Li Q, Zhou J, Lian Y, Zhang B, Co-operative V, Carr-Johnson F, Karp D, Mohan C, Wakeland E, Olsen N. Interferon signature gene expression is correlated with autoantibody profiles in patients with incomplete lupus syndromes. Clin Exp Immunol. 2010;159:281–291. doi: 10.1111/j.1365-2249.2009.04057.x. [PMC gratuitous commodity] [PubMed] [CrossRef] [Google Scholar]
• Ishii T, Onda H, Tanigawa A, Ohshima S, Fujiwara H, Mima T, Katada Y, Deguchi H, Suemura M, Miyake T, Miyatake K, Kawase I, Zhao H, Tomiyama Y, Saeki Y, Nojima H. Isolation and expression profiling of genes upregulated in the peripheral blood cells of systemic lupus erythematosus patients. DNA Res. 2005;12:429–439. doi: 10.1093/dnares/dsi020. [PubMed] [CrossRef] [Google Scholar]
• Silveira IG, Burlingame RW, von Mühlen CA, Bender AL, Staub HL. Anti-CCP antibodies accept more diagnostic affect than rheumatoid gene (RF) in a population tested for RF. Clin Rheumatol. 2007;26:1883–1889. doi: x.1007/s10067-007-0601-six. [PubMed] [CrossRef] [Google Scholar]
• GEO Accretion viewer. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24706

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132017/