Abstract
Objectives:
The gastrointestinal (GI) tract is commonly affected in systemic sclerosis (SSc). A positive association between anti-vinculin antibody levels and GI symptom severity is reported in SSc. We sought to examine whether anti-vinculin antibodies associate with measures of GI dysmotility and extra-intestinal clinical phenotype in SSc.
Methods:
88 well-characterized patients with SSc and GI disease were assayed for anti-vinculin antibodies by enzyme-linked immunosorbent assay (ELISA). Whole gut scintigraphy, GI symptom scores, and clinical features of SSc were compared between patients with and without antibodies.
Results:
Twenty out of 88 (23%) patients had anti-vinculin antibodies, which were more prevalent in patients with slow gastric transit (35% vs. 22%). In the univariate analyses, anti-vinculin antibody positive patients were more likely to have limited cutaneous disease (OR 9.60, 95% CI 1.19, 77.23) and thyroid disease (OR 4.09, 95% CI 1.27, 13.21). Such patients were also less likely to have lung involvement based on a Medsger Severity Score ≥2 (OR 0.25, 95% CI 0.07, 0.92). Higher levels of anti-vinculin autoantibodies were associated with less gastric emptying (β coefficient −3.41, 95% CI −6.72, −0.09). The association between anti-vinculin antibodies and each of these clinical features remained significant in the multi-variable model. In particular, the presence of anti-vinculin antibodies (β coefficient −6.20, 95% CI −12.33, −0.063) and higher levels of anti-vinculin antibodies (β coefficient −3.64, 95% CI −7.05, −0.23) were each significantly associated with slower gastric transit.
Conclusions:
Anti-vinculin antibodies associate with slower gastric transit in SSc and may provide insight into GI complications of SSc.
Keywords: systemic sclerosis, scleroderma complications, vinculin antibodies, gastrointestinal, SSc-related gastrointestinal disease, GI dysmotility
INTRODUCTION
Systemic sclerosis (SSc) is a rare immune-mediated connective tissue disease characterized by progressive vasculopathy, autoimmunity, and fibrosis. Over time, it can lead to multiorgan dysfunction with a highly heterogenous clinical presentation (1). The gastrointestinal (GI) tract is the most commonly affected organ system in SSc (2); any segment of the GI tract can be involved and patients may present with a variety of GI symptoms. Patients can develop severe GI disease at any time during their disease course regardless of disease duration. It remains a major challenge to identify patients at high risk for progressive GI disease, and predict which areas of the GI tract are likely to be affected (3).
Serum antibodies in SSc, such as anti-centromere antibody (ACA), anti-topoisomerase I antibody (ATA), and anti-RNA polymerase III antibody (ARA) are commonly detected early in the disease course and are associated with different patterns of skin and internal organ involvement. As a result, they serve as useful diagnostic and prognostic biomarkers in SSc (4, 5). Previous studies have identified functional autoantibodies targeting the muscarinic-3 acetylcholine receptor (anti-M3R) that contribute to severe lower GI dysmotility in SSc, although screening for these autoantibodies is not yet clinically available (6). Autoantibodies to ganglionic neuronal nicotinic acetylcholine receptor autoantibodies (anti-gAChR) are also present in a small subset of patients and may have functional implications (7). Nevertheless, the pathophysiology of GI disease in SSc remains unclear in many SSc GI subgroups and there are likely other biomarkers of GI disease that have yet to be discovered.
The protein vinculin has been previously identified as an autoantigen in patients with different GI diseases (8, 9), including irritable bowel syndrome (IBS) (10). Vinculin is a cytoskeletal protein that binds to actin and promotes cell adhesion, gut motility, and angiogenesis (11, 12). Many of the functional GI diseases are temporally associated with infectious gastroenteritis, and a model has been proposed that could explain this observation: cytolethal distending toxin (CdtB) produced by bacteria during infectious gastroenteritis (IGE) may result in the development of anti-CdtB antibodies, which then cross-react with vinculin protein found in the gut and lead to an immune response against vinculin (13, 14). Levels of anti-CdtB and anti-vinculin antibodies can distinguish patients with IBS-D (diarrhea-predominant IBS) from Inflammatory Bowel Disease (IBD) (10). More recently, a study in SSc demonstrated an association between anti-vinculin autoantibodies and GI symptom severity measured by the Gastrointestinal Visual Analogue Scale (GI-VAS) (15). A limitation of this study was that self-reported GI symptom scores were used to measure GI severity. Furthermore, these surveys are not specific to SSc and are unable to identify specific regions of dysfunction across the GI tract. In the present study, we therefore sought to determine whether the presence and titer of anti-vinculin antibodies in SSc associate with (a) abnormal GI transit measured by whole-gut transit (WGT) scintigraphy; and (b) GI symptom severity measured by the validated UCLA GIT 2.0 (University of California Los Angeles Scleroderma Clinical Trials Consortium Gastrointestinal Tract 2.0) instrument (16).
PATIENTS AND METHODS
Patients
All participants met either the 2013 ACR/EULAR classification criteria for SSc (17), the 1980 American College of Rheumatology (ACR) criteria (18), or had at least three of five features of the CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) (19) and were recruited from the Johns Hopkins Scleroderma Center during routine clinical visits. Participants with GI symptoms (including early satiety, nausea, vomiting, unintentional weight loss, distension, bloating, diarrhea, and/or constipation as determined by the treating physician) underwent the WGT study. Patients who were minimally symptomatic or asymptomatic were also recruited to capture the full spectrum of GI disease. All participants signed written, informed consent. Ethical approval was provided by the Johns Hopkins Institutional Review Board.
Clinical Phenotyping of SSc
Demographic included age, sex, and race. Race was determined with a self-questionnaire that included the following categories based on the standard data collection in our Scleroderma Center database: American Indian/Alaska Native, Asian, Black or African American, Indian Sub-Continent, Mid-East/Arabian, Native Hawaiian/Pacific Islander, White, Other/Unknown, Not Applicable. Other clinical data such as disease duration, smoking status, SSc subtype, presence (yes/no) of telangiectasia, calcinosis, arthralgia, synovitis, tendon friction rubs, thyroid disease (any), diabetes mellitus, gastric antral vascular ectasia (GAVE), and cancer history were documented by the physician at the patient’s first clinical encounter and at six-month intervals during follow-up visits. Disease duration was defined as the interval of time between the first non-Raynaud’s symptom and the WGT study. SSc subtype (diffuse cutaneous SSc or limited cutaneous SSc) was defined based on the extent of skin involvement. GI, cardiac, muscle, and lung involvement and severity were also captured at baseline and longitudinally using the Medsger severity score (20). The maximum modified Medsger GI Severity Score was used to characterize SSc GI severity. Five categories were evaluated including: (a) score 0 = normal (no GI symptoms); (b) score 1 = requiring GERD medications (including H2 blocker, proton pump inhibitor or pro-kinetic); (c) score 2 = High-dose GERD meds or A/biotics for bacterial overgrowth; (d) score 3 = episodes of pseudo-obstruction or malabsorption syndrome; (e) score 4 = severe GI dysmotility requiring enteral or total parenteral nutrition (TPN). Severe GI involvement was defined as a Medsger severity score ≥2. The presence of sicca symptoms was defined as the presence of any of the following: dry eyes for more than 3 months, the sensation of sand or gravel in the eyes, the use of artificial tears 3 times daily; dry mouth for more than 3 months, swollen salivary glands, and/or the requirement of liquids to swallow due to dry mouth. Other clinical variables are defined in the supplemental methods.
Other instruments
UCLA GIT 2.0 Survey
The University of California Los Angeles Scleroderma Clinical Trials Consortium Gastrointestinal Tract 2.0 (UCLA GIT 2.0) is a validated patient-reported outcome measure designed to quantify GI symptoms in SSc. The UCLA GIT 2.0 questionnaire contains 34 items, organized into seven subscales: reflux, distension/bloating, soilage, diarrhea, social functioning, emotional wellbeing, and constipation, and a total score is calculated (21). The maximum UCLA GIT scores were utilized in the analysis to capture phenotype.
Whole Gut Transit Scintigraphy
All participants underwent WGT scintigraphy. Patients fasted the night before the study and were instructed to avoid promotility agents, antibiotics, opiates, benzodiazepines, and stool softeners three days prior to the study. Per protocol, patients consumed a standard amount of radiolabeled In-111 water to assess esophageal and liquid gastric emptying and a radiolabeled Tc-99m egg meal to evaluate solid gastric emptying. Subsequently, a gamma camera was used to capture anterior and posterior images of the GI tract at predetermined periods of time (1/2 h, 1hr, 2hrs, 4hrs, 6hrs, 24hrs, 48hrs and 72 hrs) to evaluate transit throughout the entire GI tract. Slow gastric transit was defined as delayed solid gastric emptying at 4 hours. Slow colonic transit was defined as <14% emptying at 24hr, <41% emptying at 48hr, and <67% emptying at 72 hr (22).
Antibody Profiles
A second-generation enzyme-linked immunosorbent assay (ELISA) was used to measure anti-vinculin antibodies in the sera of patients using a validated second-generation assay established at Cedars Sinai (23). After undergoing epitope optimization, the antigen was mobilized onto high-binding plates and blocked with 3% bovine serum albumin in PBS to counter non-specific binding. Anti-vinculin antibody levels were evaluated after 70 minutes using optic densities (OD) obtained after measuring the absorbance at 370nm. The anti-vinculin antibody assay was considered positive when the OD was ≥ 1.68 per previously published studies (10, 15).
Patient sera were also screened for other autoantibodies using the commercially available Euroline immunoblot assay (Scleroderma Nucleoli Profile Uroline [IgG]; Euroimmun, Lubeck, Germany). This assay provides the ability to screen for autoantibodies to Scl-70, centromere (CENP A or CENP B), RNA polymerase-3 (RP11 or RP155), Fibrillarin (U3RNP), Th/To, Ku, PmScl75, and PmScl100. Medium to strong bands (>11) on signal intensity were considered positive and were determined based on the manufacturer’s cutoffs. Positivity to either subunit of centromere, RNA polymerase-3, and PmScl were considered positive.
Statistical Methods
We examined associations between dichotomous variables using chi-square or Fisher’s exact tests where appropriate. The student’s t-test was used to evaluate differences in the mean values of two continuous variables, and the Wilcoxon-Mann-Whitney test was used to compare the medians of highly skewed continuous variables. The Spearman rank coefficient was used to assess the linear correlation between two continuous variables. Univariable linear and logistic regression analyses were used to examine the strength of bivariate associations. Multivariable regression analyses were constructed to determine whether associations remained after adjusting for potential confounders. All statistical analyses were conducted using STATA Version 14 (StataCorp, College Station, TX). Statistical tests were two-sided, and statistical significance was defined as p<0.05 for all analyses.
RESULTS
Anti-vinculin antibodies and SSc clinical characteristics
Based on the ideal cutoff levels, 20 (23%) of 88 patients with SSc had levels over the threshold of optical density (OD) ≥1.68. The detailed clinical and demographic features of SSc patients who were positive and negative for anti-vinculin antibodies are shown in Table 1. There was no difference in age, sex, race, or disease duration between anti-vinculin antibody positive and negative patients. Other clinical features of SSc including mRSS, cardiac involvement, myopathy, cancer, severe GI involvement, Gastric Antral Vascular Ectasia (GAVE), sicca symptoms, calcinosis, tendon friction rubs, and telangiectasias were not significantly different between the anti-vinculin antibody positive and negative patients.
Table 1.
Evaluation of associations between SSc clinical characteristics and anti-vinculin antibodies
| Anti-vinculin antibodies | |||
|---|---|---|---|
| Variable | Positive | Negative | p-value |
| Age at first symptom, mean (SD) | 62 ±10.7 | 57 ±12.3 | 0.084 |
| Female subjects % (n) | 88.2% (15/17) | 89.3% (50/56) | 1.000 |
| Race | |||
| White, % (n) | 94.1% (16/17) | 72.7% (40/55) | 0.095 |
| Disease duration from first non- Raynaud’s symptom to date of WGT, median (IQR) | 14.3 (3.7,15.3) | 7.8 (4.9,16.4) | 0.520 |
| Ever smoker, % (n) | 24.4% (5/17) | 35.7% (20/56) | 0.631 |
| SSc Type | |||
| Limited cutaneous disease, % (n) | 94.1% (16/17) | 62.5% (35/56) | 0.016 † |
| Maximum mRSS, median (IQR) | 4.0 (2.0, 6.0) | 4.0 (2.0, 9.0) | 0.520 |
| Significant Raynaud’s phenomenon, (score ≥2), % (n) | 23.5% (4/17) | 39.3% (22/56) | 0.266 |
| Severe GI involvement, % (≥2), %(n) | 70.6% (12/17) | 74.6% (41/55) | 0.746 |
| GAVE, % (n) | 11.8% (2/17) | 5.5% (3/55) | 0.586 |
| Cardiac involvement (≥1), % (n) | 13.3% (2/15) | 18.0% (9/50) | 1.000 |
| Myopathy, % (n) | 11.8% (2/17) | 13% (7/56) | 1.000 |
| Lung involvement (≥2), % (n) | 26.7% (4/15) | 59.1% (26/44) | 0.039 † |
| Cancer, % (n) | 41.2% (7/17) | 19.6% (11/56) | 0.071 |
| Telangiectasia, % (n) | 88.2% (15/17) | 75.0% (42/56) | 0.329 |
| Calcinosis, % (n) | 17.7% (3/17) | 26.8% (15/56) | 0.536 |
| Arthralgia, % (n) | 76.5% (13/17) | 72.7% (40/55) | 1.000 |
| Synovitis, % (n) | 23.5% (4/17) | 10.9% (6/55) | 0.232 |
| Tendon friction rub, no. (%) | 5.9% (1/17) | 7.3% (4/55) | 1.000 |
| Sicca symptoms, % (n) | 70.6% (12/17) | 76.8% (43/56) | 0.604 |
| Pulmonary function parameters, mean (SD) | |||
| FVC % predicted | 78± 25.4 | 79 ± 25.4 | 0.897 |
| DLCO % predicted | 72 ± 24.4 | 61 ± 27.1 | 0.202 |
| Pulmonary Fibrosis, % (n) | 26.5% (4/17) | 43.6% (24/55) | 0.165 |
| RVSP by echo (mmHg), median (IQR) | 37.0 (35.0, 42.0) | 31.0 (25.0, 35.0) | 0.031 † |
| Thyroid, % (n) | 47.1% (8/17) | 17.9% (10/56) | 0.014 † |
| Diabetes, % (n) | 5.9% (1/17) | 1.8% (1/56) | 0.414 |
| Dead, % (n) | 0% (0/17) | 3.6% (2/56) | 1.000 |
| Autoantibody positive, (%) (n) | |||
| Anti-Topoisomerase-1 | 6.7% (1/15) | 16.7% (9/54) | 0.442 |
| Anti-RNA-polymerase III | 6.7% (1/15) | 0.0% (0/54) | 0.217 |
| Anti-Centromere | 60.0% (9/15) | 37.0% (20/54) | 0.144 |
| Anti- Ku | 6.7% (1/15) | 1.9% (1/54) | 0.390 |
| Anti-Th/To | 0% (0/15) | 0% (0/54) | N/A |
| Anti-U3-RNP | 6.7% (1/15) | 1.9% (1/54) | 0.390 |
| Anti-PM-Scl | 0.0% (0/15) | 7.4% (4/54) | 0.570 |
| Medications | |||
| Proton Pump Inhibitor | 85.0% (17/20) | 93.0% (53/57) | 0.367 |
| Prokinetics | 25.0% (5/20) | 23.0% (14/61) | 0.605 |
| Immunosuppressant | 60.0% (9/15) | 62.0% (28/45) | 0.878 |
Statistically significant
SSc = Systemic sclerosis; WGT= whole-gut transit scintigraphy study; mRSS = modified Rodnan Skin Score; significant Raynaud’s phenomenon = maximum Medsger severity score ≥2; GAVE = Gastric Antral Vascular Ectasia; severe GI involvement = maximum Medsger GI severity score ≥2; Cardiac involvement = maximum Medsger cardiac severity score ≥ 1; Lung involvement = maximum Medsger severity score ≥2. Normal FVC = forced vital capacity >70%, Normal DLCO = diffusing capacity of carbon monoxide >60%; RSVP = estimated right ventricular systolic pressure by echocardiogram
Patients who were anti-vinculin antibody positive were significantly more likely to have limited cutaneous disease (94.1% vs. 62.5%; p=0.02) and thyroid disease (47.1% vs. 17.9%; p=0.01). Among the anti-vinculin antibody positive patients, with thyroid disease, all of them had hypothyroidism. Anti-vinculin antibody positive patients were also less likely to have pulmonary involvement as defined by a Medsger severity score ≥2 (26.7% vs. 59.1%; p=0.04) and had a higher estimated right ventricular systolic pressure (RVSP) by echo [37.0 mmHg (35.0, 42.0) vs. 31.0 mmHg (25.0, 35.0); p=0.03)] compared to anti-vinculin antibody negative patients. Other cardiopulmonary parameters, including the mean % predicted FVC and DLCO on pulmonary function testing, were similar between the two groups. Of the anti-vinculin positive SSc patients, 60.0% (9/15) were also positive for anti-centromere antibodies, 6.7% (1/15) were also positive for anti-Topoisomerase I (Scl-70) and 6.7% (1/15) were positive for anti-RNA polymerase III.
Univariate Models
Univariate linear and logistic regression analyses were performed to examine the strength of the association between SSc-specific clinical features and positivity for anti-vinculin antibodies (Table 2). Anti-vinculin antibody positive patients were 9.6 times as likely to have limited cutaneous disease as those who were anti-vinculin antibody negative (95% CI 1.19, 77.23; p=0.03), and they had more than triple the risk of thyroid disease (OR 4.1, 95% CI 1.27, 13.21; p= 0.02). Anti-vinculin antibody positive patients also had 75% lower risk of lung disease (OR 0.25, 95% CI 0.07, 0.92; p=0.04) and had a higher estimated RVSP (OR 1.22, 95% CI 1.00, 1.49; p=0.05).
Table 2.
Measuring the association between antibody positivity and SSc clinical characteristics
| Clinical and demographic features | Odds Ratio | 95% CI | p-value |
|---|---|---|---|
| Age at first symptom | 1.05 | 0.99–1.10 | 0.089 |
| Female subjects | 0.90 | 0.16–4.93 | 0.903 |
| Race | |||
| White | 6.00 | 0.73–49.3 | 0.095 |
| Disease duration from first non-Raynaud’s symptom to date of WGT study | 1.04 | 0.97–1.12 | 0.237 |
| Ever smoker | 0.75 | 0.23–2.44 | 0.632 |
| SSc Type | |||
| Limited cutaneous disease | 9.60 | 1.19–77.23 | 0.034 † |
| Significant Raynaud’s phenomenon (score ≥2) | 0.48 | 0.14–1.65 | 0.241 |
| Severe GI involvement (≥2) | 0.82 | 0.25–2.74 | 0.747 |
| GAVE | 2.31 | 0.35–15.1 | 0.382 |
| Cardiac involvement (≥1) | 0.70 | 0.13–3.67 | 0.674 |
| Myopathy | 0.93 | 0.18–4.98 | 0.936 |
| Lung involvement (≥2) | 0.25 | 0.07–0.92 | 0.036 † |
| Cancer | 2.86 | 0.89–9.22 | 0.078 |
| Telangiectasia | 2.50 | 0.51–12.32 | 0.260 |
| Calcinosis | 0.59 | 0.15–2.33 | 0.447 |
| Arthralgia | 1.22 | 0.34–4.33 | 0.760 |
| Synovitis | 2.51 | 0.62–10.24 | 0.199 |
| Tendon friction rub | 0.80 | 0.08–7.65 | 0.844 |
| Sicca Symptoms | 0.73 | 0.22–2.44 | 0.604 |
| Pulmonary function parameters | |||
| FVC % predicted | 1.00 | 0.98–1.02 | 0.895 |
| DLCO % predicted | 1.01 | 1.00–1.04 | 0.202 |
| RVSP by echo | 1.22 | 1.00–1.49 | 0.051 |
| Thyroid | 4.09 | 1.27–13.21 | 0.019 † |
| Diabetes | 3.44 | 0.20–58.09 | 0.392 |
| Autoantibody positive | |||
| Anti-Topoisomerase-1 | 0.35 | 0.04–3.07 | 0.348 |
| Anti-Centromere | 2.55 | 0.79–8.23 | 0.117 |
| Anti-Ku | 3.79 | 0.22–64.39 | 0.357 |
| Anti-U3RNP | 3.79 | 0.22–64.39 | 0.357 |
Statistically significant
SSc = Systemic sclerosis; WGT= whole-gut transit scintigraphy study; significant Raynaud’s phenomenon = maximum Medsger severity score ≥2; GAVE = Gastric Antral Vascular Ectasia; severe GI involvement = maximum Medsger GI severity score ≥2; Cardiac involvement = maximum Medsger cardiac severity score ≥ 1; Lung involvement = maximum Medsger severity score ≥2. Normal FVC = forced vital capacity >70%, Normal DLCO = diffusing capacity of carbon monoxide >60%; RSVP = estimated right ventricular systolic pressure by echocardiogram
GI symptom scores in SSc patients with anti-vinculin antibodies
We explored whether anti-vinculin antibodies are associated with GI symptom severity as measured by the UCLA GIT 2.0 score. Patients across the cohort had median scores consistent with moderate severity for reflux [0.8 (IQR 0.3,1.3); ref mod= 0.5,1.0] (24), bowel distension [1.5 (IQR 0.8,2.1); ref mod= 1.01, 1.6), diarrhea [0.5 (IQR 0.0,0.5); ref mod=0.5,1.0], constipation [0.9 (IQR 0.5,1.3); ref mod= 0.5,1.0], and impact on emotional well-being [0.7 (IQR 0.2,1.8); ref mod=0.5,1.0] and a median score consistent with mild impairment in social functioning [0.3 (IQR 0.2,1.1); ref mod=0.0,0.49] based on the previously reported definitions of GI symptom severity (24). We found no significant association between the UCLA GIT 2.0 total score and positivity for anti-vinculin antibodies.
Association between whole gut transit and anti-vinculin antibodies
To determine whether anti-vinculin antibodies are associated with delayed GI transit and severity, we examined the associations between delayed transit in the esophagus, stomach, small bowel, and colon as measured by the WGT study and anti-vinculin antibody positivity (Table 3). Anti-vinculin antibodies were more prevalent among patients with delayed solid gastric emptying at 4 hours than in those without (35% vs. 22%), though this was not statistically significant. Anti-vinculin antibodies were not enriched among patients with delayed transit in other areas of the gut. Interestingly, a significant correlation was noted between higher anti-vinculin antibody levels and lower (worse) percent gastric emptying at 4 hours (β coefficient −3.41, 95% CI −6.72, −0.09, p=0.04). No significant correlations were identified for liquid and solid gastric emptying at 1 or 2 hours, esophageal transit time, small bowel emptying (6 hours), or colonic emptying (24, 48 and 72 hours).
Table 3.
Linear regression analyses evaluating the association between anti-vinculin antibody levels and GI transit
| Region of the gut | β coefficient | 95%CI | p-value |
|---|---|---|---|
| Esophagus | |||
| Esophageal Transit Time | −1.18 | −4.17,1.82 | 0.437 |
| Esophageal % emptying at 10 sec | 1.65 | −4.94, 8.24 | 0.619 |
| Stomach | |||
| Liquid | |||
| % Emptying at 1/2 hour | 7.03 | 9.59,23.6 | 0.399 |
| % Emptying at 1 hour | −0.83 | −5.70,4.04 | 0.735 |
| % Emptying at 2 hours | 1.48 | −5.73, 2.78 | 0.492 |
| Solid | |||
| % Emptying at 1 hour | −2.44 | −8.03, 3.13 | 0.384 |
| % Emptying at 2 hours | −1.54 | −7.94, 4.86 | 0.632 |
| % Emptying at 4 hours | −3.41 | −6.72, −0.09 | 0.044 † |
| Small Bowel | |||
| % Emptying at 6 hours | −2.47 | −8.40,3.46 | 0.409 |
| Colon | |||
| % Emptying at 24 hours | −2.90 | −9.58,3.78 | 0.389 |
| % Emptying at 48 hours | −5.68 | −25.12,13.76 | 0.562 |
| % Emptying at 72 hours | −8.38 | −28.92,12.14 | 0.419 |
Statistically significant
Normal ranges: Esophageal transit time (ETT) = >15 seconds; Esophageal emptying at 10 sec = ≥83%; Normal liquid T1/2 ≤74 min solid emptying 2 hrs ≥ 40%; solid emptying 4 hrs ≥ 90%; Normal small bowel transit time at 6 hrs ≥ 49%; Normal % colonic emptying at 72 hrs ≥67%
Multivariable Models
We then sought to determine whether the associations between anti-vinculin antibody positivity and clinical characteristics of SSc, remained after adjusting for age and sex (Table 4). In the multivariable model, anti-vinculin antibody positivity significantly associated with a higher odds of limited SSc (OR 8.99, 95%CI 1.05, 76.83; p=0.05), 78% lower risk of lung involvement (OR 0.22, 95% CI 0.06, 0.86, p=0.03), an increased odds of thyroid disease (OR 3.87, 95% CI 1.16, 12.93; p=0.03), and a higher estimated RVSP (β coefficient 6.41, 95% CI 0.37, 12.45; p=0.04). In the multivariable model, a significant association between the positivity for anti-vinculin antibodies and percent gastric emptying remained (β coefficient −6.20, 95% CI −12.33, −0.063, p=0.05). Because thyroid disease can negatively impact GI motility, we performed an additional analysis re-evaluating the association between anti-vinculin antibody positivity and percent gastric emptying, after also adjusting for thyroid disease. Importantly, the inverse association between anti-vinculin antibody levels and slow gastric transit persisted even after adjustment (β coefficient −6.94, 95% CI −13.1, −0.76, p=0.03).
Table 4.
Multivariable modeling with clinical and demographic features
| % Solid Gastric Emptying* | Thyroid | Limited cutaneous disease | Lung involvement | RVSP (mmHg) | |
|---|---|---|---|---|---|
| Anti-vinculin antibody (+/−) | β −6.20 95% CI −12.33, −0.063 (p= 0.048) † |
OR 3.87 95% CI 1.16, 12.93 (p= 0.028) † |
OR 8.99 95% CI 1.05, 76.83 (p= 0.045) † |
OR 0.22 95% CI 0.06, 0.86 (p=0.029) † |
β 6.41 95% CI 0.37, 12.45 (p=0.038) † |
| Anti-vinculin antibody levels | β −3.64 95% CI −7.05, −0.23 (p= 0.037) † |
OR 1.78 95% CI 0.87, 3.64 (p= 0.117) |
OR 2.44 95% CI 1.05, 5.70 (p= 0.038) † |
OR 0.48 95% CI 0.22, 1.02 (p= 0.057) |
β 3.38 95% CI 0.04, 6.72 (p=0.047)† |
All analysis were adjusted for Age and Sex;
at 4 hours
Statistically significant
Positive anti-vinculin antibodies had a threshold of optical density (OD) ≥ 1.68
RVSP= right ventricular systolic pressure measured by Echocardiogram; Lung involvement = maximum Medsger severity score ≥2
When examining the relationships between anti-vinculin antibody levels and clinical characteristics in the adjusted model, an inverse association between anti-vinculin antibody level and percent gastric emptying at 4 hours remained (β coefficient −3.64, 95% CI −7.05, −0.23; p=0.04). The association between higher anti-vinculin antibody levels and a higher odds of having limited cutaneous disease (OR 2.44, 95% CI 1.05, 5.70; p=0.04) and a higher RVSP (β coefficient 3.38, 95% CI 0.04, 6.72; p=0.05) also remained. Trends towards an association remained between higher anti-vinculin antibody levels and (a) less lung involvement (OR 0.48, 95% CI 0.22, 1.02; p=0.06); and (b) thyroid disease (OR 1.78; 95% CI 0.87, 3.64; p=0.12) were identified, though these associations no longer reached statistical significance.
DISCUSSION
Anti-vinculin antibodies are a novel autoantibody specificity identified in several GI diseases including IBS, functional dyspepsia and SSc. This is the first study to describe the extraintestinal clinical features of SSc patients who were positive for anti-vinculin antibodies and to examine the association between anti-vinculin antibodies and objective measures of GI transit by whole-gut scintigraphy. Using a previously established and validated ELISA, we found that anti-vinculin antibodies are common in SSc and were present in 23% of our patient cohort. This prevalence is similar to another study by Suliman et al. in which anti-vinculin antibodies were identified in 37% of a SSc cohort enriched for GI disease and in 32% of a SSc cohort enriched for vascular disease (15). While our cohort had slightly more severe GI disease compared to patients who did not complete the WGT study (25), we also included patients who were minimally symptomatic, which could explain why the prevalence of anti-vinculin antibodies in our cohort was slightly lower.
We report for the first time that anti-vinculin antibody positivity is associated with slow gastric transit in SSc, and that anti-vinculin antibody levels inversely correlate with percent gastric emptying. This finding is consistent with a recent study which demonstrated an inverse correlation between higher levels of circulating anti-vinculin antibody levels and the number of interstitial cells of Cajal (ICC) in the myenteric plexus of the human stomach (26). Vinculin is located in the ICC, which function as the pacemaker cells of the GI tract. It is not yet clear whether anti-vinculin antibodies have a direct pathogenic effect on the ICC resulting in GI dysmotility or whether they are a marker of GI dysfunction and ICC attrition. We did not find an association between anti-vinculin antibodies and percent emptying in the esophagus, small bowel or colon, despite the fact that ICC and vinculin are present throughout the GI tract. It is possible that ICC play a different role in the stomach than in other parts of the GI tract, though further studies are needed to understand why the association between anti-vinculin antibodies and GI dysmotility is specific for the stomach.
Anti-vinculin antibodies in IBS are thought to result from antibodies raised by the host against Cytolethal distending toxin B (CdtB), which is a toxin produced by Gram-negative bacteria that cause gastroenteritis, that cross-react with vinculin. This has been demonstrated in post-infectious models of IBS (14). It is not clear if this model can be extrapolated to SSc, as GI symptoms often occur later in the disease course and are not necessarily preceded by an infectious GI illness. It is possible that small intestinal bacterial overgrowth (SIBO) in SSc leads to the build-up of bacteria and the release of toxin (27), which then generates an immune response against CdtB and vinculin. It would be instructive to screen SSc sera for antibodies against both CdtB and vinculin.
We did not find an association between positivity for anti-vinculin antibodies and GI symptom scores measured by the GIT 2.0 questionnaire. This is consistent with a study by Suliman et al. which did not find an association between anti-vinculin antibodies and the UCLA GIT 2.0 (15). However, they did find a positive association between higher anti-vinculin antibody levels and the GI-visual analogue scale (GI-VAS) scores. This could be due to differences in patient reported outcome measures used to assess GI symptoms in SSc. We previously found a poor correlation between GI symptom scores measured by the GIT 2.0 and objective transit measures on the WGT study (28). Moreover, the GIT 2.0 was not administered on the same day as the blood draw and WGT study, which may have impaired our ability to determine associations with symptom scores.
We found that thyroid disease was enriched among SSc patients who were positive for anti-vinculin antibodies. Hypothyroidism is known to alter GI dysmotility and lead to an increased prevalence of SIBO (29). However, when we also adjusted for hypothyroidism in the multivariable model, the association remained between anti-vinculin antibodies and slow gastric transit, suggesting that thyroid disease is not the primary driver behind this relationship. We also found that patients with limited cutaneous disease and/or patients with less severe lung disease were more likely to be anti-vinculin antibody positive. Our findings suggest that patients with this clinical phenotype should be monitored for upper GI symptoms more vigilantly. Interestingly, Suliman et al. previously found a trend toward more pulmonary arterial hypertension among patients with anti-vinculin antibodies in a SSc group enriched for vascular disease (15). The positive association that we identified between anti-vinculin antibodies and a higher estimated RVSP further supports this finding. Of note, we did not identify an association between vinculin antibodies and sex, which is particularly notable given that slow gastric transit, functional dyspepsia, and IBS are significantly more common among women (30, 31). This suggests that the mechanisms causing GI disease in SSc are (partly) distinct from those that cause functional GI disorders in the general population.
Strengths of this study are the use of a well-characterized, prospective SSc cohort with objective transit data measured by WGT scintigraphy. There are several limitations of our study. Our cohort was enriched for GI disease so we were unable to determine the true prevalence of anti-vinculin antibodies in a general SSc cohort. Also, the GIT 2.0 questionnaire was not administered concurrently with the WGT study and the blood draw. Although prior studies have shown that GI symptoms reflect damage from the disease and are relatively stable over time (32), this still could have introduced some variability into our study. Lastly, mechanistic investigations should be done to understand the association between gastric emptying and anti-vinculin antibodies.
In conclusion, we demonstrate that anti-vinculin antibodies are common in SSc and are associated with delayed gastric emptying on the WGT study, limited cutaneous disease, a high RVSP on echo, thyroid disease, and less severe lung disease. If validated in another cohort using objective transit data, anti-vinculin antibodies could potentially be a useful marker for slow gastric transit and extraintestinal phenotype in SSc.
Supplementary Material
Significance and Innovations.
Anti-vinculin antibody positivity and anti-vinculin antibody levels are associated with slow gastric transit in SSc.
Extra-intestinal features associated with anti-vinculin antibodies include limited scleroderma, thyroid disease, a higher RVSP, and less severe lung disease.
Slower gastric transit remains significantly associated with anti-vinculin antibody positivity and higher anti-vinculin antibody levels even after adjusting for age and sex.
Acknowledgements
The authors would like to thank Dr. Jay Pasricha for his contributions.
Funding:
This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases at the National Institute of Health [K23 AR071473 to ZM], the Rheumatology Research Foundation, the Scleroderma Research Foundation, and the Jerome L. Greene Foundation. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript publication.
Disclosure of statement:
MP has received grants from Bausch Health and Synthetic Biologics, has been an advisor for Bausch Health, Synthetic Biologics, Ardelyx, Progenity and 9 meters, equity for Synthetic Biologics and Gemelli Biotech and speaker for Ardelyx, not relevant to the current study. The other authors have declared no conflict of interest.
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