A Novel Noninvasive Method Based on Salivary Inflammatory Biomarkers for the Screening of Celiac Disease.

Celiac disease (CD) is a chronic, immune-mediated disorder caused by an intolerance to ingested gluten present in some cereals such as wheat, rye, and barley. CD develops in genetically susceptible individuals and it has been estimated that it affects approximately 1% of Caucasians.,

The diagnosis of CD is based on the presence of intestinal symptoms together with the evaluation of genetic (HLA-DQ2+ and/or DQ8+), serologic (anti-endomysium and antitransglutaminase autoantibodies), and histologic markers. It has been argued that approximately 70% of CD cases are not diagnosed properly as a result of negative serologic results and the complex interpretation of the histologic findings in intestinal biopsy specimens, especially in older children and adult patients. Moreover, diagnostic endoscopy is an expensive procedure that involves sedation or anesthesia, and no more than 1:7 people with the highest HLA risk genotype are finally diagnosed with CD. However, an early and proper diagnosis of the disease is of great importance to avoid extraintestinal complications, including cardiovascular and neurologic problems or the development of certain types of cancer. Thus, additional diagnostic tests, preferentially tests that are cost effective and noninvasive, are strictly necessary.

Oral mucosa is the first part of the gastrointestinal system and it has been suggested that it could resemble its immunopathologic characteristics, being a much more accessible tissue for CD diagnosis. Given that saliva is the most accessible body fluid, it has been broadly scrutinized for biomarkers of noninvasive diagnosis of a wide range of disorders, including inflammatory bowel disease. Although some attempts for CD screening in saliva have been performed (ie, antibody detection), gene expression analyses of CD-related inflammatory cytokines have not been assessed in this fluid.

Considering that CD is highly underdiagnosed and that actual tests are expensive and invasive, our aim was to analyze the expression of inflammatory genes in the intestine and saliva of celiac patients and controls to find salivary biomarkers that resemble the status of the intestinal epithelium and that could be used for diagnosis. In addition, we wanted to leverage the saliva collection to set up HLA genotyping in this fluid, strengthening the predictive power of the gene expression signature.

To select potential biomarkers, we quantified the expression of 92 inflammatory genes in intestinal and saliva samples from 6 individuals (3 celiac patients and 3 controls). Fourteen of the genes tested were expressed in all the samples, in both saliva and small intestine (Figure 1A). The 8 inflammatory genes with the highest and most reproducible expression levels were selected for subsequent analyses in intestinal and saliva samples from another 18 celiac patients and 21 non-celiac individuals. All genes were expressed in the intestine, although in 3 of the saliva samples 1 to 3 genes could not be detected (Figure 1B and C). To evaluate if the inflammatory gene expression in saliva resembles the status of the celiac intestinal epithelia, we compared gene expression between celiac and non-celiac patients in both tissues. We found that genes CXCL1 and IL1B were up-regulated in CD biopsy specimens (Figure 1D). Likewise, CXCL1 and IL1B presented increased expression in the saliva of patients (Figure 1E). Correlation analyses between intestine and saliva showed a statistically significant correlation between the levels of these genes in both tissues (Figure 1F), suggesting that the celiac-related gene expression changes of the intestine can be assessed in saliva. Investigating whether other inflammatory conditions of the gut or mouth also present these or other alterations in saliva gene expression would be of great interest.

Figure 1

Heatmaps of Expression values were normalized against the average expression of each gene. Differential expression of the genes CXCL1 and IL1B in the () small intestine and () saliva samples from active celiac patients celiac disease (CD) (N = 18) and controls (Ctrl) (n = 21). ∗∗P < .01; ∗P < .05; +P < .1 by Student t test. (F) Pearson correlation (r) of CXCL1 (grey dots) and IL1B (black dots) expression between small intestine and saliva samples (n = 39).

Subsequently, we evaluated whether these salivary biomarkers could have clinical utility to differentiate between CD patients and non-celiac controls in the same sample set. Receiver operating characteristic curve analyses yielded area under the curve (AUC) values greater than 0.7 for both genes (Supplementary Figure 1A and B). In addition, a logistic regression model was built using the combination of both biomarkers. The combinatory logistic regression model yielded a receiver operating characteristic plot AUC value of 0.69 (Supplementary Figure 1C and D). Finally, the addition of the HLA genotyping (CD risk, HLA-DQ2/DQ8+; or nonrisk) in the same saliva samples improved all 3 models, giving a 0.84 AUC value (P < .01; 95% CI, 0.7064–0.9742) (Figure 2A and B), with 81% sensitivity and 67% specificity in distinguishing celiac patients from non-celiac individuals.

Supplementary Figure 1

Receiver operating characteristic (ROC) plot of the individual gene expression values of ( (C) ROC plot of the combinatory logistic regression model of the expression of both genes in the evaluation cohort (n = 39). (D) Comparison of the combinatory logistic regression values using gene expression between celiac disease patients (CD) and controls (Ctrl) in the evaluation cohort (n = 39). ∗∗P < .01 based on an unpaired Student t test. AUC, area under the curve.

Figure 2

Receiver operating characteristic (ROC) plot of the combinatory logistic regression model including gene expression and HLA genotype in the Comparison of the combinatory logistic regression values between celiac patients (celiac disease [CD]) and controls (Ctrl) in the () evaluation cohort (n = 39) and () confirmation cohort (n = 100). ∗∗P < .01 by Student t test.

To validate if this model could be useful for a noninvasive diagnosis of CD we performed a blind analysis of 100 saliva samples of individuals who were evaluated by the gastroenterologists as potential celiac patients. We assigned a disease/non-disease classification to each patient based on the values of the combined expression of the 2 biomarkers plus the presence of the HLA risk genotype. We correctly classified 73% of the individuals (Figure 2D) with 91% sensitivity and 51% specificity, thus confirming the validity of the technique for CD screening. If only those patients being prediagnosed as celiac using this model were subjected to endoscopy and biopsy acquisition, we would improve the positivity rate from the actual 1:7 to 2:3.

Here we present a systematic study profiling inflammatory markers in saliva samples of CD patients, enhancing the prospect of an important role for salivary diagnostics in the detection of gastrointestinal pathologies. Moreover, we have used the same starting material for both gene expression analyses and HLA genotyping, limiting the collection to a single noninvasive sample. We found that the use of this combination as a prediagnostic approach would reduce the number of patients subjected to endoscopy and biopsy acquisition and improve the positivity rate, with the subsequent savings in medical care and patient well-being.