Circulating miRNAs have recently emerged as candidate biomarkers for disease, particularly cancer (36,37,39,58). The present study is the first to demonstrate the potential of circulating miRNAs as noninvasive biomarkers of pediatric CD. An initial screen of patients with CD by microfluidic qRT-PCR array identified a significantly altered serum miRNA profile in comparison with healthy controls. These findings were subsequently validated in a much larger set of cases and controls.
All 24 miRNAs significantly altered in CD sera were elevated. The pathogenesis of IBD is a complex process involving inflammatory signaling, lymphocyte infiltration of the gut, and epithelial cell damage. Each of these may result in increases in the levels of circulating miRNA. For example, exosomes secreted in the course of inflammatory signaling may carry specific miRNAs into the circulation. The intestine is a highly vascular organ, and thus activated lymphocytes in the lamina propria may contribute to circulating miRNA. Furthermore, injury to intestinal epithelia may result in increases in epithelium-specific miRNAs in the circulation, as has been observed for tissue miRNAs in heart or liver injury (39,59). For instance, we found that circulating miR-192 is elevated in CD; miR-192 is also the most greatly expressed miRNA in intestinal epithelia (31).
Serological testing is frequently used in the diagnosis of children with suspected IBD, although evidence suggests current markers are suboptimal as screening tools for disease in this patient population, with reported sensitivities ranging from 55% to 71% (63–65). Thus, although the diagnosis of CD ultimately must be made on histopathologic grounds, the introduction of improved noninvasive testing may help to close the gap between the onset of symptoms and the final diagnosis, allowing for earlier treatment. Conversely, a negative screening test result may help reduce unnecessary endoscopy/colonoscopy.
The serum miRNAs examined here display encouraging diagnostic utility, performing favorably in comparison with some standard serological markers. MiR-484 and let-7b each possessed sensitivities > 80%, and 3 had specificities > 90% in comparison with healthy controls. In addition, each panel miRNA was unchanged in the serum of celiac patients compared with age-, race-and sex-matched controls, suggesting that these miRNAs may be specific for IBD or CD, rather than simply indicators of intestinal inflammation in general. This finding contrasts with current IBD serological markers, which are often present in non-IBD intestinal disease. For instance, ASCA is detected in a large proportion of patients with celiac disease, whereas perinuclear anti-neutrophil cytoplasmic antibodies can also be present in celiac disease or microscopic colitis (9,12,13).
Once the diagnosis of IBD is made, the current serological markers of CD are of limited use because they correlate poorly with disease activity or outcome in both adult and pediatric patients (66,67). Studies of circulating miRNA suggest that it may be a more dynamic biomarker; for example, levels of plasma miR-1, the most abundantly expressed miRNA in the heart (68), are elevated at the time of diagnosis in acute myocardial infarction and return to normal by the time of hospital discharge (38). Likewise, miRNA released from tumor cells can significantly alter circulating miRNAs levels, which normalize following tumor resection (42,69,70). We have found that after 6 months of treatment, serum miR-484 and miR-195 levels were significantly reduced from levels observed at the time of diagnosis. Reductions in panel miRNA levels did not significantly correlate with improved PCDAI scores. However, it remains possible that serum miRNAs accurately represent improvements at the mucosal level, as clinical scoring systems, as well as other surrogate markers, correlate poorly with mucosal healing (71,72).
A number of limitations to this preliminary analysis can be rectified with a larger cohort of cases and controls. The nonsignificant trend toward decreased circulating levels of several miRNAs in the longitudinal analysis may be because of lack of statistical power. In addition, it will be essential to obtain adequate numbers of CD cases reflecting different disease locations and types of complications. Likewise, it will be important to determine whether UC is characterized by a distinct set of circulating miRNAs, because these may be useful in distinguishing UC from colitis caused by CD. It will also be essential to analyze circulating miRNA levels in patients with gastrointestinal complaints not caused by CD (eg, infectious, allergic, or functional disease) because this is the group in whom noninvasive testing can be most useful. The analysis of other inflammatory conditions, such as rheumatoid arthritis, can also help establish the specificity of the CD-associated circulating miRNAs, although in practice, other clinical features can also serve to distinguish CD and nongastrointestinal disease.
In summary, this pilot study has identified a number of miRNAs significantly increased in the serum of patients with pediatric CD These CD-associated miRNAs display encouraging clinical utility that will require confirmation in large validation groups. Our findings suggest that large-scale investigations combining circulating miRNAs, laboratory, and genetic markers of CD may result in composite models with improved sensitivity and specificity for IBD in general and CD in particular.
The authors wish to thank Dr Alessio Fasano and Ms Debby Kryszak for providing sera from patients with celiac disease and associated controls.
We are grateful to all of the members of the Friedman laboratory; the Division of Gastroenterology, Hepatology, and Nutrition; those who have supported the Center for Pediatric IBD at Children's Hospital of Philadelphia, and most of all, the patients who generously agreed to participate in research on IBD.
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