Redefining the practical utility of blood transcriptome biomarkers in inflammatory bowel disease
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by altering phases of clinical relapse and remission. IBD can be defined by two major subtypes: Crohn's disease (CD) and ulcerative colitis (UC). The mechanisms underlying the development and pathophysiology of IBD are not fully understood but an overactive mucosal immune response against gut microorganisms in genetically susceptible individuals is thought to have major impact.
The abundance of available and emerging therapeutic options is good news for IBD sufferers, however, the complexity of the genetic makeup of the IBD disease in susceptible patients makes it difficult to identify and match the individual patient with the most suitable therapy option. Biomarkers that can predict genetically attributed risk and therapy response in order to indicate the right treatment for the right patient at the right time and minimize unnecessary endoscopic/radiological examinations are much needed.
Peripheral blood cells share about 80% of their transcriptome with that of other tissues and profiling of gene expression in these cells is employed routinely in descriptive and comparative analysis of autoimmune and inflammatory diseases. Previous studies, albeit on a relatively small sample of IBD patients, revealed that gene expression profiles generated by microarray analysis of whole blood cells may differentiate CD and UC from non-IBD conditions and even active/inactive states of Crohn's disease.
In this report the authors screened for IDB candidate genes using RNA sequencing of whole blood liquid biopsies taken from relatively large cohorts of both adult and pediatric patients with defined clinically active IBD.
In total 915 patients with IBD and 280 healthy control individuals were recruited and assigned to two groups: patients with remission or with mild IBD were considered to have inactive disease, whilst those with moderate or severe IBD were considered to have active disease. The control population did not suffer from inflammation or intestinal diseases. Most IBD patients were already being treated with Mesalazine, a drug used in the management of IBD, but blood samples were taken before any additional treatments were prescribed.
RNA sequencing analysis revealed 148 and 111 differentially expressed genes between pediatric IBD patients and controls, and between adult patients and controls, respectively.
For pediatric patients no single RNA was able to distinguish between CD and UC. In adult IBD 36 RNAs differentiated the two.
A 15 transcript panel of the most differentially expressed genes was investigated as a potential biomarker signature, S100A12, OPLAH, ATP9A, ANOS1, FCGR1A, ITGB4, UTS2R, MMP9, COX6B2, ANXA3, CACNA1E, GALNT14, IL18R1, PFKFB3 and KLRF. All genes, apart from KLRF1, were upregulated at least 2-fold in IDB pediatric group in comparison to the healthy controls.
S100A12, OPLAH, ATP9A, ANOS1, FCGR1A, ITGB4, UTS2R, MMP9, COX6B2 were upregulated in both adult and pediatric IBDs and the other 6 genes were upregulated in adults IBDs but not pediatric compared to controls.
Expression was confirmed by qRT-PCR using the same RNA samples as those used in the RNAseq analysis. All 15 genes were differentially expressed between pediatric patients with active IBD to controls including active UC and CD groups. None of these genes differentiated pediatric inactive IBD from controls.
In adults, ATP9A, GALNT14, KLRF1 and PFKFB3 differed between active IBD and controls. KLRF1 also differentiated inactive IBD from control patients.
The diagnostic potential of the 15 genes was investigated in a replicated dataset using blood samples collected from cohort of newly recruited 718 IBD patients and 184 healthy controls have confirmed the initial findings. This confirmed differential expression of all 15 genes between pediatric active IBD (UC and CD) and healthy controls. 13 of the 15 genes were also differentially expressed in inactive UC and CD versus controls.
In adults, UTS2R expression was significantly different in both active and inactive UC compared to controls. 10 of the genes were differentially expressed in active CD whilst 9 differentiated inactive CD from healthy patients.
Specificity and sensitivity of these 15 genes as a biomarker were assessed using ROC and AUC analysis. 9 genes were shown to have a high power to distinguish active IBD groups from controls in the pediatric cohorts but no genes showed sufficient power to discriminate between inactive IBD from controls.
None of the differentially expressed genes had enough discriminatory power to distinguish between adult IBD and health patients.
In summary, improvement in disease indexing systems, ease of access to patient-derived samples and establishing indicative IBD biomarker profiles needs to be pursued further to allow real time examination of the IBD phenotype and genotype in individual patients during a course of disease and treatment and to drive forward new treatments and possibly a cure for the IBD disorders.
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