As indicated in the new ESPGHAN diagnostic algorithm for CD [13
], small bowel biopsy may now be avoided in a sizeable proportion of patients who have clinical symptoms: anti-tTG antibodies levels 10 times the normal values, predisposing HLA genotype. Unfortunately, not all patients have such a high production of anti-tTG antibodies, and many are asymptomatic. Moreover, this new protocol is rarely applicable to at-risk relatives [13
]. Therefore, the aim of this study was to determine whether the gene expression profile of CD-associated genes in PBMs could help to differentiate patients affected by CD from controls as a step towards the molecular diagnosis of the disease. We studied the genes that have most often been associated with CD and those that show interesting functional profiles [3
]. As expected, the analysis of the expression of each single gene did not unequivocally differentiate between celiac and non-celiacs, but a multivariate combination of genes that are implicated in the pathogenesis of CD was selected in the target tissue.
We previously showed that the NF-kB complex is specifically and precociously implicated in the gluten-induced inflammatory response in celiac mucosa: in fact, the NF-kB complex is fully activated just 6 hours after gluten exposure [15
]. The cREL
and TNFAIP3 genes are both involved in the regulation of this nuclear activating complex. The former is one subunit of the complex, while the latter is a negative regulator of its activation. TNFAIP3
(also known as A20
) expression is up-regulated by NF-kB activation, as confirmed in our study, and it acts in a negative feedback loop to control NF-kB-dependent gene expression. Furthermore, the TNFAIP3
gene is a susceptibility locus for several human inflammatory and autoimmune diseases, including inflammatory bowel disease, rheumatoid arthritis, psoriasis, lupus and type 1 diabetes. TNFAIP3
is also frequently inactivated in subsets of B-lineage lymphomas that are characterized by NF-kB hyper activation and was therefore suggested to be a novel tumour suppressor [16
plays a crucial role in the activation of the gluten-induced mucosal activation through the NK system. It is most abundantly produced by CD4+ T cells and natural killer T (NKT) cells and is important for the development of the pro-inflammatory Th17 lineage. It has a protean function principally oriented to the activation of intraepithelial T-cell, which play a pivotal role in the development of the mucosal damage [17
gene controls the homing of intraepithelial lymphocytes (IELs), which are essential for the production of the gluten induced epithelial damage, and is less active in celiacs than in controls. It is the activation of IELs that drives the destruction of the intestinal epithelium. The main role of IELs is to promote immune protection by preventing the entry and spread of pathogens while avoiding unwanted and excessive inflammatory reactions capable of damaging the intestinal epithelium. To that end, IELs exert a cytolytic function to eliminate infected and damaged cells, and regulatory functions that contribute to epithelium healing and repair. Deregulated activation of IELs is a hallmark of CD and is critically involved in epithelial cell destruction and the subsequent development of villous atrophy. In addition, lymphocytic infiltration of the small intestinal epithelium in the absence of villous atrophy has been observed in patients with dermatitis herpetiformis [18
], an autoimmune skin manifestation of CD; a finding that supports the concept that intraepithelial lymphocytosis is a marker of CD even in the absence of intestinal damage [19
]. It was recently demonstrated that elevated RGS1
levels profoundly reduce T cell migration to lymphoid-homing chemokines, whereas RGS1
depletion selectively enhances such hemotaxis in gut T cells [20
]. Its capacity to limit the egress of inflammatory and/or autoimmune cells could clearly promote immunopathology.
Finally, the LPP
gene appears to have a relevant activity in modulating cell adhesion since it is an integral component of cell migration. It is not surprising that up- or down-regulation of LPP
expression results in an increase or decrease, respectively, in cell migration [21
]. Recent data showed that the over-expression of LPP
increased epidermal growth factor-stimulated migration of vascular SMCs induced by TGF-β1, suggesting the participation of LPP
in cell motility [22
Since the expression of these genes is not independent, it is not possible to give a priority of function to any of them: indeed genes that are not selected by the stringent criteria of the multivariate analysis may well have their own relevant function, but their contribution is no longer significant when other more “discriminating” genes are included in the equation. Nevertheless, it is very interesting to note that this model is built by genes that are more likely to exert a relevant function in the abnormal gluten-induced response in individuals with a specific genomic profile.
When we moved from the target tissue to PBMs, to our surprise, 3 of the 4 genes selected for their discriminating capacity are the same as those included in the multivariate model of the small intestinal mucosa tissue. Again the NF-kB complex appears implicated in development of CD, as well as the fascinating LPP
gene. The KIAA1109
gene, located in the region encompassing KIAA1109/Tenr/IL2/IL21
in chromosome 4q27, has often been replicated in association studies and provides a significant contribution to the discrimination between celiacs and non celiacs. This cluster region is involved in the differentiation of naïve human CD4+ T cells into Th17 cells [24
]. It is important to note that Th17 cells produce a variety of cytokines, among which, IL-17A, IL-17F, IL-21 and IL-22
. Genetic alterations in the 4q27 locus could result in non-functional IL-21
and hence lack of IL-17A
or vice versa. The regulation of this process may be an important factor in determining the risk for CD, as shown in such other autoimmune diseases as rheumatoid arthritis and uveitis [25
In conclusion, we report an intriguing picture of the possible relationships among the expressions of candidate CD genes in the target mucosa, but also, with a minor difference, in PBMs. The analysis of the expression of each single gene is non-informative and does not reveal the specific isolated function of any of these candidates. Indeed, only the search for functional pathways may shed light on the complex gluten-induced abnormal response in genetically predisposed individuals.
We suggest that the expression of a small set of candidate genes in PBMs can be used to distinguish CD patients from healthy controls and from disease controls (patients affected by Crohn’s disease), without considering clinical data, HLA or anti-tTG antibodies. In fact, the procedure we used resulted in a distance between groups (very low Wilk’s lambda) that is unusual with ordinary diagnostic tools. We did not add anti-tTG antibodies or HLA data to the multivariate equation because it is well recognized that the former has a very high sensitivity and specificity, and the latter has a very high negative predictive value. Since we reached a correct diagnostic classification in the validation set (above 95%) by gene expression data only, we would have shown an overoptimistic estimate by adding these strong discriminators. However, this should be done in clinical practice in order to reinforce the sensitivity and the specificity of the diagnosis when duodenal biopsy is not available or desirable.
Expression may be regulated by the specific polymorphism associated to the disease but, in the case of CD, none of the identified polymorphisms contributed greatly to the pathogenesis of the gluten-induced immune response. Indeed, expression data should be examined within the framework of a reasonable pathogenic pathway. The same gene may be over- or under-expressed and produce significant downstream stimuli. Expression is one of the several regulations that control the production of functional molecules from a specific protein-coding gene. Epigenetic mechanisms have recently emerged as important partners in this domain, and our group reported a specific microRNA that is over-expressed in celiac patients versus controls [27
Genome-wide studies have identified many encoding variants associated to CD. Several of these are implicated or are in linkage with regulatory DNA marked by deoxyribonuclease hypersensitive sites. Most of these sites are active during fetal development and associated with gestational exposure phenotype. Indeed, disease-associated variants often perturb transcription factor recognition sequences, altering the normal regulatory networks. In common human disease, and certainly in CD, regulatory DNA variations are likely to play a pivotal role, since there are no ‘missing’ or ‘failed’ genes [28
The missing variance of heredity in CD is probably due to the thousands of expression quantitative loci, expression ‘hot spots’, where a polymorphism at a locus is responsible for changes in gene expression of many other genes, and finally by gene-by-environment interactions [29
]. Gene expression is currently the best tool with which to explore the final results of genetic variance; it is quite robust and reproducible and may be tailored to specific target and non-target tissues. We cannot, therefore, predict a precise functional model of the gluten-induced immune response by studying a small, albeit important, set of genes, but we can try to obtain clues about this complexity. Interaction among genes, which is not considered in genome-wide association studies, is estimated by ordinary multivariate analysis, which is likely to provide an independent model of a possible function, or, at least, point to the genes whose expression is important in the differentiation between the affected and the unaffected.
Our discriminant function is proposed in the attempt to improve the diagnosis of CD and as a support to limit invasive techniques. Molecular analysis to discriminate a pathogenic from a healthy phenotype has become increasingly popular with the advent of innovative applications in many types of cancer and complex diseases [9
]. Esophago-gastro-duodenoscopy is still the gold standard for the diagnosis of CD, but it can decrease the patient’s compliance and is indeed a major bottleneck in developing countries: a simple blood sample, which can also be easily dispatched, may help to disseminate the diagnostic coverage to the majority of patients that cannot reach a specialized reference centre [30
In the near future, because of the new ESPGHAN protocol [13
], we may have no information about the status of the traditional target tissue in many patients: gene expression on a blood sample may well add safety and sensitivity to a biopsy-free diagnostic protocol, thereby providing a good proxy of the mucosal status.