Abstract

Background and Objectives

   Genome-wide association studies (GWAS) have identified several genetic
   regions involved in immune-regulatory mechanisms to be associated with
   celiac disease. Previous GWAS also revealed an over-representation of
   genes involved in type 2 diabetes and anorexia nervosa associated with
   celiac disease, suggesting involvement of common metabolic pathways for
   development of these chronic diseases. The aim of this study was to
   extend these previous analyses to study the gene expression in the gut
   from children with active celiac disease.

Material and Methods

   Thirty six target genes involved in type 2 diabetes and four genes
   associated with anorexia nervosa were investigated for gene expression
   in small intestinal biopsies from 144 children with celiac disease at
   median (range) age of 7.4 years (1.6–17.8) and from 154 disease
   controls at a median (range) age 11.4.years (1.4–18.3).

Results

   A total of eleven of genes were differently expressed in celiac
   patients compared with disease controls of which CD36, CD38, FOXP1,
   SELL, PPARA, PPARG, AGT previously associated with type 2 diabetes and
   AKAP6, NTNG1 with anorexia nervosa remained significant after
   correction for multiple testing.

Conclusion

   Shared genetic factors involved in celiac disease, type 2 diabetes and
   anorexia nervosa suggest common underlying molecular pathways for these
   diseases.

Introduction

   The prevalence of autoimmune disease including celiac disease has
   increased among the population in many high-income countries worldwide
   [[38]1, [39]2]. Celiac disease is an autoimmune intestinal disorder
   triggered by intolerance to gluten in genetic susceptible individuals
   carrying the HLA-DR3-DQ2 and DR4-DQ8 risk haplotypes [[40]3]. Another
   feature of celiac disease is the presence of autoantibodies directed
   against tissue transglutaminase (tTGA), the serological marker for the
   disease [[41]4, [42]5] as well as to protein kinase C delta (PRKCD)
   [[43]6]. It is well established that HLA-DQ heterodimers present gluten
   peptides to CD4^+ T-cells causing an inflammation of the gut mucosa
   leading to villous atrophy with malabsorption of vitamins and nutrients
   as a consequence [[44]5, [45]7].

   Most chronic inflammatory disorders have a multifactorial etiology
   caused by a cumulative effect of environmental factors triggering the
   disease in genetically susceptible individuals [[46]8]. To disentangle
   which of environmental and/or genetic factors that are involved in
   disease development and to provide a deeper understanding of the
   pathogenesis, linkage analysis, allele-sharing methods, genetic
   association studies in human populations, or genetic analysis of large
   crosses in model organisms have been developed [[47]9]. Genome-wide
   association studies (GWAS) enable testing of the whole genome in order
   to identify statistical association between genetic variants and a
   trait of interest to compare the frequency of genetic variants
   (alleles) in affected and unaffected individuals [[48]10].

   To date, GWAS has identified more than forty genome-wide significant
   non-HLA risk loci linked to celiac disease [[49]7, [50]11–[51]15]. Many
   of these celiac disease associated loci exhibit an overlap with those
   of other immune-related diseases such as type 1 diabetes (T1D)
   [[52]13]. In a previous family GWAS on celiac disease, an overlap
   between genes implicated in type 2 diabetes (T2D) and anorexia nervosa
   and gene-regions associated with celiac disease was revealed and a new
   model behind disease was suggested [[53]11]. Although there is an
   overlap of celiac disease and other autoimmune conditions, few studies
   have been performed on the possible common aetiology between celiac
   disease and metabolic conditions such as T2D and anorexia. Due to the
   large number of associated polymorphisms and possible bias towards
   common metabolic pathways, the overlap which was identified in the
   previous GWAS could be just a chance finding. However, there are also
   data supporting a connection between these metabolic pathways and
   celiac disease. A population study from India recently demonstrated an
   increased frequency of tTGA in T2D patients [[54]16]. Furthermore, a
   change in metabolism may affect the risk for disease. When a
   gluten-free diet was introduced in patients diagnosed with celiac
   disease, it reduced the risk of T2D, even when corrected for BMI
   [[55]17].

   Despite the fact that there can be a high variability of expression of
   genes in different tissues and between individuals making it a
   challenge to use this type of data for diagnostic purposes, targeting
   of genes and expression of them in the affected organ may lead to
   better understanding of how genes are involved in signaling pathways
   that eventually lead to chronic diseases such as celiac disease and
   T2D. The aim of this study was therefore to investigate potential
   common genetic factors contributing to the development of celiac
   disease, T2D and anorexia nervosa by analyzing gene expression in
   intestinal biopsies from children with active celiac disease as
   compared with disease controls.

Results

   A total of 46 target genes involved in T2D or in anorexia nervosa were
   identified by pathway enrichment analysis in our previous GWAS
   study[[56]11]. Six of these genes were analyzed for gene expression
   previously in our material [[57]11, [58]18] and the remaining forty
   genes were selected in this study for gene expression analysis. A pilot
   gene expression experiment was run on all 40 target genes involved in
   T2D or anorexia nervosa using 51 celiac disease cases and 64 controls.
   Out of these 40 target genes, 16 genes showed nominally significant
   differences in gene expression and were picked along with reference
   genes for the main experiment including 144 cases and 154 controls. The
   list of all 46 target genes and control genes are presented in [59]S1
   Table (including the six identified T2D genes which were analyzed for
   gene expression previously [[60]11].)

   Results from the 16 target genes from the main experiment and the fold
   change expression difference between the target genes and the reference
   “housekeeping” gene IPO8 are presented in [61]Table 1. Eleven genes
   reached a nominally significant p-value below 0.05, of which nine
   remained significant after adjusting for multiple comparisons using
   Bonferroni correction ([62]Fig 1). Of those nine genes, AKAP6 and NTNG1
   were genes associated with anorexia nervosa and the remaining seven
   (AGT, CD36, CD38, FOXP1, PPARA, PPARG and SELL) were selected for their
   relationship with T2D ([63]S1 Table).

Table 1. Results from the main gene expression analysis.

   The Delta-Delta C[T] (ΔΔC[T]) relative quantification method was used
   to estimate mRNA levels of target genes relative to a reference gene
   (IPO8) in the small intestinal biopsies from patients with celiac
   disease and compared with controls. A total of 144 cases and 154
   control samples were analyzed. The p-value is calculated using the
   independent samples t-test for equality of means (equal variances
   assumed).
   Gene P-value Mean Ct Difference Std. Error Lower Upper Bonferroni
   corrected p-value FC Percent change Direction in CD vs Control
   CD38 6.00E-19 0.74 0.08 0.59 0.89 2.40E-17 1.67 67% UP
   CD36 3.40E-14 1.19 0.15 -1.48 -0.9 1.36E-12 2.28 128% DOWN
   PPARG 8.60E-14 0.83 0.09 -1 -0.65 3.44E-12 1.77 77% DOWN
   PPARA 6.10E-10 0.33 0.05 -0.42 -0.23 2.44E-08 1.25 25% DOWN
   AGT 1.10E-08 0.59 0.09 0.42 0.76 4.40E-07 1.5 50% UP
   SELL 1.40E-08 0.88 0.15 0.59 1.17 5.60E-07 1.84 84% UP
   FOXP1 5.70E-05 0.22 0.05 -0.32 -0.12 2.28E-03 1.17 17% DOWN
   AKAP6 5.20E-04 0.29 0.08 -0.45 -0.12 0.02 1.22 22% DOWN
   NTNG1 1.10E-03 0.57 0.18 -0.92 -0.22 0.04 1.48 48% DOWN
   BCL2L11 1.70E-03 0.23 0.07 -0.38 -0.09 0.07 1.18 18% DOWN
   PIEZO2 6.70E-03 0.29 0.1 -0.49 -0.09 0.27 1.22 22% DOWN
   FTO 0.08 0.09 0.05 -0.01 0.19 1 1.07 7% UP
   HFE 0.09 0.19 0.11 -0.02 0.41 1 1.14 14% UP
   ZNF804B 0.11 0.43 0.28 -0.99 0.13 1 1.35 35% DOWN
   ADRA1D 0.18 0.36 0.22 -0.8 0.08 1 1.29 29% DOWN
   KCNJ11 0.53 0.1 0.12 -0.33 0.14 1 1.07 7% DOWN
   [64]Open in a new tab

   FC = Fold Change, CD = Celiac Disease

Fig 1. Nine target genes showing significant difference in gene expression
between celiac disease (CD) patients and control patients.

   [65]Fig 1
   [66]Open in a new tab

   The y-axis shows the fold change gene expression and the mean
   expression in control patients is set to one.

Discussion

   In this study we found that seven genes involved in T2D (CD36, CD38,
   FOXP1, SELL, PPARA, PPARG, AGT) and two genes involved in anorexia
   (AKAP6, NTNG1) were differently expressed in the small intestinal
   tissue of celiac patients compared with control patients, suggesting
   common genetic pathways leading to the disease among these
   phenotypically different chronic disorders. A plausible explanation
   could be that the genes discovered in this study are just an artifact
   of an inflamed tissue. However, except for CD38, AGT and SELL, the
   remaining genes are lower expressed in patients and therefore at least
   unlikely to be the result of an increased representation of immune
   cells in the intestinal tissue of celiac patients compared with
   controls.

   The T2D related gene, CD36, is down regulated in celiac patients in
   this study. Studies on this gene showed its various functions in many
   processes including angiogenesis, inflammation, lipid metabolism,
   atherosclerosis, and platelet activation [[67]19]. It has been shown
   that the CD36 gene is linked with increased risk of T2D [[68]20] and
   the CD36 protein level may serve as a biological marker of T2D
   [[69]21]. Transgenic mice overexpressing CD36 have reduced blood lipids
   and deficiency of CD36 could lead to insulin resistance [[70]22]. Our
   data show down regulation of CD36 in celiac patients and the CD36
   protein has also been previously shown to be significantly reduced in
   active as compared to inactive celiac disease and normal mucosal
   samples [[71]23]. If this is a consequence of inflammation or a risk
   for developing both diseases needs to be addressed.

   This study also showed that CD38 was up-regulated in children with
   celiac disease. However, when the expression of this gene was
   normalized to CD3D, which is a part of the T-cell receptor, the
   difference was only nominally significant. The CD38 protein is like
   CD36, a surface molecule and CD38 is expressed in human T and B cells
   during different stages of their development. A function of CD38 is
   mediating insulin secretion [[72]24] and an immune response with
   auto-antibodies to CD38 protein is present in T2D patients [[73]24,
   [74]25].

   Another finding from this study was the down-regulation of mRNA levels
   of both PPARA and PPARG in celiac disease children as compared with
   controls. Peroxisome Proliferator-Activated Receptors (PPAR)-alpha and
   PPAR-gamma are two of three known subtypes of PPARs acting as
   transcription factors activated by ligands. Peroxisomes contain enzymes
   necessary for the oxidation process [[75]26]. PPARs are known to
   regulate target genes of inflammatory responses as well as energy
   balance [[76]27]. Importantly, PPARs have been found to act as
   anti-inflammatory and for these reasons PPARs have been considered for
   the development of new therapies for chronic inflammatory diseases
   [[77]28]. Previous studies on PPARG revealed its contribution in
   numerous diseases, such as: obesity [[78]29], T2D [[79]30], and
   atherosclerosis [[80]27] as well as in celiac disease [[81]31]. It has
   also been shown that tTGA drives inflammation via PPARG down-regulation
   in celiac patients [[82]31] and that down-regulation of proteins
   involved in PPAR signaling are associated with the highest celiac
   disease histological score [[83]32].

   Angiotensin-2, or the AGT gene was more expressed in the small
   intestinal mucosa of celiac disease children as compared with controls.
   AGT encodes a potent vasoconstrictor and acts directly on vascular
   smooth muscle and it is associated with T1D and T2D as well as with
   cardiovascular disease [[84]33–[85]35]. Interestingly, olmesartan, an
   angiotensin II receptor antagonist, has been shown to cause a severe
   form of sprue-like enteropathy [[86]36]. SELL or L-selectin, encodes a
   cell surface adhesion protein which mediates the adherence of
   lymphocytes to endothelial cells. SELL is associated to both T2D
   [[87]37] and amyotrophic lateral sclerosis (ALS) [[88]38]. Both AGT and
   SELL are expressed at higher levels in celiac cases compared with
   controls indicating a high activity of adherence and interaction
   between leucocytes and endothelial cells.

   The forkhead box P1 (FOXP1) is an essential transcriptional regulator
   for thymocyte development and the generation of quiescent naive T cells
   [[89]39]. BCL2L11 (which was slightly above the corrected p-value of
   0.05) is important for apoptosis (cell death) and thymocytes lacking
   this pro-apoptotic Bcl-2 family member (also known as Bim) are
   refractory to apoptosis induced by TCR-CD3 stimulation [[90]40].
   BCL2L11 has been identified as an essential initiator of apoptosis in
   thymocyte-negative selection of autoreactive T-cells [[91]40]. BCL2L11
   and FOXP1 seems to have an important role in normal thymocyte apoptosis
   and are both significantly down regulated in celiac cases compared with
   controls in this study.

   AKAP6 and NTNG1 were previously reported in a GWAS of anorexia nervosa
   [[92]41]. AKAP6 is a member of A-kinase anchoring protein. Following
   the name, its main role is to attach enzymes and transport it near the
   target substrate. It is mostly expressed in brain and cardiac region as
   well as in skeletal muscles [[93]42]. NTNG1 (Netrin-G1) is a member of
   protein family playing an important role in the development of the
   human nervous system [[94]43]. Previous studies revealed that mutations
   in NTNG1 are associated with schizophrenia [[95]44] and Parkinson’s
   disease [[96]45].

   In conclusion, even though the common mechanisms for the development of
   celiac disease, T2D or anorexia nervosa remains unresolved, the present
   study shows that several genes associated with T2D and anorexia nervosa
   are differentially expressed in children with active celiac disease as
   compared with controls, indicating a connection between these diseases.

Materials and Methods

Ethics Statement

   This study has been conducted according to the principles expressed in
   the Declaration of Helsinki and approved by the Regional ethics board
   in Gothenburg. All guardians and study participants (when appropriate)
   gave their written informed consent. All personal data as well as
   results obtained from the research are coded and will remain
   confidential to all except for the treating physician at each hospital.

Biological material

   All children who were investigated at the Departments of Pediatrics
   with an upper endoscopy were consecutively asked to participate in the
   study as previously described [[97]18, [98]46].

   There were no exclusion criteria for participation. A child with a
   Marsh score >1 was considered to have CD and included as a case. A
   child with a Marsh score ≤1 was considered not to have CD and included
   as a disease control. In all, small intestinal biopsies and blood
   samples were collected from 144 cases with CD at a median age of 7.4
   years (range 1.6–17.8) and from 154 disease controls at median
   11.4.years (range 1.4–18.3). Among the disease controls, the most
   common diagnoses were gastroesophageal reflux disease and helicobacter
   pylori gastritis, whereas only eleven children had Crohn´s disease and
   four children ulcerative colitis, respectively. Another eight disease
   controls were detected with moderately elevated tTG antibody levels in
   a screening study and were considered to have potential CD.

RNA extraction

   Small intestinal biopsies were immediately put in a RNA stabilizing
   reagent, RNAlater solution (Life Technologies, CA, USA) and put in -4°C
   overnight in order to allow the reagent to penetrate the tissue. The
   biopsy was further frozen in -80°C until RNA extraction was carried
   out. Total RNA was extracted using the miRNeasy Mini Kit (QIAGEN,
   Germany) or the Maxwell^® 16 Total RNA Purification Kit (Promega)
   together with the Maxwell^®16 instrument. The RNA quality and quantity
   was checked with a NanoDrop 2000 spectrophotometer and a 2100
   Bioanalyzer (Agilent Technologies). RNA was converted to cDNA using the
   Vilo kit (Life Technologies, CA, USA).

Reference gene validation

   A total of 23 reference (housekeeping) genes were tested with the
   GeNorm algorithm [[99]47]. The IPO8 gene had the highest stability
   value (m<0.5) and was chosen as reference gene for normalization.

Gene Expression Analysis

   Quantitive gene expression analysis was performed by means of
   quantitative Polymerase Chain Reaction (qPCR) with TaqMan chemistry
   (Life Technologies, CA, USA). A total of 1 ng/reaction cDNA together
   with Master Mix was added to all genes simultaneously using a Nanodrop
   II dispenser (GC biotech, Netherlands), for a final reaction volume of
   2 μl per gene and sample. QPCR reaction was run on the real-time PCR,
   ABI PRISM 7900HT Sequence Detection System (Life Technologies, CA,
   USA). Raw data was analyzed with the SDS 2.4 and RQ manager 1.2.1
   software provided by the instrument.

Gene selection criteria

   The T2D risk genes were defined by the IPA analysis (Ingenuity Inc.,
   CA, USA) in our previous GWAS [[100]11]. All of these genes, except
   PPARG, were located in potential risk regions of celiac disease, with
   one or more nominally associated SNPs [[101]11]. PPARG was chosen due
   to its relation to PPARA. We also included the nearest gene from four
   regions associated with anorexia nervosa [[102]41] and overlapping with
   the results from our GWAS in celiac disease [[103]11].

Statistical analysis

   Gene expression data was analyzed using the Delta-Delta C[T] (ΔΔC[T])
   relative quantification method[[104]48]. This approach enables the
   expression of target genes to be normalized to reference genes, and
   then compared between cases and control samples (the ΔΔC[T] value). To
   normalize the qPCR reaction for the amount of RNA added, the use of an
   internal reference gene (housekeeping gene) is used [[105]48]. The
   threshold cycle (C[T]) or the cycle of quantification (Cq), is the PCR
   cycle when the amplification reaches a set threshold. Delta Ct (ΔC[T])
   is calculated by the difference between the cycle of quantification for
   the target gene compared with the reference gene(-s). The t-test was
   used to calculate if there was a significant difference between the
   mean delta C[T] value of the controls compared to the mean delta C[T]
   of cases.

URLs

   Protein Database—UniProtKb/SwissProt: [106]http://www.uniprot.org/

   Entrez Cross Database: [107]http://www.ncbi.nlm.nih.gov/sites/gquery

   Gene Cards: [108]http://www.genecards.org/

   A Catalog of Published Genome-Wide Association Studies:
   [109]www.genome.gov/gwastudies.

Supporting Information

   S1 File. Phenotype information.

   This file gives information regarding CD diagnosis.

   (TXT)
   [110]Click here for additional data file.^ (3.3KB, txt)
   S2 File. Ct values.

   This file gives the Ct value for each gene and patient.

   (TXT)
   [111]Click here for additional data file.^ (318.1KB, txt)
   S1 Table. All tested genes including six genes previously run in
   Östensson et al. [[112]11] and Montén et al. [[113]18].

   A list of all type 2 Diabetes and anorexia genes (including six genes
   were run as previously described. Forty-two genes were involved in type
   2 diabetes and four were associated with anorexia. A total of forty
   target genes were analyzed in this study.

   (DOCX)
   [114]Click here for additional data file.^ (44.6KB, docx)

Acknowledgments