The DMT1 IVS4+44C>A polymorphism and the risk of iron deficiency anemia in children with celiac disease

<div><p>Background</p><p>Iron deficiency anemia in celiac disease is related to impaired duodenal mucosal uptake, due to villous atrophy. Iron enters the enterocytes through an apical divalent metal transporter, DMT1. Different DMT1 transcripts have been identified, depending on the presence of an iron-responsive element that allows DMT1 up-regulation during iron starvation. An intronic DMT1 polymorphism, IVS4+44C>A, has been associated with metal toxicity, and the CC-carriers show high iron levels.</p><p>Aims</p><p>This study investigates the association between DMT1 IVS4+44C>A and anemia in a cohort of 387 Italian celiac children, and the functional role of the polymorphism.</p><p>Methods and results</p><p>By association analysis, we found that DMT1 IVS4+44-AA genotype confers a four-fold risk of developing anemia, despite of atrophy degree. By analysis of mRNA from gastroesophageal biopsies, we found that total DMT1 is significantly upregulated in presence of mild, but not severe, atrophy, independently from IVS4+44C>A variant, and in normal but not in atrophic CC-biopsies. Moreover, we found that A-allele is associated to preferential expression of the DMT1 transcripts lacking the iron-responsive element, thus limiting the DMT1 overexpression that normally occurs to respond to iron starvation.</p><p>Discussion</p><p>Possibly, the IVS4+44-AA-related dysregulation of the iron-induced changes in DMT1 expression is not able to impair iron absorption in physiological condition. However, if exacerbated by the concomitant massive loss of functional absorbing tissue paralleling worsened stages of villus atrophy, it might be ineffective in counteracting iron deficiency, despite of DMT1 overexpression.</p><p>Conclusion</p><p>We suggest, for the first time, that celiac disease may unmask the contribution of the DMT1 IVS4+44C>A polymorphism to the risk of anemia.</p></div

Clinical features of 387 Italian children with Celiac Disease stratified according to IDA.

<p>Clinical features of 387 Italian children with Celiac Disease stratified according to IDA.</p

Total DMT1 expression in gastroesophageal biopsies from potential celiac children.

<p><b>A)</b> Expression of total DMT1 mRNA from 27 gastroesophageal biopsies resulted into significant up-regulation of the iron transporter in the subgroup showing T3a degree of villous atrophy (n = 3) with respect to the subgroups with normal mucosa (T0 = 11). No significant difference in DMT1 expression was found in T3b (n = 6) and in T3c (n = 7) biopsies. On the right the result of the t-test between the ΔCt values for DMT1 expression (ΔCt = Ct _DMT1- Ct _β-actin) of T0 and T3a biopsies. <b>B)</b> Significant overexpression of total DMT1 transcript in null atrophy (T0) biopsies from CC-carriers with respect to those from AA-carriers, as shown by the t-test on the right between their ΔCt values for DMT1 expression. Data are represented as mean ± standard deviation of the fold change from at least three different assays performed in duplicate. The t-test has been used to determine the statistical significance between groups by using the ΔCt values of the DMT1 target and the β-actin reference, due to the small size number. A <i>p</i>-value less than 0.05 has been considered significant.</p

Villous atrophy degree of 27 consecutive duodenal biopsies stratified according to DMT1 IVS+44C>A polymorphism.

<p>Villous atrophy degree of 27 consecutive duodenal biopsies stratified according to DMT1 IVS+44C>A polymorphism.</p

Logistic regression analysis for 387 celiac patients presenting (134) or not (253) IDA (Hb less than 3° percentile).

<p>Logistic regression analysis for 387 celiac patients presenting (134) or not (253) IDA (Hb less than 3° percentile).</p

DMT1 IVS+44C>A polymorphism is associated to DMT1 +IRE/–IRE transcript ratio.

<p>DMT1 IVS+44C>A polymorphism is associated to DMT1 +IRE/–IRE transcript ratio.</p

Gene Expression Profile of Peripheral Blood Monocytes: A Step towards the Molecular Diagnosis of Celiac Disease?

<div><p>Aim</p><p>Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls.</p> <p>Participants</p><p>Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn’s disease patients) were enrolled.</p> <p>Results</p><p>Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%.</p> <p>Conclusions</p><p>The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.</p> </div

mRNA expression of candidate genes in peripheral blood monocytes.

<div><p>A) <i>KIAA1109</i>expression was higher in CD (<i>p</i>=0.05) and in CD-GFD patients (<i>p</i>=0.05) than in controls, but also in Crohn peripheral monocytes (<i>p</i>=0.02); B) <i>c-REL</i> expression was lower in CD peripheral monocytes than in controls (<i>p</i><0.01), but become higher than controls (<i>p</i><0.01) and CD (<i>p</i><0.01) after one year of GFD; the same profile was observed in Crohn peripheral monocytes; C) <i>SH2B3</i> expression was lower in CD versus controls (<i>p</i>=0.04) whereas it was significantly higher in Crohn and CD-GFD patients versus controls (<i>p</i><0.01) and CD (<i>p</i><0.01). D) <i>LPP</i> expression was lower in CD peripheral monocytes than in controls (<i>p</i>=0.04); E-F) <i>TNFAIP3</i> and <i>RGS1</i> genes expression were lower in CD peripheral monocytes versus controls (<i>p</i><0.01) and higher in Crohn patients versus controls (<i>p</i><0.01) and CD patients (<i>p</i><0.01). Both genes expression levels normalized after one year of GFD;.</p> <p>RQ: relative quantification; Ctr: controls; CD: celiac disease; CD-GFD: celiac patients on a gluten-free diet; * <i>p</i><0.01, **<i>p</i><0.05.</p></div

Distribution of the Discriminant Score of CD, Controls, Crohn and CD patients on gluten free diet.

<p>The D-score clearly separated the four groups of subjects evaluated. Only CD patients had a negative D-score. The D-score of CD patients on gluten free diet was intermediate between the scores of controls and Crohn patients.</p

Analysis of the levels of mRNA expression of associated genes in duodenal tissue.

<div><p>A) IL-21 is over-expressed in CD compared to controls (<i>p</i><0.01), B) IL-2 shows a very small trend of increase in CD compared to controls; C) and D) Expression of the KIAA1109 and cREL genes: the patterns are very similar and did not show any variations among the three groups.</p> <p>RQ: relative quantification; Ctr: controls; CD: celiac disease; CD-GFD: celiac patients on a gluten-free diet; * <i>p</i><0.01, **<i>p</i><0.05.</p></div