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. 2019 Dec 19;6:187.
doi: 10.3389/fnut.2019.00187. eCollection 2019.

MAGI2 Gene Region and Celiac Disease

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Free PMC article

MAGI2 Gene Region and Celiac Disease

Amaia Jauregi-Miguel et al. Front Nutr. .
Free PMC article

Abstract

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24-2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.

Keywords: MAGI2; association analysis; celiac disease; expression analysis; long non-coding RNA; tight junction.

Figures

Figure 1
Figure 1
Genomic context of CD associated SNP rs6969266. The reference sequence of MAGI2 (NCBI) and genomic annotations of a 20-kb region around candidate SNP rs6962966 are shown.
Figure 2
Figure 2
Characterization of RPL4-587D13.2 expression in human tissues and the C2BBe1 cell line. (A) mRNA expression of MAGI2 and RPL4-587D13.2 in RNA from 9 human tissues. Values are represented relative to the tissue with the highest expression levels (brain). (B) Subcellular localization of RPL4-587D13.2 in C2BBe1 cells. The box-plots show the ratio of nuclear to whole cell transcripts, represented as the maximum and minimum values and the mean (n = 3); LNC13 and RPLP0 are used as nuclear and cytoplasmic controls, respectively. (C) Representative western blot and (D) quantification of MAGI2 protein levels in C2BBe1 cells silenced for RPL4-587D13.2 (siRNA_1 and siRNA_2). The mean and standard deviation of MAGI2 expression relative to the control siRNA are shown (n = 3) (*0.01 < p < 0.05).
Figure 3
Figure 3
Effects of MAGI2 silencing and gliadin on the expression of a TJ-related genes in C2BBe1 intestinal cells. (A) Western blot of MAGI2 protein upon silencing in C2BBe1 cells. (B) Quantification of MAGI2 protein levels under different cell treatments, relative to control cells (transfected with control siRNA and incubated with PT-BSA). Mean and standard deviation are shown (n = 3). (C) Heat map of candidate gene expression in different conditions; PTG stimulation, siRNA mediated MAGI2 silencing (MAGI2_si1) and PTG stimulation of MAGI2_si1 cells. Expression levels are shown as fold-change relative to the average of control samples (silenced with a control siRNA and incubated with PT-BSA). Columns represent treatment categories and lines candidate genes (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001).
Figure 4
Figure 4
mRNA expression of (A) RPL4-587D13.2, (B) MAGI2, (C) CLDN2, and (D) ZAK in duodenal biopsies from CD patients at diagnosis (white dots), treated CD patients (gray dots) and non-Celiac controls (black dots) represented as mean and SEM, relative to the average of the control samples (*0.01 < p < 0.05; **0.001 < p < 0.01).
Figure 5
Figure 5
eQTL analyses for (A) MAGI2 and (B) RP4-587D13.2 in duodenal biopsies from patients with CD and non-CD controls. Scatter plots show expression relative to the control mean in patients lacking or harboring the tag SNP rs2691527*T allele. Spearman correlation between MAGI2 and RP4-587D13.2 expression levels according to the (C) absence or (D) presence of tag SNP rs2691527*T allele.

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