Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in European EoE cases and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replication of the 5q22 locus (meta-analysis p = 1.9×10−16), we identified association at 2p23 (encoding CAPN14, p = 2.5×10−10). CAPN14 was specifically expressed in the esophagus, dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13, and located in an epigenetic hotspot modified by IL-13. There was enriched esophageal expression for the genes neighboring the top 208 EoE sequence variants. Multiple allergic sensitization loci were associated with EoE susceptibility (4.8×10−2 < p < 5.1×10−11). We propose a model that elucidates the tissue specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13–inducible esophageal response involving CAPN14

Plasmodium falciparum erythrocytic stage parasites require the putative autophagy protein PfAtg7 for normal growth.

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite's erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth

PfAtg7 has an unusual primary structure.

<p>(A) PfAtg7 contains long insertions within the C-terminal and N-terminal domains. A schematic of PfATG7 and ScATG7 domains illustrates the presence of insertions in PfAtg7 as compared to the well-described C-terminal and N-terminal domains of ScAtg7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067047#pone.0067047-Noda1" target="_blank">[42]</a>. (B) Alignment of ScAtg7 and PfAtg7 C-terminal regions reveals conservation of key motifs. Alignment between yeast (Sc) and <i>P. falciparum</i> (Pf) ATG7 C-terminal domain illustrate similarity, with conservation of the ATP binding domain (black bar), the catalytic cysteine (box), amino acids required for hydrogen bonding (black arrowheads), and salt bridges (open arrowheads) between ATG7 and ATG8 (for complete alignment see Fig. S1C in File S1).</p

PfAtg7 is essential for normal parasite growth.

<p>Short-term growth experiment reveals slow growth phenotype upon TMP removal. PfAtg7-RFA clone A1 (A) B4 (B) and parental PM1 (C) were washed free of TMP and parasitemia was monitored by flow cytometry every 24 h for 3 days. Parasitemia in the absence of TMP (white bars/circles) for clones A1 and B4 was significantly reduced compared to growth in the presence of TMP (black bars/circles; **p<0.001, student’s T test). Parental line (PM1) shows significant growth difference at 48 h (*p<0.05, student’s T test) but no significant difference in the presence and absence of TMP at the 24 h or 72 h timepoints. (D) PfATG7 attenuation results in a sustained slow growth phenotype. Two independent PfATG7-RFA transgenic clones, A1 and B4 (not shown), were monitored every 24 h by staining with acridine orange and enumeration of parasitemia by flow cytometry for up to 8 days. Dilutions were performed on parasite cultures every three days to maintain the parasites at optimal parasitemia and avoid parasite death. Overall dilutions were factored in, resulting in “cumulative growth.” (E) PM1 parental parasites exhibit no significant change in growth over the 8 day period.</p

Western blotting confirms expression of PfAtg7 fusion proteins.

<p>(A) PfATG7-HA transgenic parasites confirm protein expression. Western blot of two independent clones detects PfAtg7-HA at ∼150 kDa and ∼65 kDa. Lane 1: PfAtg7-HA clone C1; Lane 2: PfAtg7-HA clone F2; Lane 3: UCH-HA positive control (see Materials and Methods). (B) PfAtg7-RFA transgenic parasites confirm expression of the two PfAtg7 species. Western blot detection of PfATG7-RFA fusion protein incubated in the presence of TMP reveals PfATG7 at ∼200 kDa and ∼100 kDa. RFA tag is 47 kDa.</p

PfAtgs 3, 4, 7, 8 are expressed in<i>P. falciparum erythrocytic stage parasites.</i>

<p>Genomic DNA (gDNA) and RNA was extracted from asynchronous erythrocytic stage parasites, the latter used for cDNA production. Primers flanking introns were chosen, except for PfATG8, which has no predicted introns. No RT control shows lack of gDNA contamination of RNA preparation.</p

Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in European EoE cases and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replication of the 5q22 locus (meta-analysis p = 1.9×10(−16)), we identified association at 2p23 (encoding CAPN14, p = 2.5×10(−10)). CAPN14 was specifically expressed in the esophagus, dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13, and located in an epigenetic hotspot modified by IL-13. There was enriched esophageal expression for the genes neighboring the top 208 EoE sequence variants. Multiple allergic sensitization loci were associated with EoE susceptibility (4.8×10(−2) < p < 5.1×10(−11)). We propose a model that elucidates the tissue specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13–inducible esophageal response involving CAPN14