Identification of APP As a Novel Candidate Familial Eosinophilia Predisposition Gene

Eosinophilia, such as familial eosinophilia (FE), idiopathic eosinophilia and reactive eosinophilia, is characterized by a sustained overproduction of peripheral blood eosinophils with or without end-organ damage. Germline variants in cytokine and eosinophil function genes are known to contribute to the development of the disease, suggesting common genetic basis may be shared among FE and sporadic eosinophilia. For example, FE is a rare autosomal dominant disorder with susceptible locus mapped to the chromosome 5q region in one American famliy (Rioux et al., 1998). This region (5q31-33) contains several cytokine genes which play functional roles in the development of eosinophils. Germline mutation in JAK1, which is involved in cytokine signaling, has been found in inherited eosinophilia (Kate et al., 2016). Furthermore, genetic variants in genes associated with eosinophilopoietic function (CCR3,CCL5 etc) have been identified in several idiopathic eosinophilia (Lee et al.,2017). Despite of these findings, most cases of eosinophilia have no identifiable mutations. In this new study, we collected a Chinese three-generation pedigree with documented FE. We aimed to investigate novel genetic variants of FE in this pedigree, and found that APP (amyloid-β precursor protein) might be a novel predisposition gene associated with FE.

Firstly, we performed Whole-Genome sequencing analysis on isolated eosinophils from peripheral blood of 3 patients and 3 unaffected family members. Point mutations and small indels were selected as potential pathogenic variants if they met the following criteria: 1. heterozygous mutations 2. not previously described 3. predicted to be deleterious or located in regulatory region. As a result of this analysis, 109 mutated genes were considered as candidates, of which APP was the only gene carrying nonsynonymous mutation in the coding region. APP encodes amyloid-β precursor protein that can generate a number of functional peptides. A deleterious missense mutation (c.C470G, p.T157R) was found in the protein binding domain of APP. This mutation was verified by Sanger sequencing in all 3 FE patients. APP has been shown to regulate gene expression (Kögel et al., 2005). As APP knock-out mouse exhibits decreased mRNA expression levels of several cytokines (IL-6, IL-10 and IL-1β) (Carrano et al., 2015), and IL-1β is associated with eosinophil activation (Esnault et al., 2013), we pursued further on APP as a potential eosinophilia predisposition gene.

Secondly, we explored transcriptional changes that may be associated with germline mutated APP in eosinophilia patients. We adopted RNA-seq analysis on isolated eosinophils from peripheral blood of 3 patients and 3 controls. A total of 2035 (up:1249, down:786) differentially expressed genes (DEGs) were identified in sorted patient eosinophils as compared to controls (FDR < 0.01). To functionally classify these genes, we conducted pathway analysis and identified "Lysosome", "Chemokine signaling", "Cytokine-cytokine receptor interaction" as significant pathways associated with DEGs. In addition, we found that 51 DEGs were overlapped with previously documented eosinophil-associated genes based on IPA Knowledge Base (N=324). We further determined whether these 51 eosinophil-associated DEGs are interacted with 109 mutated genes by constructing the protein-protein interaction network (PPI). Remarkably, APP was the only gene that was functional linked to 10 eosinophil-associated proteins, including several cytokines (CXCRs, CCRs), to form a sub-network (Score > 0.9). In contrast, randomly picked 51 genes failed to form PPI network. These results indicated that the interactions between APP and eosinophil-associated proteins were potentially of biological significance. Since the analysis of PPI was based on the prior knowledge, we further conducted co-expression analysis on APP and its interaction proteins using our transcriptome data. Interestingly, we found that APP expression was highly correlated with the mRNA level of several eosinophilia-related cytokines including IL-1β, CCR2,CCR3 and CXCR4 (PCC > 0.8).

Collectively, our data show that APP is a promising predisposition gene that may contributes to FE as a cytokine modulator. This study furthers our understanding of the genetic basis of familial eosinophilia and other eosinophilia and provides novel therapeutic targets for this heterogenous disease.