PIK3AP1 and SPON2 Genes Are Differentially Methylated in Patients With Periodic Fever, Aphthous Stomatitis, Pharyngitis, and Adenitis (PFAPA) Syndrome.

Nataša Toplak,Tadej Avčin,Maruša Debeljak,Tine Tesovnik,Jernej Kovač,Daša Perko,Tomaž Rozmarič,Ema Lovšin

doi:10.3389/fimmu.2020.01322

Abstract

Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome is the most common autoinflammatory disease in children and is often grouped together with hereditary periodic fever syndromes, although its cause and hereditary nature remain unexplained. We investigated whether differential DNA methylation was present in DNA from peripheral blood mononuclear cells (PBMC) in patients with PFAPA vs. healthy controls. A whole-epigenome analysis (MeDIP and MBD) was performed using pooled DNA libraries enriched for methylated genomic regions and identified candidate genes, two of which were further evaluated with methylation-specific restriction enzymes coupled with qPCR (MSRE-qPCR). The analysis showed that the PIK3AP1 and SPON2 gene regions are differentially methylated in patients with PFAPA. MSRE-qPCR proved to be a quick, reliable, and cost-effective method of confirming results from MeDIP and MBD. Our findings indicate that a B-cell adapter protein (PIK3AP1), as the PI3K binding inhibitor of inflammation, and spondin-2 (SPON2), as a pattern recognition molecule and integrin ligand, could play a role in the etiology of PFAPA. Their role and the impact of changed DNA methylation in PFAPA etiology and autoinflammation need further investigation.

Highlights

Periodic fever syndrome with adenitis, pharyngitis, and aphthous stomatitis (PFAPA) belongs to the group of autoinflammatory diseases (AID)
To identify the potentially relatively small change in the methylation patterns due to the specifics of a peripheral blood mononuclear cells (PBMC)-derived DNA sample and to confirm whether the PFAPA cohort differs in DNA methylation patterns from healthy controls, a whole-epigenome analysis was performed using pooled DNA libraries enriched for methylated genomic regions using Methylated DNA Immunoprecipitation (MeDIP) and Methyl-CpG-binding domain (MBD)
The first half (4:1163329–1163502) of the fifth intron was, less (P = 0.001) methylated and the second half (4:1163480–1163727) was more (P = 0.0191) methylated in PFAPA patients compared to the healthy controls. Results from both MeDIP and MBD were confirmed with MSRE-qPCR, with some differences

Summary

Introduction

Periodic fever syndrome with adenitis, pharyngitis, and aphthous stomatitis (PFAPA) belongs to the group of autoinflammatory diseases (AID). This group of disorders is marked by increased inflammation associated with the innate immune system, and most of the disorders are inherited in a Mendelian pattern [1, 2]. Of the various autoinflammatory diseases, many have a confirmed known genetic cause. PFAPA syndrome still has an unknown genetic background and pathogenesis [3]. PFAPA was first described by Marshall et al [4]. Its most common feature is periodic fever, while its other features are more variable: pharyngitis, aphthous stomatitis, and cervical

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