Proteomics plus genomics approaches in primary immunodeficiency: the case of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome

Abstract

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a rare syndrome due to a mutation in the forkhead box protein 3 gene (FOXP3) leading to an impaired regulatory T cell (T(reg) ) activity associated both with skewed T helper type 2 (Th2) response and autoreactive phenomena. The purpose of this study was to describe a combined proteomics and genomics approach to comprehensively evaluate clinical and immunological phenotypes of patients affected by IPEX. T cell receptor (TCR)-Vβ repertoire and peripheral blood lymphocytes phenotype from three brothers affected by IPEX were studied by flow cytometry. Specific immunoglobulin (Ig)E were evaluated by means of an allergenic molecules microarray [immuno solid-phase allergen chip (ISAC)]. Total RNA was extracted and hybridized to Affymetrix oligonucleotide arrays to obtain quantitative gene-expression levels. No FOXP3 protein was detectable within CD127(-) CD25(high) CD4(+) T cells from peripheral blood. A T cell-naive phenotype (CD62L(+) CD45R0(-)) associated with a reduction of both CD26 and CD7 expression and a TCR-Vβ 8 and 22 family expansions were found. B lymphocytes were mainly CD5(+) (B1) cells expressing a naive phenotype (tcl1(+) CD27(-)). The three IPEX patients had severe food allergy and specific IgE reactivity to cow's milk allergens, a hen's egg allergen and a wheat allergen. Gene expression profile analysis revealed a dysregulation associated mainly with Th1/Th2 pathways. The multiplexing evaluation reported in this study represents a comprehensive approach in the assessment of genetic conditions affecting the immune system such as the IPEX syndrome, paving the way for the development of diagnostic tools to improve the standard clinical and immunological profiling of the disease.

Fig. 1

T cell receptor (TCR)-Vβ repertoire analysis of CD4⁺ T lymphocytes. Expansions of TCR-Vβ8 and TCR-Vβ22 were detected in the two older brothers affected by immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, Y1 and Y2, respectively, reflecting the clinical picture of the two patients. All remaining TCR-Vβ were in the normal range. Grey bar: IPEX patient Y1; white bar: IPEX patient Y2; black bar: IPEX patient Y3.

Fig. 2

Clustered heatmap graphic representation of the 67 significant genes expressed differentially in the two youngest patients (on the left) and their parents (on the right). Details on gene-related proteins are given in Table 3. The green colour indicates down-regulated genes, red colour over-expressed genes.