Humoral Immunodeficiency with Hypotonia, Feeding Difficulties, Enteropathy, and Mild Eczema Caused by a Classical FOXP3 Mutation

Abstract

We describe here the case of a boy who presented with pulmonary infections, feeding difficulties due to velopharyngeal insufficiency and gastroesophageal reflux, myopathy, and hypotonia soon after birth. Later, he was also found to have an elevated immunoglobulin (Ig) E and mild eczema and was diagnosed with inflammatory bowel disease. Further immunological screening at the age of 7 years showed low B and NK cell numbers but normal CD4⁺ and CD8⁺ T cells and notably, normal numbers of CD4⁺ regulatory T (Treg) cells. Serum IgG, IgA, and IgM were low to normal, but he had a deficient response to a pneumococcal polysaccharide vaccine and thus a humoral immunodeficiency. To our surprise, whole exome sequencing revealed a mutation in forkhead box protein 3 (FOXP3), encoding an essential transcription factor for the development and function of Treg cells. This classical mutation is associated with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Further in vitro studies indeed showed defective function of Treg cells despite normal FOXP3 protein expression and nuclear localization. The boy underwent hematopoietic stem cell transplantation at 11 years of age and despite the temporary development of diabetes while on prednisone is now doing much better, IgE levels have declined, and his fatigue has improved. This case illustrates that a classical pathogenic mutation in FOXP3 can lead to a clinical phenotype where the diagnosis of IPEX syndrome was never considered because of the lack of diabetes and the presence of only mild eczema, in addition to the normal Treg cell numbers and FOXP3 expression.

Keywords: FOXP3; IPEX syndrome; Treg; WES; autoimmunity; immunodeficiency.

Figure 1

(A) The immunophenotyping of the T and B lymphocyte subsets of the patient was compared with the maturation of his 2-year-old brother who did not carry the mutation. (B) Standard screening of CD4⁺ regulatory T cells using CD127 and CD25 immunostaining (upper panel) was further supported by the assessment of a normal expression of intracellular forkhead box protein 3 (lower panel). (C) Function of the patient’s helper cell differentiation was tested by measuring the cytokines for the Th1 (IFNγ, TNFα), Th2 (IL-13), and Th17 (IL-17) in the supernatant of anti-CD3/anti-CD28-stimulated T cells as determined by Luminex (eBioscience, San Diego, CA, USA).

Figure 2

(A) Mutation c.1010G>A; p.(Arg337Gln) in the forkhead box protein 3 (FOXP3) gene identified using whole exome sequencing (gene on reverse strand). The index patient is hemizygous, and the mother is a heterozygous carrier (left panel). Mutation c.1010G>A; p.(Arg337Gln) in the FOXP3 gene is not present in the grandmother, I:1 (right panel). (B) The cosegregation study in the family showing that the mutation had occurred as a de novo change in the mother of the patient. Arrow indicates index patient.

Figure 3

(A) FACS panels showing the intracellular staining for forkhead box protein 3 (FOXP3) and Helios in the in vitro expanded regulatory T (Treg) cells from a healthy donor (in black) and the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome patient (in red). (B) FACS plots displaying the expression of CD25 and CTLA-4. (C) FACS plots of CD4 and CD8 staining. Gate encloses the CD4⁺CD8⁺ double-positive (DP) cells. (D) Immunophenotyping for CD3 and TCRαβ of the cells in the gate in panel (C). (E) Representative images of ImageStream analysis from Treg and conventional T cells. Nuclear localization was confirmed by colocalization of the FOXP3 staining (purple) with the nuclear DAPI signal (in blue). (F) Graphs representing the proliferation of responder CD4⁺ or CD8⁺ T cells gated from total allogeneic peripheral blood mononuclear cells normalized to the proliferation of the responders only condition (0:1).