Results

Within our cohort of patients with very early onset inflammatory bowel disease, 4 a 3-year-old girl (P1) born from healthy non-consanguineous parents presented with severe chronic secretory diarrhea and eczema since the age of 3 months. Immunological work-up was normal except for high titers of serum anti-AIE75 antibody. Duodenal biopsies showed severe villous atrophy with massive lymphocyte infiltration. Diarrhea improved on parenteral nutrition and treatment with steroids, azathioprine, and tacrolimus, but clinical and histological remission was observed only after switching to sirolimus (Figure 1 A). Targeted sequencing excluded a molecular defect in FOXP3, CTLA4, LRBA, STAT3, and CD25, known causes of autoimmune enteropathy. 4 Whole exome sequencing was performed on parents-proband-trio’s DNA and identified a de novo and rare heterozygous missense variant in exon 6 of the PTPN2 gene (NM_002828. 3 c. 646T> G, chr18: 12,785,478-12,929,643) replacing a cysteine residue with a glycine in position 216 (Supplementary Table 1). Sanger sequencing confirmed familial segregation (Figure 1 B–D). Overexpression of C216G-PTPN2 and of the wild-type (WT) allele in HEK293T cells yielded comparable amount of protein (Figure 1 E). Expression of endogenous PTPN2 protein was also comparable in patient and control-derived Epstein-Barr virus–transformed B-cell lines (B-EBV)(Figure 1 F), indicating that the c. 646T> G mutation does not impact PTPN2 expression. The variant was indicated to be damaging by all prediction tools (Combined Annotation Dependent Depletion score: 25.2)(Supplementary Table 1). Cys216 is strongly conserved across species and part of the “PTP signature motif”(HC [X5] R)(Figure 1 G), which forms the phosphate-binding loop in the core of the catalytic site in all members of the PTP family. 5 Cys216 initiates catalysis, via its thiolate group, by mounting nucleophilic attack of phosphate substrates (Figure 1 H). Replacement of cysteine with glycine should impair catalysis, as glycine lacks the polar side chain to interact with phospho-tyrosyl residues. WT and mutant C-terminally tagged PTPN2 were transiently expressed in HEK293T cells, immunoprecipitated, and de-phosphorylation of STAT1 peptides, which are putative substrates of PTPN2, 2 was measured. Compared with WT-PTPN2, the mutant enzyme was, as anticipated, totally deprived of catalytic activity (Figure 1 I). Lentiviral constructs encoding WT-or C216G-PTPN2 were next used to transduce HEK293T cells stably expressing a luciferase reporter gene under the control of STAT3 transcriptional response elements (TRE). Following interleukin (IL)-6 stimulation, overexpression of WT-PTPN2 led to significant downregulation of STAT3-TRE activity over control, whereas the mutant form of PTPN2 failed to do so (Figure 1 J). Mixes of recombinant WT and C216G-PTPN2 displayed in vitro phosphatase activity proportional to the amount of WT PTPN2 (Figure 1 K) and co-transfection of C216G-PTPN2 and WT-PTPN2 repressed STAT3 transcriptional activity in IL-6–stimulated HEK293T-STAT3-TRE cells similarly as transfection by WT-PTPN2 alone (Figure 1 L), overall indicating that C216G-PTPN2 does not exert a dominant negative effect. We next compared the impact of PTPN2 deficiency on either T-cell receptor (TCR) activation or JAK-STAT signaling. Although TCR ligation with anti-CD3 antibody induced comparable Ca 2+ mobilization, same global pattern of tyrosine phosphorylation and comparable ERK1/2 and LCK phosphorylation in control and patient T-cell blasts, phosphorylation of STAT3 in response to IL-15 was increased in …