A boy born at term of Caucasian non-consanguineous parents presented with respiratory insufficiency immediately after birth due to tracheomalacia and a diaphragmatic paresis. A diaphragmatic plication was performed at the second month after birth. The patient suffered from severe pulmonary infections necessitating respiratory support repeatedly in the first years of life. Feeding difficulties started shortly after birth due to velopharyngeal insufficiency and gastroesophageal reflux and led to the placement of a percutaneous endoscopic gastrostomy tube in the first year after birth and veloplastic surgery at the age of 5 years. There was a persistence of loose stools, and an allergy was suspected. With the differential diagnosis of cow's milk allergy, a hydrolysate was started (Neocate). Due to bloody diarrhea, a combined gastro-duodenoscopy and colonoscopy was performed when the boy was 3 years old. Histopathology showed macroscopic colitis and microscopic crypt abscesses and active chronic inflammation in the colon, duodenum, stomach, and esophagus. Therefore, inflammatory bowel disease was diagnosed. Immunosuppressive medication (including prednisolone) was started which improved his stools and clinical well-being. In the years that followed, the symptoms of diarrhea were under control by azathioprine with occasional flares. Attempts to reduce azathioprine dose led to an immediate increase in diarrhea. However, despite immunosuppressive medication and surgical corrective interventions, the feeding difficulties persisted and pneumonias recurred. From the first day of life, there was a severe hypotonia and proximal muscle weakness causing a delayed motor development. A muscle biopsy suggested a mild deficiency of complex 1 of the mitochondrial respiratory chain, but this was never supported by genetic testing.
At the age of 7 years, the boy was sent to our clinical immunology unit because of the recurrent infections and allergy with an elevated IgE of 2,275 kU/ml in the presence of a normal white blood cell count and Ig spectrum (Table 1). We met a cooperative boy with a normal height, weight and head circumference, normal heart rate, respiration and saturation, and blood pressure. He had a slightly flaccid face and was sitting in a wheel chair, yet able to walk for a small distance, and he had generally low muscle tone, muscular weakness, and Gower's sign upon physical examination. We found normal eye movement, vision, and hearing and normal chewing, swallowing, and tongue movements. No signs of neuropathy, no ataxia, dysarthria, or speech impairment were found. Psychomotor development was normal otherwise, although he had low yet symmetrical reflexes.
Apart from a clear periodontitis, some pulmonary fine crackles at the end-inspiratory inhalation, and mild hepatomegaly, no other remarkable findings could be found, in particular, no splenomegaly or lymphadenopathy. The boy had a dry skin without obvious eczema, also not around the PEG insertion site for enteral feeding. Apart from 350 ml daily enteral tube feeding (Nutrini Energy Multifiber), medication consisted of daily azathioprine (1.5 mg/kg body weight, bid).
Upon laboratory investigations, complete blood count and differentials were normal, and enzymatic liver tests, kidney, and thyroid function tests were within normal ranges. The initial immunological screening tests were abnormal with low to normal serum IgG, IgA, and IgM and very high IgE levels. Antibody response to pneumococcal polysaccharide vaccination (anti-PPS23) was deficient (Table 1). Fecal elastase levels were within the normal range excluding exocrine pancreatic dysfunction; calprotectin was elevated [ranging from normal (<50) to 1,159 mug/g of feces], and microbiological stool tests (bacterial, viral, and parasitic) were repeatedly normal.
Lymphocyte enumeration was routinely done by flow cytometry and showed low numbers of B and NK cells with normal T cell numbers but a low CD4/CD8 ratio and increased CD4+CD8+ double-positive (DP) T cells (Table 1). The percentage of naive and switched memory B cells was within age-specific reference ranges. CD4+ and CD8+ T cell differentiation into memory/effector memory cells was normal (Figure 1A). The percentage of CD4+ regulatory T (Treg) cells determined by CD25 and CD127 was found to be unremarkable, and the intracellular FOXP3 expression was comparable to healthy controls (Figure 1B). Lymphocyte activation using T cell stimuli such as anti-CD3/anti-CD28 or IL-15 induced adequate proliferative responses of CD4+ and CD8+ T cells as assessed by carboxyfluorescein succinimidyl ester dilution (data not shown), although the cytokine release into the supernatant was considered high to normal, in particular for the Th2 signature cytokine IL-13 (Figure 1C).
Whole exome sequencing (WES) was performed in a trio diagnostic approach (patient and both parents). In short, libraries were prepared using the Kapa HTP kit (Illumina, San Diego, CA, USA), and capture was performed using the SeqCap EZ Exome v3.0 (Roche Nimblegen Madison, WI, USA). Sequencing was done on an Illumina HiSeq2500 HTv4 (Illumina, San Diego, CA, USA) with paired-end 125-bp reads. Read alignment to hg19 and variant calling were done with a pipeline based on BWA-MEM0.7 and GATK 3.3. Variant annotation and prioritizing were done using Cartagenia NGS Bench (Cartagenia Inc., Cambridge, MA, USA). This revealed a hemizygous mutation c.1010G>A; p.(Arg337Gln) in the FOXP3 gene (NM_014009.3) in the patient. His mother was found heterozygous for the mutation (Figure 2A). This mutation has been described as causal for IPEX syndrome and is not present in the Exac database (). Subsequent cosegregation showed that the mutation occurred de novo in the mother (Figure 2B).
Once the mutation in FOXP3 was found in the presence of normal FOXP3 protein expression, we analyzed the Treg cells more closely by investigating their functional capacities in vitro. Naive CD45+CD4+CD25+ Treg cells from the patient and a healthy donor were expanded upon stimulation with anti-CD3/anti-CD28. The cells were subsequently stained for the expression of the quintessential Treg cell transcription factors FOXP3 and Helios (Figure 3A) and the surface molecules CD25 and CTLA-4 (Figure 3B). In both cases, the expression of those proteins was comparable to a healthy donor. Also, the Treg cells of the patient were able to normally produce both IL-10 and TGF-beta and did not produce effector cytokines such as IFNgamma (data not shown). Remarkably, the patient's Treg cells presented an elevated presence of CD4+CD8+ DP cells that were also CD3+TCRalphabeta+ (Figures 3C,D).
Some FOXP3 mutations have been described to affect the nuclear localization of the protein; we therefore analyzed the nuclear localization of FOXP3 in the Treg cells of the patient by ImageStream (Figure 3E). Results showed a normal FOXP3 nuclear distribution comparable to that of the healthy donor. Finally, we assessed the functional capacity of the Treg cells to suppress the proliferation of CD4+ and CD8+ T cells. Undoubtedly, the Treg cells were unable to fully suppress the proliferation of neither CD4+ nor CD8+ T cells, although the Treg cells still retained a limited capacity to suppress CD8+ T cell proliferation (Figure 3F).
Immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (OMIM304790) is an X-linked genetic disorder characterized by enteropathy, insulin-dependent type-1 autoimmune diabetes mellitus (IDDM1), and eczema. Severe enteropathy manifesting as watery or bloody diarrhea is an early feature of the disease present in all IPEX syndrome patients. Furthermore, IDDM1 is common (>80%) and is diagnosed early, often within the first 1-3 months of life. It was found in 4% of neonatal permanent diabetes among males. Other prominent symptoms due to profound immune dysregulation in males with IPEX syndrome include eczema/dermatitis (~65%), failure to thrive (~50%), thyroiditis (~30%), and recurrent infections (~20%). Autoimmune phenomena such as nephropathy, pneumonitis, hepatitis, vasculitis, arthritis, myositis, alopecia, and autoimmune cytopenias may be present. The severe watery diarrhea and life-threatening infections reduce the lifespan of these patients, and they usually die in the first years of life.
In case of elevated IgE levels, the first entities to be considered are atopic disease and allergy, parasitic infestations, Aspergillus infection (allergic bronchopulmonary aspergillosis) in cystic fibrosis, and secondary to HIV infection. Although allergy was indicated by positive RAST tests against milk protein, soja protein, casein, lactalbumin, lactoglobulin, and chicken protein, all other causes for the increased IgE levels were excluded in our patient. Primary immunodeficiency disorders (PIDs) such as hyper-IgE syndrome, Wiskott-Aldrich syndrome, leaky severe combined immunodeficiency/Omenn syndrome, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, or other rare disorders such as IPEX-like disease (for instance due to FOXP3, WASP, CD25, RAG1/2, AIRE, carbohydrate defect in glycosylation-variant PGM3 mutations, or STAT3 or STAT1 gain-of-function mutations) were considered.
In patients with PIDs, a breakdown of self-tolerance mechanisms or immune dysregulation often exists, with high incidence of autoimmunity and autoinflammation as a consequence. In the patients with IPEX syndrome, the lack of FOXP3 in lymphoid tissues (thymus, spleen, and lymph nodes), as encoded by the FOXP3 gene at Xp11.23-Xq13.3, leads to impaired development and suppressed function of CD4+CD25+ Treg cells. FOXP3 functions as the master transcription factor for the development of Treg cells, in both human and mice, together with STAT5a, and the high-affinity IL-2 receptor CD25. Beyond its role in Treg cell differentiation, continuous FOXP3 expression is also required in mature Treg cells for their suppressive function and the full manifestation of the aforementioned key features of these Treg cells. The formation of Treg cell identity, characterized by its unique transcriptional program, requires the activation of additional genes. Indeed, enforced expression of the genes encoding IKAROS family zinc finger 4 (Eos), interferon regulatory factor 4, SATB homeobox 1, lymphoid enhancer-binding factor 1, or GATA-binding protein 3 cooperate with FOXP3 to activate the expression of most, but still not all, of the Treg cell signature genes.
The protein FOXP3 has four functional domains: an N-terminal proline-rich repressor domain, a zinc finger domain, a leucine zipper domain, and a C-terminal DNA-binding forkhead/winged helix domain. Mutations that affect any of these key functional domains may alter or abrogate the ability of the transcription factor to regulate gene expression in Treg cells. Mature naive B cells from IPEX patients often express autoreactive antibodies, suggesting an important role for Treg cells in maintaining peripheral B cell tolerance and B cell anergy.
The B cell numbers in our patients were rather low and showed an impaired pneumococcal polysaccharide vaccination response. Remarkably, there were no autoreactive antibodies detectable in our patient. Although anti-GAD or anti-islet cell antibodies were absent, a systematic investigation of all possible organ-specific autoantibodies was not performed. There was no evidence of autoimmune cytopenia. Also autoantibodies against nuclear antigens (ANA, ENA), neutrophil cytoplasmic proteins (ANCA-IFT, MPO, and PR3), and cardiolipin were all repeatedly negative (data not shown). Still, the complete lack of autoantibodies cannot be confirmed definitely.
The pathogenicity of substitutions at position 337 (R337P and R337Q), similar to the one found in our patient, was reported previously in IPEX syndrome patients. In normal FOXP3, the side chain of arginine at position 337 is predicted to make close contact with the DNA backbone. The presence of a strong basic residue at this position is conserved in all forkhead transcription factors and contributes to DNA binding through both hydrogen bonding and electrostatic interactions. The R337Q substitution destroys this close contact with the DNA backbone and is predicted to cause a significant loss of positive charge at the DNA-binding surface of FOXP3, previously suggested to result in a significant loss of DNA-binding affinity which leads to severe IPEX syndrome. Nonetheless our case is noticeably moderately affected:even though he is carrying the same deleterious mutation at the arginine position 337.