Thomas DiMaggio

Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number

Elevated basal serum tryptase levels are present in 4–6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase–encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

Germline hypomorphic CARD11 mutations in severe atopic disease

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor–induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.

ERBIN deficiency links STAT3 and TGF-β pathway defects with atopy in humans

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-&bgr; activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-&bgr; signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut) have evidence of deregulated TGF-&bgr; signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3–ERBIN–SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-&bgr;. In turn, cell-intrinsic deregulation of TGF-&bgr; signaling is associated with increased functional IL-4R&agr; expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4R&agr;/GATA3 axis in vitro. These findings link increased TGF-&bgr; pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

B-Cell Activating Factor (BAFF) is elevated in Chronic Granulomatous Disease

Chronic Granulomatous Disease (CGD) is an inherited defect in superoxide production leading to life-threatening infections, granulomas, and, possibly, abnormal immunoglobulin concentrations. We investigated whether factors controlling antibody production, such as B-cell activating factor (BAFF), were altered in CGD. CGD subjects had significantly increased mean (2.3-fold, p < 0.0001) plasma concentrations of BAFF compared to healthy donors. Patients on IFN-γ treatment had significantly higher BAFF concentrations compared with CGD patients not taking IFN-γ (1.6-fold, p < 0.005). Leukocytes from CGD subjects produced normal amounts of BAFF in response to IFN-γ or G-CSF in vitro. Expression of BAFF-R and TACI was significantly reduced on CGD B cells. Elevated BAFF in CGD correlated with CRP (R = 0.44), ESR (R = 0.49), and IgM (R = 0.47) and increased rapidly in healthy subjects following intravenous endotoxin administration. These findings suggest that elevated BAFF in CGD subjects and healthy donors is a consequence of acute and chronic inflammation.

A common haplotype containing functional CACNA1H variants is frequently coinherited with increased TPSAB1 copy number

PurposeCaV3.2 signaling contributes to nociception, pruritus, gastrointestinal motility, anxiety, and blood pressure homeostasis. This calcium channel, encoded by CACNA1H, overlaps the human tryptase locus, wherein increased TPSAB1 copy number causes hereditary α-tryptasemia. Germ-line CACNA1H variants may contribute to the variable expressivity observed with this genetic trait.MethodsTryptase-encoding sequences at TPSAB1 and TPSB2, and TPSG1 and CACNA1H variants were genotyped in 46 families with hereditary α-tryptasemia syndrome. Electrophysiology was performed on tsA201 HEK cells transfected with wild-type or variant CACNA1H constructs. Effects on clinical phenotypes were interrogated in families with TPSAB1 duplications and in volunteers from the ClinSeq cohort.ResultsThree nonsynonymous variants in CACNA1H (rs3751664, rs58124832, and rs72552056) cosegregated with TPSAB1 duplications in 32/46 families and were confirmed to be in linkage disequilibrium (LD). In vitro, variant CaV3.2 had functional effects: reducing current densities, and altering inactivation and deactivation properties. No clinical differences were observed in association with the CACNA1H haplotype.ConclusionA previously unrecognized haplotype containing three functional CACNA1H variants is relatively common among Caucasians, and is frequently coinherited on the same allele as additional TPSAB1 copies. The variant CACNA1H haplotype, which in vitro imparts partial gain of function, does not result in detectable phenotypic differences in the heterozygous state.

Hypomorphic caspase activation and recruitment domain 11 (CARD11) mutations associated with diverse immunologic phenotypes with or without atopic disease

Background Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor &kgr;B, c‐Jun N‐terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B‐cell expansion with nuclear factor &kgr;B and T‐cell anergy (heterozygous, gain‐of‐function mutations), and severe atopic disease (loss‐of‐function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole‐exome sequencing. Objectives We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss‐of‐function alleles. Methods Cell transfections and primary T‐cell assays were used to evaluate signaling and function of CARD11 variants. Results Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X‐linked–like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled‐coil domains of the CARD11 protein. Conclusion These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.