Presenter of 2 Presentations
RAC2 MUTATIONS AND IMMUNE DEFICIENCY – FUNCTIONAL SPECTRUM OF AN INTERNATIONAL COHORT
Abstract
Background and Aims
Mutations in RAC2, were first identified in neonates carrying RAC2 dominant negative, p.D57N, who presented with severe neutrophil defects and T-cell lymphopenia. Subsequently, dominant-activating mutations were reported in patients with combined immunodeficiency/immunodysregulation (CIID) or SCID. We assembled an international cohort of 48 published and unpublished patients with RAC2 mutations to characterize the spectrum of disease.
Methods
Clinical and immunologic phenotypes of 48 germline-mutant RAC2 patients were obtained from referring physicians and literature reports. Heterologous expression assays including superoxide production, PAK1 binding, AKT activation were used to characterize RAC2 variant effects. Protein stability was determined by western blot.
Results
Patient presentation varied by mutation; five patients presented as SCID with reticular dysgenesis, five as neonatal neutrophil deficiency (D57N), and 38 as CIID. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with SCID and absent lymphocytes. Activating variants with lower stability (I21S, E62K, N92K, N92S) presented as CIID with lymphopenia, detectable by newborn screening but presenting later with recurrent sinopulmonary and viral infections. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Severity of phenotype and initial clinical presentation was correlated with protein stability - increased stability led to neonatal presentation and dominant mutations with decreased stability had later clinical onset.
Conclusions
Clinical presentation of RAC2 mutation-bearing patients is determined by a combination of protein activity and stability. Heterologous expression and analysis is useful for deciphering the molecular basis of the clinical phenotype.
IMMUNOGENETICS ASSOCIATED WITH SEVERE COCCIDIOIDOMYCOSIS
Abstract
Background and Aims
Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to Western United States and Mexico. While exposure is common, pneumonia is thought to occur in ~3% of residents annually with <1% of those developoing disseminated disease.
Methods
We enrolled DCM patients, performed whole-exome sequencing and assessed cytokine production in peripheral blood mononuclear cells. Confocal microscopy co-localized DECTIN-1 and fungal endospores. Transfection demonstrated DECTIN-1, DUOX1 and DUOXA1 roles in β-glucan-stimulated H2O2 production. RNA was sequenced from STAT3-mutated, autosomal-dominant Hyper-IgE syndrome (AD-HIES) patients' respiratory tissues. Duox1-/- mice were infected with Coccidioides.
Results
In an exploratory set of 67 DCM patients, two had haploinsufficient STAT3 mutations. Defects in β-glucan sensing and response were seen in 34/67 (50.7%) cases. Damaging CLEC7A (n=14) and PLCG2 (n=11) variants were found and PBMC from patients with these variants produced less β-glucan-stimulated TNF-α than healthy controls (P<0.005). Using ancestry matched controls, damaging variants in CLEC7A and PLCG2 were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). In a validation cohort of 112 DCM patients PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044) and CLEC7a Y238* (P=0.0656) were confirmed. Fifteen discovery cohort patients had heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production in transfected cells. AD-HIES patient airway epithelial cells had decreased DUOX1/DUOXA1 transcripts. Duox1-/- mice had increaed morbidity and mortaligy following Coccidoiodes infection.
Conclusions
Patients with DCM have impaired β-glucan sensing or responsiveness affecting H2O2 production. Genetically impaired Coccidioides recognition and cellular response decrease inflammatory cytokine production and underlie susceptibility to disseminated coccidioidomycosis.