Author Correction: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

A Correction to this paper has been published: https://doi.org/10.1038/s41590-021-00929-x.

A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

Elucidating the mechanisms that sustain asthmatic inflammation is critical for precision therapies. We found that interleukin-6- and STAT3 transcription factor-dependent upregulation of Notch4 receptor on lung tissue regulatory T (Treg) cells is necessary for allergens and particulate matter pollutants to promote airway inflammation. Notch4 subverted Treg cells into the type 2 and type 17 helper (TH2 and TH17) effector T cells by Wnt and Hippo pathway-dependent mechanisms. Wnt activation induced growth and differentiation factor 15 expression in Treg cells, which activated group 2 innate lymphoid cells to provide a feed-forward mechanism for aggravated inflammation. Notch4, Wnt and Hippo were upregulated in circulating Treg cells of individuals with asthma as a function of disease severity, in association with reduced Treg cell-mediated suppression. Our studies thus identify Notch4-mediated immune tolerance subversion as a fundamental mechanism that licenses tissue inflammation in asthma.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

Unique variants in CLCN3, encoding an endosomal anion/proton exchanger, underlie a spectrum of neurodevelopmental disorders.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl- channels and Cl-/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3-/- mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

High number of candidate gene variants are identified as disease-causing in a period of 4 years.

Advances in bioinformatic tools paired with the ongoing accumulation of genetic knowledge and periodic reanalysis of genomic sequencing data have led to an improvement in genetic diagnostic rates. Candidate gene variants (CGVs) identified during sequencing or on reanalysis but not yet implicated in human disease or associated with a phenotypically distinct condition are often not revisited, leading to missed diagnostic opportunities. Here, we revisited 33 such CGVs from our previously published study and determined that 16 of them are indeed disease-causing (novel or phenotype expansion) since their identification. These results emphasize the need to focus on previously identified CGVs during sequencing or reanalysis and the importance of sharing that information with researchers around the world, including relevant functional analysis to establish disease causality.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Severe allergic dysregulation due to a gain of function mutation in the transcription factor STAT6.

BACKGROUND: Inborn errors of immunity have been implicated in causing immune dysregulation, including allergic diseases. STAT6 is a key regulator of allergic responses. OBJECTIVES: This study sought to characterize a novel gain-of-function STAT6 mutation identified in a child with severe allergic manifestations. METHODS: Whole-exome and targeted gene sequencing, lymphocyte characterization, and molecular and functional analyses of mutated STAT6 were performed. RESULTS: This study reports a child with a missense mutation in the DNA binding domain of STAT6 (c.1114G>A, p.E372K) who presented with severe atopic dermatitis, eosinophilia, and elevated IgE. Naive lymphocytes from the affected patient displayed increased TH2- and suppressed TH1- and TH17-cell responses. The mutation augmented both basal and cytokine-induced STAT6 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reversed STAT6 hyperresponsiveness to IL-4, normalized TH1 and TH17 cells, suppressed the eosinophilia, and improved the patient's atopic dermatitis. CONCLUSIONS: This study identified a novel inborn error of immunity due to a STAT6 gain-of-function mutation that gave rise to severe allergic dysregulation. Janus kinase inhibitor therapy could represent an effective targeted treatment for this disorder.

De novo EIF2AK1 and EIF2AK2 Variants Are Associated with Developmental Delay, Leukoencephalopathy, and Neurologic Decompensation.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

Atypical Localization of Eczema Discriminates DOCK8 or STAT3 Deficiencies from Atopic Dermatitis.

PURPOSE: Autosomal recessive dedicator of cytokinesis 8 (DOCK8-/-) and autosomal dominant signal transducer and activator of transcription 3 (STAT3-/+) deficiencies are inborn errors of immunity (IEI) disorders present with the classic features of eczema and create a dilemma during differentiation from atopic dermatitis (AD). Therefore, an appropriate approach is required for eczema to diagnose DOCK8-/- and STAT3-/+ early. Here, we described a set of clinical and immunological variables, including atypical AD localizations and lymphocyte subsets, to differentiate DOCK8-/- or STAT3-/+ from AD. METHODS: This multicenter study involved 100 patients with DOCK8-/- and STAT3-/+ and moderate/severe AD. We recruited disease manifestations, including detailed localizations of eczema, infections, and allergy. Principle component analysis (PCA) was used to discriminate DOCK8-/- or STAT3-/+ from AD. RESULTS: There were 43 patients with DOCK8-/-, 23 with STAT3-/+, and 34 with AD. Pneumonia, severe infections, mucocutaneous candidiasis, and skin abscesses were commonly observed in DOCK8 and STAT3 deficiencies. Atypical skin involvement with neonatal rash, retro auricular, axillary, sacral, and genital eczema discriminate DOCK8-/- and STAT3-/+ from AD with high specificity ranges between 73.5 and 94.1% and positive predictive index ranges between 55 and 93.1%. Together with using absolute numbers of CD3+, CD4+, and CD8+ T cells, the combined clinical and laboratory features showed perfect differentiation between DOCK8-/- or STAT3-/+ and AD via PCA. CONCLUSIONS: The described features can be easily implemented by physicians providing early diagnosis of DOCK8 and STAT3 deficiencies.

Evolution and long-term outcomes of combined immunodeficiency due to CARMIL2 deficiency.

BACKGROUND: Biallelic loss-of-function mutations in CARMIL2 cause combined immunodeficiency associated with dermatitis, inflammatory bowel disease (IBD), and EBV-related smooth muscle tumors. Clinical and immunological characterizations of the disease with long-term follow-up and treatment options have not been previously reported in large cohorts. We sought to determine the clinical and immunological features of CARMIL2 deficiency and long-term efficacy of treatment in controlling different disease manifestations. METHODS: The presenting phenotypes, long-term outcomes, and treatment responses were evaluated prospectively in 15 CARMIL2-deficient patients, including 13 novel cases. Lymphocyte subpopulations, protein expression, regulatory T (Treg), and circulating T follicular helper (cTFH ) cells were analyzed. Three-dimensional (3D) migration assay was performed to determine T-cell shape. RESULTS: Mean age at disease onset was 38 ± 23 months. Main clinical features were skin manifestations (n = 14, 93%), failure to thrive (n = 10, 67%), recurrent infections (n = 10, 67%), allergic symptoms (n = 8, 53%), chronic diarrhea (n = 4, 27%), and EBV-related leiomyoma (n = 2, 13%). Skin manifestations ranged from atopic and seborrheic dermatitis to psoriasiform rash. Patients had reduced proportions of memory CD4+ T cells, Treg, and cTFH cells. Memory B and NK cells were also decreased. CARMIL2-deficient T cells exhibited reduced T-cell proliferation and cytokine production following CD28 co-stimulation and normal morphology when migrating in a high-density 3D collagen gel matrix. IBD was the most severe clinical manifestation, leading to growth retardation, requiring multiple interventional treatments. All patients were alive with a median follow-up of 10.8 years (range: 3-17 years). CONCLUSION: This cohort provides clinical and immunological features and long-term follow-up of different manifestations of CARMIL2 deficiency.

Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

Combined immunodeficiency caused by a loss-of-function mutation in DNA polymerase delta 1.

BACKGROUND: Mutations affecting DNA polymerases have been implicated in genomic instability and cancer development, but the mechanisms by which they can affect the immune system remain largely unexplored. OBJECTIVE: We sought to establish the role of DNA polymerase δ1 catalytic subunit (POLD1) as the cause of a primary immunodeficiency in an extended kindred. METHODS: We performed whole-exome and targeted gene sequencing, lymphocyte characterization, molecular and functional analyses of the DNA polymerase δ (Polδ) complex, and T- and B-cell antigen receptor repertoire analysis. RESULTS: We identified a missense mutation (c. 3178C>T; p.R1060C) in POLD1 in 3 related subjects who presented with recurrent, especially herpetic, infections and T-cell lymphopenia with impaired T-cell but not B-cell proliferation. The mutation destabilizes the Polδ complex, leading to ineffective recruitment of replication factor C to initiate DNA replication. Molecular dynamics simulation revealed that the R1060C mutation disrupts the intramolecular interaction between the POLD1 CysB motif and the catalytic domain and also between POLD1 and the Polδ subunit POLD2. The patients exhibited decreased numbers of naive CD4 and especially CD8 T cells in favor of effector memory subpopulations. This skewing was associated with oligoclonality and restricted T-cell receptor ß-chain V-J pairing in CD8+ but not CD4+ T cells, suggesting that POLD1R1060C differentially affects peripheral CD8+ T-cell expansion and possibly thymic selection. CONCLUSION: These results identify gene defects in POLD1 as a novel cause of T-cell immunodeficiency.

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice.

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.

ClinPhen extracts and prioritizes patient phenotypes directly from medical records to expedite genetic disease diagnosis.

PURPOSE: Diagnosing monogenic diseases facilitates optimal care, but can involve the manual evaluation of hundreds of genetic variants per case. Computational tools like Phrank expedite this process by ranking all candidate genes by their ability to explain the patient's phenotypes. To use these tools, busy clinicians must manually encode patient phenotypes from lengthy clinical notes. With 100 million human genomes estimated to be sequenced by 2025, a fast alternative to manual phenotype extraction from clinical notes will become necessary. METHODS: We introduce ClinPhen, a fast, high-accuracy tool that automatically converts clinical notes into a prioritized list of patient phenotypes using Human Phenotype Ontology (HPO) terms. RESULTS: ClinPhen shows superior accuracy and 20× speedup over existing phenotype extractors, and its novel phenotype prioritization scheme improves the performance of gene-ranking tools. CONCLUSION: While a dedicated clinician can process 200 patient records in a 40-hour workweek, ClinPhen does the same in 10 minutes. Compared with manual phenotype extraction, ClinPhen saves an additional 3-5 hours per Mendelian disease diagnosis. Providers can now add ClinPhen's output to each summary note attached to a filled testing laboratory request form. ClinPhen makes a substantial contribution to improvements in efficiency critically needed to meet the surging demand for clinical diagnostic sequencing.

Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination.

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2's isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.

A recurring mutation in the respiratory complex 1 protein NDUFB11 is responsible for a novel form of X-linked sideroblastic anemia.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.

De novo variant in KIF26B is associated with pontocerebellar hypoplasia with infantile spinal muscular atrophy.

KIF26B is a member of the kinesin superfamily with evolutionarily conserved functions in controlling aspects of embryogenesis, including the development of the nervous system, though its function is incompletely understood. We describe an infant with progressive microcephaly, pontocerebellar hypoplasia, and arthrogryposis secondary to the involvement of anterior horn cells and ventral (motor) nerves. We performed whole exome sequencing on the trio and identified a de novo KIF26B missense variant, p.Gly546Ser, in the proband. This variant alters a highly conserved amino acid residue that is part of the phosphate-binding loop motif and motor-like domain and is deemed pathogenic by several in silico methods. Functional analysis of the variant protein in cultured cells revealed a reduction in the KIF26B protein's ability to promote cell adhesion, a defect that potentially contributes to its pathogenicity. Overall, KIF26B may play a critical role in the brain development and, when mutated, cause pontocerebellar hypoplasia with arthrogryposis.

The Epithelial Sodium Channel Is a Modifier of the Long-Term Nonprogressive Phenotype Associated with F508del CFTR Mutations.

Cystic fibrosis (CF) remains the most lethal genetic disease in the Caucasian population. However, there is great variability in clinical phenotypes and survival times, even among patients harboring the same genotype. We identified five patients with CF and a homozygous F508del mutation in the CFTR gene who were in their fifth or sixth decade of life and had shown minimal changes in lung function over a longitudinal period of more than 20 years. Because of the rarity of this long-term nonprogressive phenotype, we hypothesized these individuals may carry rare genetic variants in modifier genes that ameliorate disease severity. Individuals at the extremes of survival time and lung-function trajectory underwent whole-exome sequencing, and the sequencing data were filtered to include rare missense, stopgain, indel, and splicing variants present with a mean allele frequency of <0.2% in general population databases. Epithelial sodium channel (ENaC) mutants were generated via site-directed mutagenesis and expressed for Xenopus oocyte assays. Four of the five individuals carried extremely rare or never reported variants in the SCNN1D and SCNN1B genes of the ENaC. Separately, an independently enriched rare variant in SCNN1D was identified in the Exome Variant Server database associated with a milder pulmonary disease phenotype. Functional analysis using Xenopus oocytes revealed that two of the three variants in δ-ENaC encoded by SCNN1D exhibited hypomorphic channel activity. Our data suggest a potential role for δ-ENaC in controlling sodium reabsorption in the airways, and advance the plausibility of ENaC as a therapeutic target in CF.