Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease.

BACKGROUND: Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders. METHODS: We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function. RESULTS: We identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The gene UBA1 lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. CONCLUSIONS: Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).

A Recurrent De Novo Heterozygous COG4 Substitution Leads to Saul-Wilson Syndrome, Disrupted Vesicular Trafficking, and Altered Proteoglycan Glycosylation.

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

Hermansky-Pudlak syndrome: Mutation update.

Hermansky-Pudlak syndrome (HPS) is a group of 10 autosomal recessive multisystem disorders, each defined by the deficiency of a specific gene. HPS-associated genes encode components of four ubiquitously expressed protein complexes: Adaptor protein-3 (AP-3) and biogenesis of lysosome-related organelles complex-1 (BLOC-1) through -3. All individuals with HPS exhibit albinism and a bleeding diathesis; additional features occur depending on the defective protein complex. Pulmonary fibrosis is associated with AP-3 and BLOC-3 deficiency, immunodeficiency with AP-3 defects, and gastrointestinal symptoms are more prevalent and severe in BLOC-3 deficiency. Therefore, identification of the HPS subtype is valuable for prognosis, clinical management, and treatment options. The prevalence of HPS is estimated at 1-9 per 1,000,000. Here we summarize 264 reported and novel variants in 10 HPS genes and estimate that ~333 Puerto Rican HPS subjects and ~385 with other ethnicities are reported to date. We provide pathogenicity predictions for missense and splice site variants and list variants with high minor allele frequencies. Current cellular and clinical aspects of HPS are also summarized. This review can serve as a manifest for molecular diagnostics and genetic counseling aspects of HPS.

Mutations in GET4 disrupt the transmembrane domain recognition complex pathway.

The transmembrane domain recognition complex (TRC) targets cytoplasmic C-terminal tail-anchored (TA) proteins to their respective membranes in the endoplasmic reticulum (ER), Golgi, and mitochondria. It is composed of three proteins, GET4, BAG6, and GET5. We identified an individual with compound heterozygous missense variants (p.Arg122His, p.Ile279Met) in GET4 that reduced all three TRC proteins by 70% to 90% in his fibroblasts, suggesting a possible defect in TA protein targeting. He presented with global developmental delay, intellectual disabilities, seizures, facial dysmorphism, and delayed bone age. We found the TA protein, syntaxin 5, is poorly targeted to Golgi membranes compared to normal controls. Since GET4 regulates ER to Golgi transport, we hypothesized that such transport would be disrupted in his fibroblasts, and discovered that retrograde (but not anterograde) transport was significantly reduced. Despite reduction in the three TRC proteins, their mRNA levels were unchanged, suggesting increased degradation in patient fibroblasts. Treating fibroblasts with the FDA-approved proteasome inhibitor, bortezomib (10 nM), restored syntaxin 5 localization and nearly normalized the levels of all three TRC proteins. Our study identifies the first individual with GET4 mutations.

A comprehensive, multidisciplinary, precision medicine approach to discover effective therapy for an undiagnosed, progressive, fibroinflammatory disease.

Precision medicine has generated diagnoses for many patients with challenging undiagnosed disorders. Some individuals remain without a diagnosis despite comprehensive testing, and this impedes their treatment. This report addresses the role of personalized medicine in identifying effective therapy for an undiagnosed disease. A 22-year-old woman presented with chronic severe recurrent trismus, facial pain, progressive multicentric inflammatory and fibrotic masses, and high C-reactive protein. Sites of disease included the pterygomaxillary region, masseter muscles, mandible, lung, pericardium, intrabdominal cavity, and retroperitoneum. A diagnosis was not established after an extensive assessment, including multiple biopsies. The patient was subsequently evaluated under the Undiagnosed Diseases Program at the National Institutes of Health. Large scale genotyping, proteomic studies, and in vitro and gene expression analyses of fibroblasts obtained from a major disease locus were performed. Germline genetic testing did not identify strong candidate genes; proteomic studies of the patient's serum and bronchoalveolar lavage fluid and gene expression analyses of her cells were consistent with dysregulation of the tumor necrosis factor-alpha pathway. The patient's cultured fibroblasts were incubated with selected drugs, and cell proliferation was inhibited by hydroxychloroquine. Treatment of the patient with hydroxychloroquine conferred prolonged beneficial clinical effects, including stabilization of trismus and reduction of corticosteroid dose, C-reactive protein, and size of masses. This case represents an example of precision medicine applied to discover effective treatments for individuals with enigmatic undiagnosed disorders.

Rationale and Design for a Phase 1 Study of N-Acetylmannosamine for Primary Glomerular Diseases.

INTRODUCTION: Sialic acids are important contributors to the polyanionic component of the glomerular filtration barrier, which regulates permeability selectivity. Pathologic glomerular hyposialylation, associated with podocyte effacement, has been implicated in human and mouse glomerulopathies. Oral treatment with N-acetylmannosamine (ManNAc), the uncharged precursor of sialic acid, ameliorates glomerular pathology in different models of glomerular disease. METHODS: Here we explore the sialylation status of kidney biopsies obtained from 27 subjects with various glomerular diseases using lectin histochemistry. RESULTS: We identified severe glomerular hyposialylation in 26% of the biopsies. These preliminary findings suggest that this condition may occur relatively frequently and may be a novel target for therapy. We describe the background, rationale, and design of a phase 1 study to test safety, tolerability, and pharmacokinetics of ManNAc in subjects with primary podocyte diseases. CONCLUSION: We recently demonstrated that ManNAc was safe and well tolerated in a first-in-human phase 1 study in subjects with UDP-N-acetylglucosamine (GlcNAc) 2-epimerase/ManNAc kinase (GNE) myopathy, a disorder of impaired sialic acid synthesis. Using previous preclinical and clinical data, we propose to test ManNAc therapy for subjects with primary glomerular diseases. Even though the exact mechanisms, affected cell types, and pathologic consequences of glomerular hyposialylation need further study, treatment with this physiological monosaccharide could potentially replace or supplement existing glomerular diseases therapies.