Detailed analysis of Japanese patients with adenosine deaminase 2 deficiency reveals characteristic elevation of type II interferon signature and STAT1 hyperactivation.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive inflammatory disease caused by loss-of-function mutations in both alleles of the ADA2 gene. Most patients with DADA2 exhibit systemic vasculopathy consistent with polyarteritis nodosa, but large phenotypic variability has been reported, and the pathogenesis of DADA2 remains unclear. OBJECTIVES: This study sought to assess the clinical and genetic characteristics of Japanese patients with DADA2 and to gain insight into the pathogenesis of DADA2 by multi-omics analysis. METHODS: Clinical and genetic data were collected from 8 Japanese patients with DADA2 diagnosed between 2016 and 2019. ADA2 variants in this cohort were functionally analyzed by in vitro overexpression analysis. PBMCs from 4 patients with DADA2 were subjected to transcriptome and proteome analyses. Patient samples were collected before and after introduction of anti- TNF-α therapies. Transcriptome data were compared with those of normal controls and patients with other autoinflammatory diseases. RESULTS: Five novel ADA2 variants were identified in these 8 patients and were confirmed pathogenic by in vitro analysis. Anti-TNF-α therapy controlled inflammation in all 8 patients. Transcriptome and proteome analyses showed that upregulation of type II interferon signaling was characteristic of DADA2. Network analysis identified STAT1 as a key regulator and a hub molecule in DADA2 pathogenesis, a finding supported by the hyperactivation of STAT1 in patients' monocytes and B cells after IFN-γ stimulation. CONCLUSIONS: Type II interferon signaling and STAT1 are associated with the pathogenesis of DADA2.

Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants.

PURPOSE: Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants. METHODS: We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFVP257L variant, for comparison of IL-1ß secretion using a cell-based assay and a novel THP-1-based assay. RESULTS: Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1ß secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFVP257L variants, the results were consistent between the cell-based assay and the THP-1-based assay. CONCLUSION: Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.

Haploinsufficiency of A20 with a novel mutation of deletion of exons 2-3 of TNFAIP3.

OBJECTIVES: Haploinsufficiency of A20 (HA20) due to loss-of-function mutations of TNFAIP3 leads to an autoinflammatory disease. These mutations produce a premature termination codon in most cases of HA20. However, exon deletion has not been reported. METHODS: Genomic DNA was extracted from the peripheral blood of the patient clinically suspected of HA20. We examined autoinflammatory disease-causing genes and performed a multiplex ligation-dependent probe amplification (MLPA) assay for copy number analysis. Next, to determine the disconnection point, genomic DNA was amplified with long-range PCR and sequenced. Finally, western blotting was carried out to measure A20 protein expression in mitogen phytohaemagglutinin (PHA)-induced T-cell blasts from the patient and a healthy volunteer. RESULTS: Targeted next-generation sequencing found no pathogenic mutation, but copy number variation (CNV) analysis suggested a heterozygous deletion of exons 2-3. The MLPA assay and long-range PCR confirmed the mutation. Western blotting analysis indicated a marked decrease in expression of A20 protein from the patient compared to a normal control. The results showed that this deletion was a pathogenic mutation. CONCLUSION: This study demonstrates a novel mutation resulting in a deletion of exons 2-3 of TNFAIP3. MLPA analysis is a useful initial screening method for HA20 patients.

Clinical characteristics and treatment of 50 cases of Blau syndrome in Japan confirmed by genetic analysis of the NOD2 mutation.

OBJECTIVES: To collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis. METHODS: Fifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians. RESULTS: The study population comprised 26 males and 24 females aged 0-61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment. CONCLUSIONS: In patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

Increased response to granulocyte-macrophage colony-stimulating factor in peripheral blood cells and transient manifestations mimicking juvenile myelomonocytic leukemia in a male patient with NEMO deficiency caused by a deep intronic pathogenic variant of IKBKG.

X-linked NF-κB essential modulator (NEMO) deficiency is a primary immunodeficiency characterized by combined immunodeficiency and ectodermal dysplasia. Monocytes from the patients demonstrate a severely impaired response to tissue necrosis factor or lipopolysaccharide, whereas hyper-inflammation is found in some patients. Juvenile myelomonocytic leukemia (JMML) is a pediatric malignancy caused by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and aberrant RAS signaling activation. Patients with JMML demonstrate characteristic manifestations such as splenomegaly, monocytosis and the presence of myeloid or erythroid precursors in the peripheral blood. Here, we present the case of a male infant with ectodermal dysplasia, bacterial septicemia, Pneumocystis pneumonia, severe inflammation and transient manifestations mimicking JMML. Genetic analysis revealed a deep intronic germline variant of IKBKG. Full-length IKBKG cDNA and NEMO protein expression were almost inexistent. Peripheral mononuclear cells (PBMCs) from the patient showed increased RAS signaling activation with GM-CSF or Phorbol 12-myristate 13-acetate without the RAS-associated gene variant, although the increased RAS signaling activation in induced-pluripotent stem cell-derived myeloid lineage and bone marrow-derived mesenchymal cells was not evident. The patient with NEMO deficiency demonstrated JMML-like manifestation and severe inflammation. PBMCs of the patient demonstrated increased RAS signaling activation with unknown pathophysiology.

Determination of α-hydroxy acids and their enantiomers in fruit juices by ligand exchange CE with a dual central metal ion system.

The content of α-hydroxy acids and their enantiomers can be used to distinguish authentic and adulterated fruit juices. Here, we investigated the use of ligand exchange CE with two kinds of central metal ion in a BGE for the simultaneous determination of enantiomers of dl-malic, dl-tartaric and dl-isocitric acids, and citric acid. Ligand exchange CE with 100 mM d-quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl-tartaric acid but not dl-malic acid or dl-isocitric acid. Addition of 1.8 mM Sc(III) ion to the BGE with 10 mM Cu(II) ion to create a dual central metal ion system permitted the simultaneous determination of these α-hydroxy acid enantiomers and citric acid. The proposed ligand exchange CE was thus well suited for detecting adulteration of fruit juices.

Glycemic variability in preterm infants receiving intermittent gastric tube feeding: report of three cases.

Late-onset hypoglycemia (day 12-16, blood glucose <50 mg/dL) was detected in three preterm infants (birthweight 998-1780 g; gestational age 27-30 weeks) by routine screening. All infants showed high serum insulin levels and extremely low ketone levels at the time of hypoglycemia. Continuous glucose monitoring was conducted at 31-34 weeks' postconceptual age when the infants were receiving intermittent gastric tube feeding with no intravenous glucose infusion. The continuous glucose monitoring results showed characteristic postprandial glucose increases and subsequent sharp deceases along with many hyper- and hypoglycemic events. This fluctuating pattern disappeared at 38-40 weeks' postconceptual age. These observations suggest that prolonged insulin oversecretion may be associated with early aggressive intravenous nutrition, and that large glycemic variability is a common feature of tube-fed preterm infants that can be explained by immature glucose homeostasis.

The environmental degradability of DEMNUM, a typical PFPE polymer.

The three environmental degradation tests of hydrolysis, indirect photolysis and Zahn-Wellens microbial degradation were conducted according to the OECD and the US EPA guidelines on DEMNUM, a typical linear perfluoropolyether polymer. Low mass degradation products that formed in each test were structurally characterized and indirectly quantified by liquid chromatography mass spectrometry (LC/MS) using a reference compound and an internal standard of similar structure. The degradation of the polymer was assumed to directly correlate with the appearance of lower mass species. The hydrolysis experiment at 50 °C showed the appearance of less than a dozen low mass species with increasing pH but at the negligible total estimated amount of ∼2 ppm relative to polymer. A dozen low mass perfluoro acid entities also appeared following the indirect photolysis experiment in synthetic humic water. Their maximum total amount was at ∼150 ppm relative to polymer. The largest total amount of low mass species formed during the Zahn-Wellens biodegradation test amounted to only ∼80 ppm relative to polymer. The Zahn-Wellens conditions tended to produce larger low mass molecules than the ones formed under photolysis. The results from all three tests indicate that the polymer is stable and non-degradable in the environment.

Human RELA dominant-negative mutations underlie type I interferonopathy with autoinflammation and autoimmunity.

Inborn errors of the NF-κB pathways underlie various clinical phenotypes in humans. Heterozygous germline loss-of-expression and loss-of-function mutations in RELA underlie RELA haploinsufficiency, which results in TNF-dependent chronic mucocutaneous ulceration and autoimmune hematological disorders. We here report six patients from five families with additional autoinflammatory and autoimmune manifestations. These patients are heterozygous for RELA mutations, all of which are in the 3' segment of the gene and create a premature stop codon. Truncated and loss-of-function RelA proteins are expressed in the patients' cells and exert a dominant-negative effect. Enhanced expression of TLR7 and MYD88 mRNA in plasmacytoid dendritic cells (pDCs) and non-pDC myeloid cells results in enhanced TLR7-driven secretion of type I/III interferons (IFNs) and interferon-stimulated gene expression in patient-derived leukocytes. Dominant-negative mutations in RELA thus underlie a novel form of type I interferonopathy with systemic autoinflammatory and autoimmune manifestations due to excessive IFN production, probably triggered by otherwise non-pathogenic TLR ligands.

Enantioseparation of α-hydroxy acids by chiral ligand exchange CE with a dual central metal ion system.

Using two kinds of central metal ions in a background electrolyte, ligand exchange CE was investigated for the simultaneous enantioseparation of dl-malic, dl-tartaric, and dl-isocitric acids. Ligand exchange CE with 100 mM d-quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl-tartaric acid but not dl-malic acid or dl-isocitric acid. A dual central metal ion system containing 0.5 mM Al(III) ion in addition to 10 mM Cu(II) ion in the background electrolyte enabled the simultaneous enantioseparation of the three α-hydroxy acids. These results suggest that the use of a dual central metal ion system can be useful for enantioseparation by ligand exchange CE.

Direct enantioseparation of lipoic acid in dietary supplements by capillary electrophoresis using trimethyl-ß-cyclodextrin as a chiral selector.

Lipoic acid, an antioxidant, naturally occurs as the (R)-enantiomer, while synthetic lipoic acid is racemic. It is thus of interest to know the (R)-enantiomer content of lipoic acid supplements. Here, we used capillary electrophoresis to directly enantioseparate lipoic acid in dietary supplements by using a sulfonated capillary with an effective voltage of +18 kV and direct detection at 200 nm. Factors affecting migration time and resolution of lipoic acid were investigated. The optimum background electrolyte was found to be 100 mM phosphate buffer (pH 7.0) containing 8 mM trimethyl-ß-cyclodextrin as a chiral selector at 20°C. Under the proposed conditions, direct chiral resolution of lipoic acid in dietary supplements was conducted successfully.

Induced Pluripotent Stem Cell-Derived Monocytes/Macrophages in Autoinflammatory Diseases.

The concept of autoinflammation, first proposed in 1999, refers to a seemingly unprovoked episode of sterile inflammation manifesting as unexplained fever, skin rashes, and arthralgia. Autoinflammatory diseases are caused mainly by hereditary abnormalities of innate immunity, without the production of autoantibodies or autoreactive T cells. The revolutionary discovery of induced pluripotent stem cells (iPSCs), whereby a patient's somatic cells can be reprogrammed into an embryonic pluripotent state by forced expression of a defined set of transcription factors, has the transformative potential to enable in vitro disease modeling and drug candidate screening, as well as to provide a resource for cell replacement therapy. Recent reports demonstrate that recapitulating a disease phenotype in vitro is feasible for numerous monogenic diseases, including autoinflammatory diseases. In this review, we provide a comprehensive overview of current advances in research into autoinflammatory diseases involving iPSC-derived monocytes/macrophages. This review may aid in the planning of new studies of autoinflammatory diseases.

Allograft dysfunction after lung transplantation for COPA syndrome: A case report and literature review.

Coatomer subunit alpha (COPA) syndrome is an autoinflammatory disease with autoimmune and autoinflammatory manifestations affecting lungs, joints, and kidneys. COPA syndrome is caused by heterozygous loss-of-function mutations in COPA gene, encoding α subunit of coatmer protein complex I (COP-I) coated vesicles. Mutant COPA induces constitutive activation of stimulator of interferon genes, leading to systemic inflammation and elevated type I interferon response. We have previously reported a Japanese family of COPA syndrome with a novel V242G mutation. Two out of four patients required lung transplantation due to intractable interstitial lung disease and respiratory failure. Both of them deceased after lung transplantation, one due to sepsis and the other due to allograft dysfunction possibly caused by the reccurent interstitial lung disease. The literature review indentified unfavorable outcome of the solid organ transplant in COPA syndrome and its related disease, however, precise clinico-pathological description of these cases has been scarce. Here, we report in detail the clinical course of our cases to clarify the pathophysiology of allograft dysfunction in COPA syndrome and propose potential therapeutic approaches to improve post-transplant graft survival.