Low remission rates and high incidence of adverse events in a prospective VEXAS syndrome registry.

OBJECTIVE: We aimed to gather real-world clinical evidence of detailed disease activity, treatments, remission rates, and adverse events (AEs) associated with vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome in a prospective study. METHODS: Patients in Japan suspected of having VEXAS syndrome were enrolled in a registry study. A novel disease activity measure (VEXASCAF) assessing 11 symptoms associated with VEXAS syndrome was evaluated at enrolment and after 3 months. AEs, survival, CRP levels, and treatments were also recorded at enrolment and 3 months after enrolment. All exons of UBA1 were sequenced using a next-generation sequencer to determine the variant allele frequencies of pathogenic variants in the peripheral blood of all patients. RESULTS: Of the 55 registered patients, 30 patients were confirmed to have pathogenic variants of UBA1. All patients were male, with a median age of 73.5 years. VEXASCAF and CRP levels decreased significantly at 3 months post-enrolment, but the oral prednisolone dose did not change. Only two patients achieved complete remission according to FRENVEX at 3 months after enrolment. During the observation period of 6 months, 28 AEs were observed, including 3 deaths, 4 malignancies from two cases, 2 thromboses, and 13 infections (including 4 mycobacterial infections). Inflammation of the lung and cervical region (i.e. parotid and submandibular gland swelling, tonsillitis, cervical swelling, and pain) were the most common AEs. CONCLUSIONS: Patients with VEXAS syndrome required high-dose glucocorticoids to achieve remission, and complications-such as malignancy, thrombosis, and infection-occurred frequently within a short observation period.

Novel STAT1 Variants in Japanese Patients with Isolated Mendelian Susceptibility to Mycobacterial Diseases.

PURPOSE: Heterozygous dominant-negative (DN) STAT1 variants are responsible for autosomal dominant (AD) Mendelian susceptibility to mycobacterial disease (MSMD). In this paper, we describe eight MSMD cases from four kindreds in Japan. METHODS: An inborn error of immunity-related gene panel sequencing was performed using genomic DNA extracted from whole blood samples. The identified variants were validated using Sanger sequencing. Functional analysis was evaluated with a luciferase reporter assay and co-transfection assay in STAT1-deficient cells. RESULTS: Patient 1.1 was a 20-month-old boy with multifocal osteomyelitis and paravertebral abscesses caused by Mycobacterium bovis bacillus Calmette-Guérin (BCG). Although the paravertebral abscess was refractory to antimycobacterial drugs, the addition of IFN-γ and drainage of the abscess were effective. Intriguingly, his mother (patient 1.2) showed an uneventful clinical course except for treatment-responsive tuberculous spondylitis during adulthood. Patient 2.1 was an 8-month-old boy with lymphadenopathy and lung nodules caused by BCG. He responded well to antimycobacterial drugs. His mother (patient 2.2) was healthy. Patient 3.1 was a 11-year-old girl with suspected skin tuberculosis. Her brother (patient 3.2) had BCG-osis, but their mother (patient 3.3) was healthy. Patient 4 was an 8-month-old girl with left axillary and supraclavicular lymphadenopathy associated with BCG vaccination. Kindreds 1, 2, and 3 were shown to have novel heterozygous variants (V642F, R588C, and R649G) in STAT1, respectively. Kindred 4 had previously reported heterozygous variants (Q463H). A luciferase reporter assay in STAT1-deficient cells followed by IFN-γ stimulation confirmed that these variants are loss-of-function. In addition, with co-transfection assay, we confirmed all of these variants had DN effect on WT STAT1. CONCLUSION: Four kindred MSMD subjects with 3 novel variants and 1 known variant in STAT1 were identified in this study. AD STAT1 deficiency might be prevalent in Japanese patients with BCG-associated MSMD.

Promoting awareness of terminology related to unmet medical needs in context of rheumatic diseases in Japan: a systematic review for evaluating unmet medical needs.

To optimize patient prognosis, patient needs, including unmet needs, should be adequately assessed. However, such needs are more challenging to report and, consequently, more likely to go unmet compared with the needs reported by physicians. We aimed to determine the appropriate direction of future research on unmet medical needs in rheumatic diseases in Japan by conducting a literature review. We searched PubMed and Web of Science using 23 terms linked to unmet medical needs for major rheumatic diseases in Japan. Further, we collected articles on health-related quality of life and investigated the scales used for assessment, as well as whether the terms "unmet needs" or "unmet medical needs" were used. We identified 949 papers on 10 diseases, including systemic lupus erythematosus, systemic sclerosis, dermatomyositis, juvenile idiopathic arthritis, adult-onset Still's disease, antiphospholipid syndrome, mixed connective tissue disease, Takayasu arteritis, Sjögren's syndrome, and Behçet's disease; 25 of the 949 papers were selected for full-text review. Fifteen articles on five diseases were related to health-related quality of life. The term "unmet needs" was used in only one article. Six out of 15 studies used the 36-item short form survey, whereas the scales used in other studies differed. The optimal treatment plan determined by a physician may not necessarily align with the best interests of the patient. In clinical research, cross sectional and standardized indicators of health-related quality of life should be employed along with highly discretionary questionnaires to assess and optimize resource allocation in healthcare and simultaneously achieve patient-desired outcomes.

Systemic inflammation caused by an intracranial mesenchymal tumor with a EWSR1::CREM fusion presenting associated with IL-6/STAT3 signaling.

Pediatric neoplastic diseases account for about 10% of cases of fever of unknown origin (FUO), and most neoplastic disease cases are leukemia, lymphoma, and neuroblastoma. Brain tumors are rarely reported as the cause of FUO, although craniopharyngioma, metastatic brain tumor, and Castleman's disease have been reported. We report a case of intracranial mesenchymal tumor (IMT) with a FET:CREB fusion gene, which had inflammatory phenotype without neurological signs. A 10-year-old girl was admitted with a 2-month history of intermittent fever and headache, whereas her past history as well as her family history lacked special events. Sepsis work-up showed no pathological organism, and empirical antibiotic therapy was not effective. Bone marrow examination showed a negative result. Cerebrospinal fluid examination showed elevated protein as well as cell counts, and head magnaetic resonance imaging showed a hypervascular mass lesion with contrast enhancement in the left cerebellar hemisphere. The patient underwent tumor excision, which made the intermittent fever disappear. Pathological examinations resembled those of classic angiomatoid fibrous histiocytoma (AFH), but the morphological features were distinct from the AFH myxoid variant; then we performed break-apart fluorescence in situ hybridization and confirmed the tumor harbored the rare EWSR1::CREM fusion gene (Ewing sarcoma breakpoint region 1 gene (EWSR1) and cAMP response element binding (CREB) family gene). Consequently, we diagnosed the condition as IMT with EWSR1::CREM fusion. Elevated serum concentration of interleukin 6 (IL-6) was normalized after tumor resection, which suggested the fever could be caused by tumor-derived IL-6. This is the first case of IMT with EWSR1::CREM fusion that showed paraneoplastic symptoms associated with the IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Although brain tumors are rarely diagnosed as a responsible disease for FUO, they should be considered as a cause of unknown fever even in the absence of abnormal neurological findings.

Immunoglobulin G deposition on proximal tubules and the tubular basement membrane in acute tubular injury complicated with focal segmental glomerulosclerosis (FSGS): A possible prediction tool for subclinical FSGS.

Immunofluorescent deposition of immunoglobulin G (IgG) in the tubular basement membrane (TBM) has been evaluated in the diagnosis of various diseases; however, few studies have investigated the immunofluorescence of acute tubular injury (ATI). Herein, we attempted to clarify IgG expression in the proximal tubular epithelium and TBM in ATI due to various causes. Patients with ATI with nephrotic-range proteinuria, including focal segmental glomerulosclerosis (FSGS, n = 18) and minimal change nephrotic syndrome (MCNS, n = 8), ATI with ischemia (n = 6), and drug-induced ATI (n = 7), were enrolled. ATI was evaluated by light microscopy. CD15 and IgG double staining and IgG subclass staining were performed to evaluate immunoglobulin deposition in the proximal tubular epithelium and TBM. IgG deposition was identified in the proximal tubules only in the FSGS group. Furthermore, IgG deposition in the TBM was observed in the FSGS group showing severe ATI. IgG3 was predominantly deposited by the IgG subclass study. Our results indicate that IgG deposition in the proximal tubular epithelium and TBM suggests the leaking of IgG from the glomerular filtration barrier and its reabsorption by proximal tubules, which may predict disruption of the glomerular size barrier, including subclinical FSGS. FSGS with ATI should be included as a differential diagnosis when IgG deposition in TBM is observed.

Intracellular virus sensor MDA5 mutation develops autoimmune myocarditis and nephritis.

Mutations in IFIH1 gene encoding viral RNA sensor MDA5 have been reported responsible for many interferonopathies, including Aicardi-Goutières syndrome (AGS) and monogenic lupus, however, the pathological link between IFIH1 mutations and various autoimmune symptoms remains unclear. Here, we generated transgenic mice expressing human MDA5 R779H mutant (R779H Tg), reported in AGS and monogenic lupus patient. Mice spontaneously developed myocarditis and nephritis with upregulation of type I IFNs in the major organs. R779H Tg Mavs-/- and R779H Tg Ifnar-/- showed no phenotypes, indicating direct MDA5-signaling pathway involvement. Rag-2 deficiency and bone marrow cells transfer from wild type to adult mice did not prevent myocarditis development, while mice with cardiomyocyte-specific expression of hMDA5 R779H showed cardiomegaly and high expression of inflammatory cytokines. Taken together, our study clarifies that type I IFNs production and chemokines from cardiomyocytes starts in neonatal period and is critical for the development of myocarditis. Activated lymphocytes and auto-antibodies exacerbate the pathogenesis but are dispensable for the onset.

Aicardi-Goutières syndrome-like encephalitis in mutant mice with constitutively active MDA5.

MDA5 is a cytoplasmic sensor of viral RNA, triggering type I interferon (IFN-I) production. Constitutively active MDA5 has been linked to autoimmune diseases such as systemic lupus erythematosus, Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome (AGS), a genetically determined inflammatory encephalopathy. However, AGS research is challenging due to the lack of animal models. We previously reported lupus-like nephritis and SMS-like bone abnormalities in adult mice with constitutively active MDA5 (Ifih1G821S/+), and herein demonstrate that these mice also exhibit high lethality and spontaneous encephalitis with high IFN-I production during the early postnatal period. Increases in the number of microglia were observed in MDA5/MAVS signaling- and IFN-I-dependent manners. Furthermore, microglia showed an activated state with an increased phagocytic capability and reduced expression of neurotrophic factors. Although multiple auto-antibodies including lupus-related ones were detected in the sera of the mice as well as AGS patients, Ifih1G821S/+Rag2-/- mice also exhibited up-regulation of IFN-I, astrogliosis and microgliosis, indicating that auto-antibodies or lymphocytes are not required for the development of the encephalitis. The IFN-I signature without lymphocytic infiltration observed in Ifih1G821S/+ mice is a typical feature of AGS. Collectively, our results suggest that the Ifih1G821S/+ mice are a model recapitulating AGS and that microglia are a potential target for AGS therapy.

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.

Enzyme activity in dried blood spot as a diagnostic tool for adenosine deaminase 2 deficiency.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by mutations in the adenosine deaminase 2 (ADA2) gene. Loss of functional ADA2 activity results in vasculitis syndrome, immunodeficiency, and hematopoietic disorders. Early diagnosis is required for effective treatment. METHODS: We developed a dried blood spot (DBS)-based ADA2 activity colorimetric assay. Heparin-affinity purification was used during sample preparation to improve the assay more efficiently. The stability of ADA2 during DBS storage and ADA2 activity of DADA2 patients and healthy controls were examined. RESULTS: Active ADA2 was extracted from the DBS of healthy controls. ADA2 activity in DBS, stored either frozen or refrigerated, remained stable for at least 90 days. A significant difference in ADA2 activity was observed between healthy controls and patients. No ADA2 activity was detected in DBS from patients. CONCLUSIONS: Our new DBS ADA2 activity assay is experimentally simple, highly adaptable, and requires no special equipment except for a microplate reader. A low background was achieved with heparin-affinity purification. The method differentiates clearly between healthy controls and patients. ADA2 activity can be reliably measured in DBS, providing an opportunity to diagnose DADA2 at an early stage.

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.

MEFV E148Q variant is more associated with familial Mediterranean fever when combined with other non-exon 10 MEFV variants in Japanese patients with recurrent fever.

OBJECTIVE: To investigate the genetic characteristics of one of the MEFV gene variants, p.Glu148Gln (E148Q), in patients with familial Mediterranean fever (FMF) and examine its significance in Japanese patients with recurrent fever. METHODS: The clinical phenotype and genomic variants of systemic autoinflammatory diseases (SAIDs), including MEFV, were analyzed in 211 Japanese patients with recurrent fever. Genetic analysis was performed via next-generation sequencing of exons, including exon-intron boundaries. RESULTS: Twelve patients met the diagnostic criteria for SAIDs other than FMF. Considering 199 patients with recurrent fever, 137 cases (68.8%) were clinically diagnosed with FMF. Although Bonferroni-adjusted p-value did not reach significance level, the group containing heterozygous E148Q and other variants tended to be at higher risk of developing the FMF phenotype (nominal p = .036) than the group with heterozygous E148Q only. Comparison between the group with heterozygous E148Q and other variants and the heterozygous group containing non-E148Q showed no statistically significant difference in FMF phenotype expression (nominal p = 1.00). CONCLUSION: Patients with heterozygous E148Q and other variants exhibited higher expression of FMF phenotype than those with heterozygous E148Q only, and suggested that other variants than E148Q as well as exon 10 variants might contribute to the FMF phenotype.

Clinical phenotypes and genetic analyses for diagnosis of systemic autoinflammatory diseases in adult patients with unexplained fever.

OBJECTIVE: To make an accurate diagnosis of systemic autoinflammatory diseases (SAIDs), clinical and genetic analyses were performed in patients with unexplained fever. METHODS: The clinical phenotype and genomic variants of 11 genes responsible for SAIDs were analyzed in 179 Japanese patients with unexplained fever. Genetic analysis was performed by next generation sequencing (NGS) on exons including exon-intron boundaries. RESULTS: Three cases met the diagnostic criteria for SAIDs other than familial Mediterranean fever (FMF). Considering 176 patients with unexplained fever, 43 cases (24.0%) were clinically diagnosed as FMF. Gene variants were found in 53 cases (30.1%) when searching for variants in the 10 disease genes other than the MEFV gene. Among them, the most frequently-identified genes were NLRP3, NOD2, NLRP12, NLRC4, and PLCG2, which accounted for 14, 7, 17, 7, and 6 cases, respectively. These variants were less than 1% of healthy individuals or novel variants, but not regarded as pathogenic since the patients did not meet the diagnostic criteria of SAIDs caused by their identified variants clinically. CONCLUSION: Twenty four percent of Japanese patients with unexplained fever were clinically diagnosed as FMF in this study. Low frequency but not pathogenic variants in genes other than MEFV were identified in 30.1% of the cases. It is not clear how much these gene variants contribute to the inflammatory phenotypes; therefore, further analysis would uncover their autoinflammatory phenotypes that cause fever.

Simple and Sensitive Analysis for Dried Blood Spot Proteins by Sodium Carbonate Precipitation for Clinical Proteomics.

Dried blood spots (DBS) are widely used for screening biomolecular profiles, including enzymatic activities. However, detection of minor proteins in DBS by liquid chromatography-mass spectrometry (LC-MS/MS) without pre-enrichment remains challenging because of the coexistence of large quantities of hydrophilic proteins. In this study, we address this problem by developing a simple method using sodium carbonate precipitation (SCP). SCP enriches hydrophobic proteins from DBS, allowing substantial removal of soluble proteins. In combination with SCP, we used quantitative LC-MS/MS proteome analysis in a data-independent acquisition mode (DIA) to enhance the sensitivity and quantification limits of proteome analysis. As a result, identification of 1977 proteins in DBS is possible, including 585 disease-related proteins listed in the Online Mendelian Inheritance in Man.

Immunophenotyping of A20 haploinsufficiency by multicolor flow cytometry.

Haploinsufficiency of A20 (HA20) causes inflammatory disease resembling Behçet's disease; many cases have been reported, including some that are complicated with autoimmune diseases. This study aims to clarify the immunophenotype of patients with HA20 by analyzing lymphocyte subsets using multicolor flow cytometry. The patients with HA20 previously diagnosed in a nationwide survey were compared by their cell subpopulations. In total, 27 parameters including regulatory T cells (Tregs), double-negative T cells (DNTs), and follicular helper T cells (TFHs) were analyzed and compared with the reference values in four age groups: 0-1, 2-6, 7-19, and ≥20 years. The Tregs of patients with HA20 tended to increase in tandem with age-matched controls at all ages. In addition, patients ≥20 years had increased DNTs compared with controls, whereas TFHs significantly increased in younger patients. In HA20 patients, the increase in DNTs and TFHs may contribute to the development of autoimmune diseases.

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.

ISSAID/EMQN Best Practice Guidelines for the Genetic Diagnosis of Monogenic Autoinflammatory Diseases in the Next-Generation Sequencing Era.

BACKGROUND: Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. METHODS: The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. RESULTS: In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. CONCLUSIONS: These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

Human CTL-based functional analysis shows the reliability of a munc13-4 protein expression assay for FHL3 diagnosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D, we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening.

Low-frequency mosaicism in cryopyrin-associated periodic fever syndrome: mosaicism in systemic autoinflammatory diseases.

Autoinflammatory disease is an 'inborn error of immunity', resulting in systemic inflammation. Cryopyrin-associated periodic syndrome (CAPS) is a prototypical autoinflammatory disease caused by gain-of-function mutations in the NLRP3 (NLR family pyrin domain containing 3) gene; these mutations activate the NLRP3 inflammasome, resulting in overproduction of IL-1ß. The first case of CAPS caused by somatic NLRP3 mosaicism was reported in 2005 after identification of variant small peaks by Sanger sequencing. An international collaborative study revealed that the majority of mutation-negative CAPS cases are due to low-level NLRP3 mosaicism, suggesting that central nervous system involvement in somatic mosaicism patients is milder than in genotype-matched heterozygous patients. Recent advances in next-generation sequencing have expanded the number of NLRP3 somatic mosaicism cases and identified a new entity called 'late-onset CAPS with myeloid-specific NLRP3 mosaicism'; however, no mosaic-specific clinical features have been identified/confirmed yet. With respect to NLRP3 mosaicism in CAPS, a prospective longitudinal study on the variant genotype, its allele frequency and its tissue distribution (along with a comprehensive clinical phenotype) would provide better understanding of NLRP3 mosaicism, resulting in more appropriate patient care and genetic counseling.

Hematopoietic stem cell transplantation in 29 patients hemizygous for hypomorphic IKBKG/NEMO mutations.

X-linked recessive ectodermal dysplasia with immunodeficiency is a rare primary immunodeficiency caused by hypomorphic mutations of the IKBKG gene encoding the nuclear factor κB essential modulator (NEMO) protein. This condition displays enormous allelic, immunological, and clinical heterogeneity, and therapeutic decisions are difficult because NEMO operates in both hematopoietic and nonhematopoietic cells. Hematopoietic stem cell transplantation (HSCT) is potentially life-saving, but the small number of case reports available suggests it has been reserved for only the most severe cases. Here, we report the health status before HSCT, transplantation outcome, and clinical follow-up for a series of 29 patients from unrelated kindreds from 11 countries. Between them, these patients carry 23 different hypomorphic IKBKG mutations. HSCT was performed from HLA-identical related donors (n = 7), HLA-matched unrelated donors (n = 12), HLA-mismatched unrelated donors (n = 8), and HLA-haploidentical related donors (n = 2). Engraftment was documented in 24 patients, and graft-versus-host disease in 13 patients. Up to 7 patients died 0.2 to 12 months after HSCT. The global survival rate after HSCT among NEMO-deficient children was 74% at a median follow-up after HSCT of 57 months (range, 4-108 months). Preexisting mycobacterial infection and colitis were associated with poor HSCT outcome. The underlying mutation does not appear to have any influence, as patients with the same mutation had different outcomes. Transplantation did not appear to cure colitis, possibly as a result of cell-intrinsic disorders of the epithelial barrier. Overall, HSCT can cure most clinical features of patients with a variety of IKBKG mutations.