Publications by authors named "Ryuta Nishikomori"

140 Publications

Hematopoietic Cell Transplantation Ameliorates Autoinflammation in A20 Haploinsufficiency.

J Clin Immunol 2021 Aug 24. Epub 2021 Aug 24.

Department of Child Health and Development, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.

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http://dx.doi.org/10.1007/s10875-021-01124-1DOI Listing
August 2021

Multiomic technologies for analyses of inborn errors of immunity: from snapshot of the average cell to dynamic temporal picture at single-cell resolution.

Inflamm Regen 2021 Jul 1;41(1):19. Epub 2021 Jul 1.

Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, 292-0818, Japan.

Advances in DNA sequencing technology have significantly impacted human genetics; they have enabled the analysis of genetic causes of rare diseases, which are usually pathogenic variants in a single gene at the nucleotide sequence level. However, since the quantity of data regarding the relationship between genotype and phenotype is insufficient to diagnose some rare immune diseases definitively, genetic information alone cannot help obtain a mechanistic understanding of the disease etiology. For such cases, exploring the molecular phenotype using multiomic analyses could be the approach of choice. In this review, we first overview current technologies for multiomic analysis, particularly focusing on RNA and protein profiling of bulk cell ensembles. We then discuss the measurement modality and granularity issue because it is critical to design multiomic experiments properly. Next, we illustrate the importance of bioimaging by describing our experience with the analysis of an autoinflammatory disease, cryopyrin-associated periodic fever syndrome, which could be caused by low-frequency somatic mosaicism and cannot be well characterized only by multiomic snapshot analyses of an ensemble of many immune cells. We found it powerful to complement the multiomic data with bioimaging data that can provide us with indispensable time-specific dynamic information of every single cell in the "immune cell society." Because we now have many measurement tools in different modalities and granularity to tackle the etiology of rare hereditary immune diseases, we might gain a deeper understanding of the pathogenic mechanisms of these diseases by taking full advantage of these tools in an integrated manner.
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http://dx.doi.org/10.1186/s41232-021-00169-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247241PMC
July 2021

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

Anal Biochem 2021 Sep 24;628:114292. Epub 2021 Jun 24.

Division of Life Science, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-15-4-1 Maeda, Teine, Sapporo, Hokkaido, 006-8585, Japan.

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.
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http://dx.doi.org/10.1016/j.ab.2021.114292DOI Listing
September 2021

Gene Mutation Presenting at Age 2 Years With Autoimmune Retinopathy and Steroid-Responsive Acute Liver Failure: A Case Report and Literature Review.

Front Immunol 2021 28;12:687280. Epub 2021 May 28.

Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare monogenic autosomal recessive disorder caused by mutation in the autoimmune regulator (AIRE) gene. Patients usually are diagnosed at ages between 5 and 15 years when they show 3 or more manifestations, most typically mucocutaneous candidiasis, Addison's disease, and hypoparathyroidism. APECED-associated hepatitis (APAH) develops in only 10% to 40% of patients, with severity varying from subclinical chronic active hepatitis to potentially fatal acute liver failure (ALF). Ocular abnormalities are fairly common, most often keratopathy but sometimes retinopathy. Here we report a 2-year-old Japanese girl with an gene mutation who developed APAH with ALF, preceded by autoimmune retinopathy associated with anti-recoverin antibody before major symptoms suggested a diagnosis of APECED. Intravenous pulse methylprednisolone therapy followed by a corticosteroid combined with azathioprine treatment resolved ALF and achieved control of APAH. To our knowledge, our patient is the youngest reported to have ALF resulting from an gene mutation. Pulse methylprednisolone induction therapy followed by treatment with corticosteroid plus azathioprine may well be effective in other children with APAH and gene mutations.
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http://dx.doi.org/10.3389/fimmu.2021.687280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194255PMC
May 2021

Case Report: Rituximab Improved Epileptic Spasms and EEG Abnormalities in an Infant With West Syndrome and Anti-NMDAR Encephalitis Associated With APECED.

Front Neurol 2021 20;12:679164. Epub 2021 May 20.

Department of Paediatrics, St Mary's Hospital, Kurume, Fukuoka, Japan.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is a rare autosomal recessive disorder caused by a mutation in the autoimmune regulator gene. Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy typically exhibit hypoparathyroidism, adrenocortical failure, and chronic mucocutaneous candidiasis. There are only a few case reports of autoimmune encephalitis during autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, but not as an initial manifestation. Furthermore, there are no reports of patients with infantile spasms/West syndrome with autoimmune encephalitis, partly because the median age for paediatric patients with anti-N-methyl-D-aspartate receptor encephalitis, which is the most frequent and best characterised in paediatric autoimmune encephalitides, is 13-14 years. Herein, we present a case of a 3-month-old infant with autoimmune encephalitis as an initial manifestation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy who later developed infantile spasms/West syndrome. A 3-month-old girl was admitted to our hospital with a fever, involuntary movements in all four limbs, and right-side facial palsy. Acute central nervous system demyelination diseases were suspected from neuroimaging findings and the presence of the cerebrospinal fluid oligoclonal band. She did not respond to multiple methylprednisolone pulse therapies and later developed infantile spasms/West syndrome and diabetes mellitus. Rituximab, a chimeric mouse/human monoclonal antibody directed against human CD20 which depletes B cells, was initially administered as a treatment for autoimmune encephalitis. Unexpectedly, this treatment resulted in complete spasm cessation and resolution of hypsarrhythmia. The patient eventually showed severely delayed developmental milestones, and her electroencephalography findings showed periodic generalised slow spike-and-wave pattern. Despite the limited ability to extrapolate findings from a single case, rituximab's effects may suggest that B cells play a crucial role in infantile spasms/West syndrome mechanisms; use of rituximab as an aetiology-specific treatment for infantile spasms/West syndrome patients with autoimmune encephalitis or its effectiveness for infantile spasms/West syndrome patients with other underlying mechanisms warrants further investigation.
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http://dx.doi.org/10.3389/fneur.2021.679164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176955PMC
May 2021

Necrotizing Funisitis as an Intrauterine manifestation of Cryopyrin-Associated Periodic Syndrome: a case report and review of the literature.

Pediatr Rheumatol Online J 2021 May 31;19(1):77. Epub 2021 May 31.

Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Background: Cryopyrin-associated periodic syndrome (CAPS) is a life-long, autoinflammatory disease associated with a gain-of-function mutation in the nucleotide-binding domain, leucine-rich repeat family, pyrin domain containing 3 (NLRP3) gene, which result in uncontrolled production of IL-1β and chronic inflammation. Chronic infantile neurologic cutaneous and articular (CINCA) syndrome/neonatal-Onset multisystem inflammatory disease (NOMID) is the most severe form of CAPS. Although the first symptoms may be presented at birth, there are few reports on the involvement of the placenta and umbilical cord in the disease. Therefore, we present herein a preterm case of CINCA/NOMID syndrome and confirms intrauterine-onset inflammation with conclusive evidence by using fetal and placental histopathological examination.

Case Presentation: The female patient was born at 33weeks of gestation by emergency caesarean section and weighted at 1,514 g. The most common manifestations of CINCA/NOMID syndrome including recurrent fever, urticarial rash, and ventriculomegaly due to aseptic meningitis were presented. She also exhibited atypical symptoms such as severe hepatosplenomegaly with cholestasis. The genetic analysis of NLRP3 revealed a heterozygous c.1698 C > G (p.Phe566Leu) mutation, and she was diagnosed with CINCA/NOMID syndrome. Further, a histopathological examination revealed necrotizing funisitis, mainly inflammation of the umbilical artery, along with focal neutrophilic and lymphocytic villitis.

Conclusions: The necrotizing funisitis, which only involved the artery, was an unusual observation for chorioamnionitis. These evidences suggest that foetal inflammation, probably due to overproduction of IL-1β, caused tissue damage in utero, and the first symptom of a newborn with CINCA/NOMID.
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http://dx.doi.org/10.1186/s12969-021-00578-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165790PMC
May 2021

Augmentation of STING-induced type I interferon production in COPA syndrome.

Arthritis Rheumatol 2021 May 13. Epub 2021 May 13.

Department of Immunology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan.

Objectives: COPA syndrome, also known as an autoinflammatory interstitial lung, joint, and kidney (AILJK) disease, is caused by heterozygous mutations in the coatomer subunit alpha (COPA) gene. We found a novel COPA variant in four patients in one family. We aimed to elucidate whether and how the variant causes manifestations of COPA syndrome by studying these four patients and in a gene-targeted mouse model.

Method: We performed whole exome sequencing in seven family members and measured type I interferon (IFN) signature of the peripheral blood cells. We analyzed the effects of COPA variants in in vitro experiments and Copa mutant mice we generated.

Results: We identified a heterozygous variant of COPA gene in the four affected members of the family (c.725T>G, p.Val242Gly). IFN score was high in the members carrying the variant. In vitro analysis revealed that COPA V242G as well as the previously reported disease-causing variants augmented the stimulator of interferon genes (STING)-induced type I IFN promoter activities. Copa mice manifested interstitial lung disease and STING-dependent elevation of IFN-stimulated genes (ISGs) expression. In Copa dendritic cells, the STING pathway was not constitutively activated, but hyperactivated upon stimulation and led to increased type I IFN production.

Conclusion: V242G, a novel COPA variant, was found in four patients from one family. The gene-targeted mice with V242G variant recapitulated the interstitial lung disease and showed augmented responses of the STING pathway leading to increase of type I IFN production.
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http://dx.doi.org/10.1002/art.41790DOI Listing
May 2021

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

J Clin Immunol 2021 Aug 17;41(6):1187-1197. Epub 2021 Mar 17.

Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan.

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 MEFV variant, for comparison of IL-1β 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-1β 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 MEFV 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.
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http://dx.doi.org/10.1007/s10875-021-01021-7DOI Listing
August 2021

A novel mutation in early-onset sarcoidosis/Blau syndrome: an association with Propionibacterium acnes.

Pediatr Rheumatol Online J 2021 Feb 18;19(1):18. Epub 2021 Feb 18.

Department of Pediatrics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, 755-8505, Ube, Yamaguchi, Japan.

Background: Early-onset sarcoidosis (EOS) and Blau syndrome (BS) are systemic inflammatory granulomatous diseases without visible pulmonary involvement, and are distinguishable from their sporadic and familial forms. The diseases are characterized by a triad of skin rashes, symmetrical polyarthritis, and recurrent uveitis. The most common morbidity is ocular involvement, which is usually refractory to conventional treatment. A gain-of-function mutation in the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene has been demonstrated in this disease; however, little is known about the relationship between the activation of NOD2 and the pathophysiology of EOS/BS. Here we describe EOS/BS with a novel mutation in the NOD2 gene, as well as detection of Propionibacterium acnes (P. acnes) in the granulomatous inflammation.

Case Presentation: An 8-year-old Japanese girl presented with refractory bilateral granulomatous panuveitis. Although no joint involvement was evident, she exhibited skin lesions on her legs; a skin biopsy revealed granulomatous dermatitis, and P. acnes was detected within the sarcoid granulomas by immunohistochemistry with P. acnes-specific monoclonal (PAB) antibody. Genetic analyses revealed that the patient had a NOD2 heterozygous D512V mutation that was novel and not present in either of her parents. The mutant NOD2 showed a similar activation pattern to EOS/BS, thus confirming her diagnosis. After starting oral prednisolone treatment, she experienced an anterior vitreous opacity relapse despite gradual prednisolone tapering; oral methotrexate was subsequently administered, and the patient responded positively.

Conclusions: We presented a case of EOS/BS with a novel D512V mutation in the NOD2 gene. In refractory granulomatous panuveitis cases without any joint involvement, EOS/BS should be considered as a differential diagnosis; genetic analyses would lead to a definite diagnosis. Moreover, this is the first report of P. acnes demonstrated in granulomas of EOS/BS. Since intracellular P. acnes activates nuclear factor-kappa B in a NOD2-dependent manner, we hypothesized that the mechanism of granuloma formation in EOS/BS may be the result of NOD2 activity in the presence of the ligand muramyl dipeptide, which is a component of P. acnes. These results indicate that recognition of P. acnes through mutant NOD2 is the etiology in this patient with EOS/BS.
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http://dx.doi.org/10.1186/s12969-021-00505-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890802PMC
February 2021

2 Mutation-Associated Case with Blau Syndrome Triggered by BCG Vaccination.

Children (Basel) 2021 Feb 6;8(2). Epub 2021 Feb 6.

Department of Dermatology, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 663-8501, Japan.

We describe a patient who developed multiple granulomatous skin lesions after Bacille de Calmette et Guérin (BCG) vaccination without significant effect by topical corticosteroid, followed by painless cystic tumors on the bilateral knees and hands and inflammatory changes on ophthalmologic examination. A functional mutation in 2 was detected by a genetic analysis, and he was diagnosed as sporadic Blau syndrome. Since 2 acts as a sensor for the BCG component, it is possible that BCG vaccination may trigger granuloma formation in Blau syndrome patients with such genetic background.
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http://dx.doi.org/10.3390/children8020117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915141PMC
February 2021

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

J Allergy Clin Immunol 2021 Aug 30;148(2):550-562. Epub 2021 Jan 30.

Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan.

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.
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http://dx.doi.org/10.1016/j.jaci.2021.01.018DOI Listing
August 2021

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

Mod Rheumatol 2021 Mar 16:1-7. Epub 2021 Mar 16.

Division of Respirology, Neurology and Rheumatology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan.

Objective: To investigate the genetic characteristics of one of the 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 , 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  = .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  = 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.
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http://dx.doi.org/10.1080/14397595.2021.1880534DOI Listing
March 2021

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

Int Immunol 2021 Mar;33(4):225-240

Laboratory of Regulatory Information, Institute for Frontier Life and Medical Science.

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.
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http://dx.doi.org/10.1093/intimm/dxaa073DOI Listing
March 2021

Case of cryopyrin-associated periodic syndrome who recovered from growth delay by treatment with canakinumab.

J Dermatol 2021 Feb 8;48(2):e98-e99. Epub 2020 Nov 8.

Department of Dermatology, University of Tsukuba, Tsukuba, Japan.

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http://dx.doi.org/10.1111/1346-8138.15681DOI Listing
February 2021

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

Ann Rheum Dis 2020 11 9;79(11):1492-1499. Epub 2020 Jul 9.

Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.

Objectives: To collect clinical information and mutation data on patients with Blau syndrome and to evaluate their prognosis.

Methods: Fifty patients with mutations were analysed. The activity of each 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 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.
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http://dx.doi.org/10.1136/annrheumdis-2020-217320DOI Listing
November 2020

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

Mod Rheumatol 2021 May 24;31(3):704-709. Epub 2020 Jul 24.

Division of Respirology, Neurology, and Rheumatology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan.

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 gene. Among them, the most frequently-identified genes were , , , , and , 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 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.
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http://dx.doi.org/10.1080/14397595.2020.1784542DOI Listing
May 2021

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

J Proteome Res 2020 07 4;19(7):2821-2827. Epub 2020 Jun 4.

Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.

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.
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http://dx.doi.org/10.1021/acs.jproteome.0c00271DOI Listing
July 2020

Immunophenotyping of A20 haploinsufficiency by multicolor flow cytometry.

Clin Immunol 2020 07 23;216:108441. Epub 2020 Apr 23.

Department of Pediatrics, Gifu University Graduate School of Medicine, Gifu, Japan.

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.
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http://dx.doi.org/10.1016/j.clim.2020.108441DOI Listing
July 2020

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

Clin Chem 2020 04;66(4):525-536

Department of Medical Genetics, Rare Diseases and Personalized Medicine, Reference Center CEREMAIA, CHU Montpellier, University of Montpellier, Montpellier, France.

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.
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http://dx.doi.org/10.1093/clinchem/hvaa024DOI Listing
April 2020

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

Mod Rheumatol 2021 Mar 3;31(2):493-497. Epub 2020 Feb 3.

Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan.

Objectives: Haploinsufficiency of A20 (HA20) due to loss-of-function mutations of 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 . MLPA analysis is a useful initial screening method for HA20 patients.
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http://dx.doi.org/10.1080/14397595.2020.1719595DOI Listing
March 2021

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

Int Immunol 2019 09;31(10):649-655

Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

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-1β. 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.
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http://dx.doi.org/10.1093/intimm/dxz047DOI Listing
September 2019

Plasma infliximab monitoring contributes to optimize Takayasu arteritis treatment: a case report.

J Pharm Health Care Sci 2019 2;5. Epub 2019 May 2.

1Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan.

Background: Infliximab (IFX), a mouse-human chimeric monoclonal antibody against human tumor necrosis factor alpha, is used in refractory cases of Takayasu arteritis. Several factors influence the pharmacokinetics of therapeutic antibodies including IFX. Monitoring plasma levels of IFX could be a useful approach in optimizing treatment via individual dose adjustment.

Case Presentation: Here, we report the case of a 4-year-old Takayasu arteritis girl who was resistant to standard therapy. IFX was started at 5 mg/kg (day 0). C-reactive protein (CRP) levels decreased from 8.7 (day 0) to 1.6 mg/dL (day 10). CRP levels were thereafter elevated again on day 23 (9.0 mg/dL), and body fluid leakage at the inflammation site in the legs was observed. Trough IFX levels decreased from 23.6 (day 10) to 2.5 μg/mL (day 23). Based on the trough levels, IFX was given biweekly at 8 mg/kg. Plasma IFX levels gradually increased, and CRP levels decreased to around 2 mg/dL. A similar pattern -initial decreases followed by increases- was observed between clinical course of IFX and IgG levels. It was speculated that IgG and IFX losses were due to fluid leakage from the patient's necrotizing legs.

Conclusions: Monitoring of plasma IFX levels can be a potential tool to optimize the treatment in Takayasu arteritis patients.
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http://dx.doi.org/10.1186/s40780-019-0136-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498684PMC
May 2019

A Rare Case of Cryopyrin-associated Periodic Syndrome in an Elderly Woman with NLRP3 and MEFV Mutations.

Intern Med 2019 Apr 18;58(7):1017-1022. Epub 2018 Dec 18.

Department of General Medicine, Nagasaki University Graduate School of Biomedical Sciences, Japan.

We herein report a case of a 75-year-old woman who presented with a low-grade fever, repeated cold-induced urticaria, and painful leg edemas with neutrocytosis. Because her mother also had cold-induced urticaria and her skin lesions histologically showed neutrophilic dermatitis, we suspected that she had familial cold autoinflammatory syndrome, a subtype of cryopyrin-associated periodic syndromes. Sequencing of the NLRP3 and MEFV genes revealed that she carried both the p.A439V missense mutation and p.E148Q homozygous mutation, which is commonly detected in familial Mediterranean fever patients. The administration of colchicine reduced the frequency and severity of her skin rash and leg edema.
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http://dx.doi.org/10.2169/internalmedicine.1401-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478996PMC
April 2019

Rescue of recurrent deep intronic mutation underlying cell type-dependent quantitative NEMO deficiency.

J Clin Invest 2019 02 18;129(2):583-597. Epub 2018 Dec 18.

Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan.

X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic IKBKG (also known as NEMO) mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose 3 boys with EDA-ID died from ID. We identify the same private variant in an intron of IKBKG, IVS4+866 C>T, which was inherited from and occurred de novo in the European mother and Japanese mother, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudoexon (PE) generating a frameshift. Its leakiness accounts for NF-κB activation being impaired but not abolished in the boys' cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in induced pluripotent stem cell-derived (iPSC-derived) macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the PE, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.
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http://dx.doi.org/10.1172/JCI124011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355244PMC
February 2019

Characterization of a large UNC13D gene duplication in a patient with familial hemophagocytic lymphohistiocytosis type 3.

Clin Immunol 2018 06 26;191:63-66. Epub 2018 Mar 26.

Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Familial hemophagocytic lymphohistiocytosis (FHL) type 3 is a life-threatening immune dysregulation syndrome caused by mutations in the UNC13D gene, encoding the munc13-4 protein, which is important for function of cytotoxic lymphocytes. FHL3 accounts for 30-40% of FHL cases, and more than 100 mutations in the UNC13D gene have been described to date. We describe the first case of FHL3 carrying an intragenic duplication of UNC13D, apparently mediated by recombination of Alu elements. NK cell degranulation and munc13-4 protein expression assays are useful for early identification of such mutations, which may be missed by analysis of genomic DNA alone.
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http://dx.doi.org/10.1016/j.clim.2018.03.012DOI Listing
June 2018

Influence of post-transplant mucosal-associated invariant T cell recovery on the development of acute graft-versus-host disease in allogeneic bone marrow transplantation.

Int J Hematol 2018 Jul 26;108(1):66-75. Epub 2018 Mar 26.

Human Health Sciences, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are T cell subpopulations that possess innate-like properties. We examined the impact of post-hematopoietic stem cell transplantation (HSCT) MAIT and iNKT cell recovery on the clinical outcomes of 69 patients who underwent allogeneic HSCT at Kyoto University Hospital. Multivariate analyses identified the absolute number of MAIT cells (< 0.48/μL on day 60 post-HSCT) as the sole independent risk factor for grade I-IV and grade II-IV acute graft-versus-host disease (aGVHD) among patients who underwent bone marrow transplantation; no correlation was observed between post-HSCT iNKT cell recovery and the development of aGVHD. Six of the 15 patients in the MAIT (≥ 0.48/μL) group developed aGVHD, five within the first 30 days post HSCT. In contrast, 13 of the 15 patients in the MAIT (< 0.48/μL) group developed aGVHD, seven after day 30 post HSCT. The overall survival of the MAIT group was slightly shorter than that of the MAIT group. Thus, the post-HSCT recovery of MAIT cells is closely related to the development of delayed onset aGVHD and the outcome of post-HSCT, suggesting its utility for identifying a subset of patients that requires more prolonged and/or intense GVHD prophylaxis.
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http://dx.doi.org/10.1007/s12185-018-2442-2DOI Listing
July 2018

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

Blood 2018 05 16;131(18):2016-2025. Epub 2018 Mar 16.

Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

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 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 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 , we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by derived from FHL3 patients. We then performed a comprehensive functional analysis of variants. Transient expression of complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported 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.
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http://dx.doi.org/10.1182/blood-2017-10-812503DOI Listing
May 2018

[Pediatric acute lymphoblastic leukemia presenting with bone and joint pain].

Rinsho Ketsueki 2018;59(2):167-173

Human Health Sciences, Graduate School of Medicine, Kyoto University.

We report on three cases of pediatric acute lymphoblastic leukemia presenting with bone pain and arthralgia as initial symptoms. At the first visit, their primary signs were recurrent bone pain and arthralgia, without significant peripheral blood abnormalities. It took 2-4 months to confirm the diagnosis from the onset of arthralgia due to this atypical presentation of the disease. Definitive diagnosis was obtained by bone marrow examination, and in all cases, complete remission was achieved by chemotherapy. As a feature of imaging, MRI exhibited diffuse bone marrow signal changes in T1-weighted images, and FDG-PET showed extensive abnormal bone marrow uptakes. In cases 2 and 3, it was difficult to diagnose by bone marrow aspiration from the iliac bone, but definitive diagnosis was obtained by bone marrow aspiration from the tibia, in which FDG-PET showed increased uptake. FDG-PET was therefore considered useful for the selection of bone marrow aspiration sites. In cases presenting with recurrent migratory bone pain and arthralgia, we need to consider performing bone marrow aspiration and imaging, such as MRI and FDG-PET, for early diagnosis and treatment of leukemia.
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http://dx.doi.org/10.11406/rinketsu.59.167DOI Listing
February 2019
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