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OBJECTIVE Coatomer subunit alpha (COPA) syndrome, also known as autoinflammatory interstitial lung, joint, and kidney disease, is caused by heterozygous mutations in COPA. We identified a novel COPA variant in 4 patients in one family. We undertook this study to elucidate whether and how the variant causes manifestations of COPA syndrome by studying these 4 patients and by analyzing results from a gene-targeted mouse model. METHODS We performed whole-exome sequencing in 7 family members and measured the type I interferon (IFN) signature of the peripheral blood cells. We analyzed the effects of COPA variants in in vitro experiments and in Copa mutant mice that were generated. RESULTS We identified a heterozygous variant of COPA (c.725T>G, p.Val242Gly) in the 4 affected members of the family. The 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 stimulator of interferon genes (STING)-induced type I IFN promoter activities. CopaV242G/+ mice manifested interstitial lung disease and STING-dependent elevation of IFN-stimulated gene expression. In CopaV242G/+ dendritic cells, the STING pathway was not constitutively activated but was hyperactivated upon stimulation, leading to increased type I IFN production. CONCLUSION V242G, a novel COPA variant, was found in 4 patients from one family. In gene-targeted mice with the V242G variant, interstitial lung disease was recapitulated and augmented responses of the STING pathway, leading to an increase in type I IFN production, were demonstrated.

Objective To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) who does not carry any NLRP3 mutation. Methods A Japanese male diagnosed as NOMID was recruited. The patient had no NLRP3 mutation even as low frequency mosaicism. We performed whole exome sequencing (WES) of the patient and his parents. Induced pluripotent stem cells (iPSCs) were established from the fibroblasts of the patient. iPSCs were then differentiated into monocytic lineage to evaluate the cytokine profile. Results We established multiple iPSC clones from an NOMID patient and incidentally found that the phenotype of monocytes from iPSC clones were heterogeneous, and could be grouped into “diseased” and “normal” phenotype. Because each iPSC clone was derived from a single somatic cell, we hypothesized the patient had somatic mosaicism of an IL-1β-related gene. WES of both representative iPSC clones and patient's blood identified a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in “diseased” iPSC clones. Knockout of the NLRC4 gene using CRISPR/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. Conclusion We concluded the patient as having somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing somatic NLRC4 mutation causes autoinflammatory symptoms compatible to NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotypic dissection for individualized diagnoses. This article is protected by copyright. All rights reserved.

Objective Gain-of-function NLRP3 mutations cause cryopyrin-associated periodic syndrome (CAPS), with gene mosaicism playing a relevant role in the pathogenesis. This study was undertaken to characterize the genetic cause underlying late-onset but otherwise typical CAPS. Methods We studied a 64-year-old patient who presented with recurrent episodes of urticaria-like rash, fever, conjunctivitis, and oligoarthritis at age 56 years. DNA was extracted from both unfractionated blood and isolated leukocyte and CD34+ subpopulations. Genetic studies were performed using both the Sanger method of DNA sequencing and next-generation sequencing (NGS) methods. In vitro and ex vivo analyses were performed to determine the consequences that the presence of the variant have in the normal structure or function of the protein of the detected variant. Results NGS analyses revealed the novel p.Gln636Glu NLRP3 variant in unfractionated blood, with an allele frequency (18.4%) compatible with gene mosaicism. Sanger sequence chromatograms revealed a small peak corresponding to the variant allele. Amplicon-based deep sequencing revealed somatic NLRP3 mosaicism restricted to myeloid cells (31.8% in monocytes, 24.6% in neutrophils, and 11.2% in circulating CD34+ common myeloid progenitor cells) and its complete absence in lymphoid cells. Functional analyses confirmed the gain-of-function behavior of the gene variant and hyperactivity of the NLRP3 inflammasome in the patient. Treatment with anakinra resulted in good control of the disease. Conclusion We identified the novel gain-of-function p.Gln636Glu NLRP3 mutation, which was detected as a somatic mutation restricted to myeloid cells, as the cause of late-onset but otherwise typical CAPS. Our results expand the diversity of CAPS toward milder phenotypes than previously reported, including those starting during adulthood.

Objective To elucidate the clinical and genetic features of patients with TNFRSF1A variants in Japan using data obtained from a nationwide survey conducted by the Ministry of Health, Labor, and Welfare of Japan study group for tumor necrosis factor receptor–associated periodic syndrome (TRAPS). Methods Inquiries were sent to 2,900 departments of internal medicine and pediatrics in all hospitals with more than 200 beds in Japan, asking whether they had patients in whom TRAPS was suspected. Genetic tests for TNFRSF1A, MEFV, and MVK were performed on 169 patients. Cell surface expression of TNFRSF1A variants was assessed using 293T cells. Results Ten patients from 10 independent families were found to have TNFRSF1A variants. We collected clinical and genetic information on 41 additional patients with TNFRSF1A variants and symptoms of inflammation from 23 independent families; 17 of these patients had not been described in the literature. The common clinical features of Japanese patients were fever of >38°C (100% of patients), arthralgia (59%), and rash (55%). The prevalence of abdominal pain (36%), myalgia (43%), and amyloidosis (0%) was significantly lower in Japanese patients than in Caucasian patients. The most common variant was T61I (appearing in 49% of patients), and it was identified in 7 of 363 healthy controls. Defects in cysteine residues and the T50M variant were associated with decreased cell surface expression, while other variants, including T61I, were not. Conclusion Patients with TNFRSF1A variants are very rare in Japan, as in other countries, but there are a number of clinical and genetic differences between Japanese and Caucasian patients. The pathogenic significance of the T61I variant remains unclear.

BACKGROUND AND AIM Acute gastrointestinal graft-versus-host disease (GI-GVHD) is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). There are very few studies on specific endoscopic findings in pediatric acute GI-GVHD. The aim of this retrospective case-control study was to elucidate the characteristic endoscopic findings in pediatric acute GI-GVHD that improve the diagnostic accuracy of endoscopy. METHODS All consecutive patients under 18 years of age who underwent allogeneic HSCT in Kyoto University Hospital from May 2003 to October 2014 were identified retrospectively. Patients who underwent GI endoscopy as a result of sustained GI symptoms were identified. Intestinal villous patterns were evaluated by magnification endoscopy with the water-immersion technique. The patients were diagnosed with acute GI-GVHD and non-GVHD on the basis of biopsy histology. Endoscopic findings of the two groups were compared. RESULTS Of the 171 patients who underwent HSCT, 30 underwent GI endoscopy. Of these, 17 and nine were diagnosed with acute GI-GVHD and non-GVHD, respectively. Compared with non-GVHD, acute GI-GVHD was associated significantly more often with short blunt villi in the duodenum (P = 0.013), variable defect villi and short blunt villi in the ileum (P = 0.009 and 0.035, respectively), and edema, erosion, and tortoiseshell-like mucosae in the colon (P = 0.017, 0.023, and 0.017, respectively). CONCLUSION Pediatric acute GI-GVHD was associated with several characteristic features on magnifying endoscopy with the water-immersion technique. These features will be useful for endoscopic diagnosis of pediatric acute GI-GVHD.

Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome of immune dysregulation and is classified as primary or secondary according to the underlying aetiology. The treatment strategies recommended for these two groups differ substantially; however, it is thought to be impossible to predict the underlying causes of HLH using conventional laboratory tests. Recent studies show that serum levels of soluble interleukin-2 receptor (sIL2R) and ferritin are useful for differentiating some forms of HLH. The present study reports that combinations of common laboratory parameters, such as the percentage of total lymphocytes within the peripheral blood leucocyte population, serum levels of lactate dehydrogenase and the sIL2R/ferritin ratio, are useful for identifying patients with familial haemophagocytic lymphohistiocytosis and for differentiating the underlying aetiology of paediatric HLH during the early course of the disease. These findings suggest that the pathogenesis of HLH differs greatly in terms of innate and adaptive immunity depending on the aetiology and may provide a new approach to unravelling the complex pathophysiology underlying this syndrome.

Objective Neonatal-onset multisystem inflammatory disease (NOMID) is a dominantly inherited autoinflammatory disease caused by NLRP3 mutations. NOMID pathophysiology is explained by the NLRP3 inflammasome, which produces interleukin-1β (IL-1β). However, epiphyseal overgrowth in NOMID is resistant to anti–IL-1 therapy and may therefore occur independently of the NLRP3 inflammasome. This study was undertaken to investigate the effect of mutated NLRP3 on chondrocytes using induced pluripotent stem cells (iPSCs) from patients with NOMID. Methods We established isogenic iPSCs with wild-type or mutant NLRP3 from 2 NOMID patients with NLRP3 somatic mosaicism. The iPSCs were differentiated into chondrocytes in vitro and in vivo. The phenotypes of chondrocytes with wild-type and mutant NLRP3 were compared, particularly the size of the chondrocyte tissue produced. Results Mutant iPSCs produced larger chondrocyte masses than wild-type iPSCs owing to glycosaminoglycan overproduction, which correlated with increased expression of the chondrocyte master regulator SOX9. In addition, in vivo transplantation of mutant cartilaginous pellets into immunodeficient mice caused disorganized endochondral ossification. Enhanced chondrogenesis was independent of caspase 1 and IL-1, and thus the NLRP3 inflammasome. Investigation of the human SOX9 promoter in chondroprogenitor cells revealed that the CREB/ATF-binding site was critical for SOX9 overexpression caused by mutated NLRP3. This was supported by increased levels of cAMP and phosphorylated CREB in mutant chondroprogenitor cells. Conclusion Our findings indicate that the intrinsic hyperplastic capacity of NOMID chondrocytes is dependent on the cAMP/PKA/CREB pathway, independent of the NLRP3 inflammasome.

Background Food allergy (FA) in childhood has been shown to be more prevalent in those born in autumn and winter. The mechanisms of this season-of-birth effect remain unclear, although shortage of vitamin D during infancy has been considered one possible mechanism. The purpose of this study was to investigate the effect of eczema on the season-of-birth effect on FA in infancy. Methods A questionnaire survey on the prevalence of allergic diseases was completed by the parents of 14 669 Japanese schoolchildren, aged 7–15 years, in Kyoto City, Japan. Logistic regression models were constructed to compare the prevalence of FA in infancy according to season of birth. Results Those born in autumn and winter had a significantly higher prevalence of FA in infancy compared to those born in spring and summer in a multivariate model (4.8% vs 3.6%, P = 0.001). The difference, however, was no longer significant when eczema before 6 months was included as either an additional or only confounding factor. The difference among those with and without eczema before 6 months was further analyzed, and it was found that, in both groups, there was no difference between those born in spring and summer and those born in autumn and winter. Conclusions The season-of-birth effect on FA in infancy was significantly affected by the existence of eczema before 6 months in Japanese children. Eczema before 6 months may be the factor directly related to the season-of-birth effect on FA in infancy.

To cite this article: Kusunoki T, Mukaida K, Morimoto T, Sakuma M, Yasumi T, Nishikomori R, Heike T. Birth order effect on childhood food allergy. Pediatric Allergy Immunology 2012: 23: 250–254. Abstract Higher birth order is associated with a smaller risk of allergy (birth order effect). The purpose of this study was to compare the significance of the birth order effect on the prevalence of specific allergic diseases [bronchial asthma (BA), atopic dermatitis (AD), allergic rhinitis (AR), allergic conjunctivitis (AC), and food allergy (FA)] among schoolchildren. A questionnaire survey dealing with the prevalence of allergic diseases was administered to the parents of 14,669 schoolchildren aged 7–15 yr. Based on the data, the prevalence of each allergic disease was compared according to birth order (1st, 2nd, and 3rd or later). Multiple regression analysis was performed to test the significance of the differences. There was no significant difference in the prevalence of BA or AD according to birth order. The prevalence of AR, AC, and FA decreased significantly as birth order increased. The prevalence of FA among those with 1st, 2nd, and 3rd or later birth order was 4.0%, 3.4%, and 2.6%, respectively (p = 0.01). With respect to symptoms in infancy, the prevalence of wheeze increased significantly and that of FA and eczema in infancy decreased significantly as birth order increased. The present data show a significant birth order effect on FA. The effect was also observed for the prevalence of FA and eczema in infancy. These data support the concept of early, non-allergen-specific programming of IgE-mediated immunity.

Acute liver failure (ALF) with macrophage activation syndrome (MAS) is well known as a complication of systemic-onset juvenile idiopathic arthritis (S-JIA). However, liver failure without overt MAS is rare in S-JIA. We encountered two Japanese children with S-JIA in whom ALF developed during the remission of clinical manifestations. ALF without MAS was improved with plasma exchange and cyclosporine A combined with pulse methylprednisolone.

Although children allergic to eggs, milk or wheat in infancy tend to become tolerant by school age, the allergic status of these children at school age has not been well evaluated. To investigate the allergic status of schoolchildren who avoided eggs, milk or wheat because of an immediate-type allergic reaction at

Objective Blau syndrome and its sporadic counterpart, early-onset sarcoidosis (EOS), share a phenotype featuring the symptom triad of skin rash, arthritis, and uveitis. This systemic inflammatory granulomatosis is associated with mutations in the NOD2 gene. The aim of this study was to describe the clinical manifestations of Blau syndrome/EOS in Japanese patients and to determine whether the NOD2 genotype and its associated basal NF-κB activity predict the Blau syndrome/EOS clinical phenotype. Methods Twenty Japanese patients with Blau syndrome/EOS and NOD2 mutations were recruited. Mutated NOD2 was categorized based on its basal NF-κB activity, which was defined as the ratio of NF-κB activity without a NOD2 ligand, muramyldipeptide, to NF-κB activity with muramyldipeptide. Results All 9 mutations, including E383G, a novel mutation that was identified in 20 patients with Blau syndrome/EOS, were detected in the centrally located NOD region and were associated with ligand-independent NF-κB activation. The median age of the patients at disease onset was 14 months, although in 2 patients in Blau syndrome families (with mutations R334W and E383G, respectively) the age at onset was 5 years or older. Most patients with Blau syndrome/EOS had the triad of skin, joint, and ocular symptoms, the onset of which was in this order. Clinical manifestations varied even among familial cases and patients with the same mutations. There was no clear relationship between the clinical phenotype and basal NF-κB activity due to mutated NOD2. However, when attention was focused on the 2 most frequent mutations, R334W and R334Q, R334W tended to cause more obvious visual impairment. Conclusion NOD2 genotyping may help predict disease progression in patients with Blau syndrome/EOS.

A 10-year-old female was diagnosed as having early-onset sarcoidosis (EOS) after a prolonged skin disease. A granuloma emerged on the face at age 2 and massive lesions extended to the rest of the body. Repeated biopsies indicated histiocytic proliferation. At age 7, fever, disseminated macular eruptions, and multinucleated giant cells in the bone marrow prompted vinblastine and prednisolone therapy. Five months after stopping therapy, hypercalcemic crisis occurred along with fever, cytopenias, and interferon-γ-nemia indicating a macrophage activation syndrome. A biopsy of nodules confirmed the diagnosis of sarcoidosis. The atypical EOS should be differentiated from histiocytosis. Pediatr Blood Cancer 2008;50:723–726. © 2007 Wiley-Liss, Inc.

Chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome) is a severe inflammatory disease that was recently found to be associated with mutations in CIAS1. However, CIAS1 mutations have been detected in only half of CINCA syndrome patients, and it remains unclear which genes are responsible for the syndrome in the remaining patients. We describe here a patient with CINCA syndrome who exhibited CIAS1 somatic mosaicism. We genetically analyzed the CIAS1 gene in various blood cells and the buccal mucosa of the patient. The production of interleukin-1β (IL-1β) by peripheral blood mononuclear cells (PBMCs) was measured by enzyme-linked immunosorbent assay, and the ability of the mutant CIAS1 gene to enhance ASC-dependent NF-κB activation was assessed to confirm that the mutations of CIAS1 found were responsible for the patient's clinical manifestations of the CINCA syndrome. The patient had 1 heterologous single-nucleotide polymorphism, 587G>A (S196N), and 1 heterologous mutation, 1709A>G (Y570C), in exon 3 of CIAS1. The latter mutation was found to occur as somatic mosaicism. The patient's PBMCs produced a large amount of IL-1β in the absence of stimulation, unlike those from controls or from his mother, who also bore the S196N polymorphism. In addition, the Y570C mutation (with or without the S196N polymorphism) increased the ability of CIAS1 to induce ASC-dependent NF-κB activation, unlike the wild-type gene or the gene bearing the S196N polymorphism alone. The findings in this patient indicate that somatic mosaicism is one reason CIAS1 mutations have not been detected in some patients with CINCA syndrome.

Systemic and inhalation therapy of granulocyte-macrophage colony-stimulating factor (GM-CSF) is usually effective in controlling autoimmune pulmonary alveolar proteinosis (PAP), but some cases are refractory to GM-CSF therapy and subjected to whole lung lavage (WLL). A 9-year-old girl developed severe respiratory failure due to autoimmune PAP was treated with inhalational 250 microg of GM-CSF daily, however, it was ineffective. Unilateral WLL was performed three times and subsequent GM-CSF inhalation therapy yielded marked physiological and radiological improvement and was continued for 1 year.