Gut microbiota of one-and-a-half-year-old food-allergic and healthy children.

BACKGROUND: Intestinal bacteria may play a role in the development of food allergies. This study aimed to analyze and compare the gut microbiota of food-allergic children with that of healthy children of the same age. METHODS: Stool samples were collected from one-and-a-half-year-old food-allergic (FA group, n = 29) and healthy controls (HC group, n = 19). A questionnaire was provided to examine the children's birth, dietary, medical, and social histories. The gut microbiota was profiled by 16S rRNA sequencing. Differences in taxonomic composition were assessed using linear discriminant analysis effect size (LEfSe), and microbial functional profiles were predicted with Tax4Fun2. RESULTS: No significant difference in the alpha diversity index between the two groups; however, a negative correlation was observed between the Shannon diversity index and the relative abundance of Bacteroides. A significant difference was observed in beta diversity (permutational multivariate analysis of variance) in the bacterial composition between the FA and HC groups (P < 0.05). The FA group had a higher abundance of Escherichia and Anaeromassilibacillus and a lower abundance of Bacteroides, Oscillibacter, Ruminococcus, Hungateiclostridium and Anaerotaenia than the HC group (LEfSe: linear discriminant analysis score >2). The FA group showed a predicted increase in the expression levels of genes associated with intestinal pathogenicity compared with that in the HC group. CONCLUSIONS: The gut microbiota of food-allergic children has a higher abundance of bacteria involved in intestinal inflammation and a lower abundance of bacteria involved in immune tolerance than that of healthy children. This dysbiosis may also be associated with food allergies.

Differences in Characteristics Between Patients Who Met or Partly Met the Diagnostic Criteria for Food Protein-Induced Enterocolitis Syndrome (FPIES).

BACKGROUND: Some patients with food protein-induced enterocolitis (FPIES)-like allergy do not completely fulfill the diagnostic criteria of the international consensus guideline for FPIES. However, it is unclear whether such FPIES-like patients represent a completely different population from FPIES. OBJECTIVE: This study aimed to clarify differences in characteristics between patients with FPIES who fully met diagnostic criteria and those who partly met them. METHODS: This was a cross-sectional study using data at the time of registration in multicenter, prospective studies of patients with FPIES in Japan. Children who had delayed emesis within 1 to 4 hours and/or diarrhea within 5 to 10 hours after ingestion of food were recruited between March 2020 and February 2022. We examined their compatibility with the diagnostic criteria of the international consensus guideline and their detailed clinical characteristics, including trigger foods, the serving size that elicited symptoms, and antigen-specific IgE antibody titers. RESULTS: Of the 225 patients with FPIES, 140 fully met the diagnostic criteria whereas 79 patients did not fully meet them but demonstrated reproducible symptoms. The frequencies of pallor, lethargy, and diarrhea were significantly higher in those who met the criteria fully, whereas the age at onset, trigger foods, comorbidity, and perinatal information were comparable. Analysis of patients with FPIES to hen's egg revealed significantly higher levels of egg white- and egg yolk-specific IgE in patients who partly met criteria, whereas the serving size eliciting symptoms was comparable. CONCLUSIONS: Patients who partly met the diagnostic criteria may have a milder phenotype of FPIES, but this needs to be validated in further studies using biomarkers reflecting the pathophysiology.

Influence of household pet ownership and filaggrin loss-of-function mutations on eczema prevalence in children: A birth cohort study.

BACKGROUND: The association between pet exposure in infancy, early childhood eczema, and FLG mutations remains unclear. METHODS: This was a birth cohort study performed in Tokyo, Japan. The primary outcome was current eczema based on questionnaire responses collected repeatedly from birth to 5 years of age. Generalized estimating equations and generalized linear modeling were used to evaluate the association. RESULTS: Data from 1448 participants were used for analyses. Household dog ownership during gestation, early infancy, and 18 months of age significantly reduced the risk of current eczema. Household cat ownership also reduced the risk of current eczema, albeit without statistical significance. The combined evaluation of children from households with pets, be it cats, dogs or both, the risk of current eczema at 1-5 years of age was lower in those with household pet exposure ownership during gestation (RR = 0.59, 95 % CI 0.45-0.77) and at 6 months (RR = 0.49, 95 % CI 0.36-0.68). , Reduced risks of eczema were also observed at 2-5 (RR = 0.52, 95 % CI 0.37-0.73) and 3-5 years of age (RR = 0.50 95 % CI 0.35-0.74) when the respective household pet ownership were evaluated at 18 months and 3 years of age. These protective associations of reduced risk of eczema were only observed in children without FLG mutations. CONCLUSIONS: Household dog and pet (dog, cat, or both) ownership was protective against early childhood eczema in a birth cohort dataset. This protective association was observed only in children without FLG mutations, which should be confirmed in studies with larger cohorts.

A genome-wide association study for allergen component sensitizations identifies allergen component-specific and allergen protein group-specific associations.

Background: Allergic diseases are some of the most common diseases worldwide. Genome-wide association studies (GWASs) have been conducted to elucidate the genetic factors of allergic diseases. However, no GWASs for allergen component sensitization have been performed. Objective: We sought to detect genetic variants associated with differences in immune responsiveness against allergen components. Methods: The participants of the present study were recruited from the Tokyo Children's Health, Illness, and Development study, and allergen component-specific IgE level at age 9 years was measured by means of allergen microarray immunoassays. We performed GWASs for allergen component sensitization against each allergen (single allergen component sensitization, number of allergen components analyzed, n = 31), as well as against allergen protein families (allergen protein group sensitization, number of protein groups analyzed, n = 16). Results: We performed GWAS on 564 participants of the Tokyo Children's Health, Illness, and Development study and found associations between Amb a 1 sensitization and the immunoglobulin heavy-chain variable gene on chromosome 14 and between Phl p 1 sensitization and the HLA class II region on chromosome 6 (P < 5.0 × 10-8). A GWAS-significant association was also observed between the HLA class II region and profilin sensitization (P < 5.0 × 10-8). Conclusions: Our data provide the first demonstration of genetic risk for allergen component sensitization and show that this genetic risk is related to immune response genes including immunoglobulin heavy-chain variable gene and HLA.

Middle meatus microbiome in patients with eosinophilic chronic rhinosinusitis in a Japanese population.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease and is subdivided into eosinophilic and noneosinophilic forms. There are few reports investigating the nasal microbiome and its pathological functions in patients with CRS. OBJECTIVE: We sought to analyze factors contributing to variations of the nasal microbiome in CRS, and on the basis of these factors, to elucidate whether the bacterial metabolites were related to the pathogenesis. METHODS: Nasal swabs were collected, and the V3 to V4 variable region of the 16S ribosomal RNA gene was amplified and sequenced. Factors contributing to variations of the nasal microbiome in patients with CRS were compared. The most influential factor was whether CRS was eosinophilic, and we compared α- and ß-diversity, bacterial species, and predictive bacterial functions between the 2 patient groups. In addition, the metabolites of the key bacteria were extracted, and we evaluated the predicted bacterial functions in airway epithelial cells. RESULTS: In total, 110 patients with CRS and 33 control subjects were enrolled. On the basis of the factors of variation, it was found that patients with eosinophilic CRS (n = 65) had different microbiomes with weighted UniFrac ß-diversity and lower α-diversity compared with those with noneosinophilic CRS (n = 45). A higher abundance of Fusobacterium nucleatum and an increased LPS pathway were observed in patients with noneosinophilic CRS compared with those with eosinophilic CRS. In airway epithelial cells, LPS derived from F nucleatum suppressed the expression levels of ALOX15 induced by TH2 cytokines. CONCLUSIONS: The differences in the nasal microbiome may play a key role in the pathophysiology of CRS.

A novel de-novo RB1 mutation identified in a patient with bilateral retinoblastoma.

Retinoblastoma manifests as ocular malignancy due to mutations in the RB1 gene. A 17-month-old girl with bilateral retinoblastoma having no family history was admitted to our hospital. The right eye was enucleated but the other was preserved with systemic chemotherapy and topical treatment. The patient has been tumor-free for over 7 years since diagnosis. All exons of RB1 were sequenced and a novel 1-base pair deletion (NM_000321.2:c.2409del, p.Asn803Lysfs*7) was detected.

Maternal BMI and allergy in children until 3 years of age (JECS).

Background: Maternal prepregnancy body mass index (BMI) may influence allergic diseases in the children who are the product of those pregnancies. Objective: The purpose of our study was to investigate the association between mothers' prepregnancy BMI and the risk of physician-diagnosed asthma, food allergy (FA), and atopic dermatitis (AD) in their children during the first 3 years of life. Methods: Data on mothers' prepregnancy BMI and physician-diagnosed asthma, FA, and AD in their children until the age of 3 years were obtained from the Japan Environment and Children's Study, a nationwide birth cohort study that has recruited 103,099 pregnant women between 2011 and 2014. Logistic regression analysis was used to analyze the results. Results: We analyzed 67,204 mother-child pairs with available information on physician-diagnosed allergic diseases. The risk of asthma was significantly higher in children born to overweight mothers (adjusted OR [aOR] =1.17 [95% CI = 1.07-1.28]) and obese mothers (aOR = 1.28 [95% CI = 1.08-1.50]), whereas the risk of FA, cow's milk allergy, and egg allergy decreased significantly in children born to overweight mothers (aOR = 0.84 [95% CI = 0.76-0.92]; aOR = 0.78 [95% CI = 0.64-0.93]; and aOR = 0.83 [95% CI = 0.74-0.94]) and obese mothers (aOR = 0.81 [95% CI = 0.67-0.97]; aOR = 0.58 [95% CI = 0.36-0.87]; and aOR = 0.73 [95% CI = 0.56-0.93]) compared with in children born to normal weight mothers, respectively. Associations between AD and maternal BMI were not detected. Conclusion: Our study showed that an increase in mothers' prepregnancy BMI was associated with an increase in asthma prevalence and a decrease in FA prevalence in their children. Further studies are needed to reveal the mechanisms associated with maternal BMI and pediatric allergic diseases.

Cancer predisposition genes in Japanese children with rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children. Germline mutations in cancer-predisposition genes have been detected in approximately 10% of pediatric cancers. However, the genetic background of RMS is still unclear, especially in Asian children. DNA was extracted from the peripheral blood of children with RMS and cancer-associated genes analyzed using targeted re-sequencing. Twenty patients participated in this study. There were three deaths due to RMS. One patient developed a second neoplasm. Nine patients had long-term co-morbidities. Six pathogenic variants were found in five patients: one nonsense variant of DICER1, one exon deletion of TP53, and three missense variants of BUB1B, LIG4, and MEN1. Two of the five patients had a family history of cancer. Two patients with missense variants of LIG4 had long-term co-morbidities of drug-induced cardiomyopathy. The missense variants of LIG4, essential for DNA double-strand break repair, were detected in two unrelated patients. While this is the first report of the germline genetic analysis of Japanese children with RMS with detailed clinical information, the frequency of the variant was almost equivalent to that of previous reports from western countries. Unbiased exon sequencing may be useful to clarify the pathogenesis of RMS in children and in predicting the clinical course of these patients.

Cancer-Predisposition Genetic Analysis in Children with Brain Tumors Treated at a Single Institution in Japan.

Brain tumors affect one-third of all children with cancer. Approximately 10% of children with cancer carry variants in cancer-predisposition genes. However, germline analyses in large cohorts of Asian children have not been reported. Thirty-eight Japanese patients with pediatric brain tumors were included in this study (19 boys, 19 girls). DNA was extracted from the patients' peripheral blood, and cancer-associated genes were analyzed using targeted resequencing. Rare variants with allele frequencies <0.1% in the general population and variants suspected to be pathogenic were extracted and analyzed. Pathogenic variants were found in 7 patients (18%): 2 nonsense variants of CHEK2 and FANCI; 2 frameshift deletions in SMARCB1 and PTCH1; and 3 missense variants of TSC1, WRN, and MLH1. The median age at diagnosis was 9.1 years, and three of the 7 patients had a family history of cancer. One patient diagnosed with basal cell nevus syndrome, also called Gorlin syndrome, developed a second neoplasm, and another with an SMARCB1 variant and an atypical teratoid/rhabdoid tumor developed a thyroid adenomatous nodule. This is the first cancer-related germline analysis with detailed clinical information reported in Japanese children with brain tumors. The prevalence was almost equivalent to that in white children.

Genome-wide association study reveals an association between the HLA-DPB1∗02:01:02 allele and wheat-dependent exercise-induced anaphylaxis.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a life-threatening food allergy triggered by wheat in combination with the second factor such as exercise. The identification of potential genetic risk factors for this allergy might help high-risk individuals before consuming wheat-containing food. We aimed to identify genetic variants associated with WDEIA. A genome-wide association study was conducted in a discovery set of 77 individuals with WDEIA and 924 control subjects via three genetic models. The associations were confirmed in a replication set of 91 affected individuals and 435 control individuals. Summary statistics from the combined set were analyzed by meta-analysis with a random-effect model. In the discovery set, a locus on chromosome 6, rs9277630, was associated with WDEIA in the dominant model (OR = 3.95 [95% CI, 2.31-6.73], p = 7.87 × 10-8). The HLA-DPB1∗02:01:02 allele displayed the most significant association with WDEIA (OR = 4.51 [95% CI, 2.66-7.63], p = 2.28 × 10-9), as determined via HLA imputation following targeted sequencing. The association of the allele with WDEIA was confirmed in replication samples (OR = 3.82 [95% CI, 2.33-6.26], p = 3.03 × 10-8). A meta-analysis performed in the combined set revealed that the HLA-DPB1∗02:01:02 allele was significantly associated with an increased risk of WDEIA (OR = 4.13 [95% CI, 2.89-5.93], p = 1.06 × 10-14). Individuals carrying the HLA-DPB1∗02:01:02 allele have a significantly increased risk of WDEIA. Further validation of these findings in independent multiethnic cohorts is needed.

Involvement of Activation of Mast Cells via IgE Signaling and Epithelial Cell-Derived Cytokines in the Pathogenesis of Pollen Food Allergy Syndrome in a Murine Model.

Murine models to elucidate the pathogenesis of pollen food allergy syndrome (PFAS), characterized by oral hypersensitivity symptoms induced by specific foods in patients previously sensitized with a pollen, are lacking. The study aimed to examine PFAS pathogenesis in a novel murine model. Birch pollen-immunized mice were orally administered apple extract, and oral symptoms were evaluated based on oral rubbing frequency following the challenge. The birch pollen-immunized mice orally challenged with apple extract exhibited PFAS-like symptoms, including oral rubbing and positive reaction of swelling by the prick test. The apple extract administered with a protease inhibitor reduced the oral rubbing frequency, which was also significantly reduced in the immunized Fcer1a -/- and mast cell-deficient mice compared with the immunized control mice. The oral rubbing frequency, serum IgE levels, and Th2-cytokine production by the cervical lymph node cells were significantly reduced in the immunized Il-33 -/- and thymic stromal lymphopoietin receptor-deficient (Crlf2 -/-) mice as compared with the immunized wild-type mice. IL-33 and thymic stromal lymphopoietin involve the pathogenesis of PFAS. The apple-extract stimulation did not lead to increased Th2-cytokine production in the oral mucosa or number of group 2 innate lymphoid cells or eosinophils. PFAS involves an early-phase response by mast cell degranulation via IgE signaling after the cross-reactivity of Bet v 1-specific IgE and the food allergen, and exacerbation of allergic symptom via proteases in food; PFAS does not involve a late phase with local Th2/eosinophilic inflammation in the oral mucosa. This novel murine model might be used for elucidating the pathogenesis and assessing new therapeutic strategies for PFAS.

Analysis of patient factors associated with hospital visits for allergic rhinitis in Japanese adult patients: A cross-sectional study.

OBJECTIVE: Allergic rhinitis (AR) is one of the most common diseases in Japan. However, several AR patients do not seek optimal treatments at clinics/hospitals. This may affect the patient's quality of life and labor productivity. In this study, we assessed the characteristics of the outpatients' AR and factors associated with their hospital visit, using the dataset obtained from a nation-wide survey in Japan. METHODS: In this cross-sectional study, we used data from the nation-wide 2013 and 2016 Comprehensive Survey of Living Conditions (CSLC) in Japan. We analyzed the data of AR outpatients through logistic regression, using the outcome as the dependent variable, and age groups, sex, household size, educational status, smoking history, alcohol use, household expenditure, psychological distress, quality of sleep, asthma and atopic dermatitis outpatients as explanatory variables. RESULTS: Among the data of 317,984 outpatients aged between 20 and 79 years in 2016 CSLC survey, the proportion of AR outpatients was significantly less among current smokers (odds ratio (OR); 0.47, 95% confidence interval (CI); 0.43-0.51, P < 0.001) and those with large household sizes (OR; 0.80, 95% CI; 0.72-0.89, P < 0.001). Conversely, the proportion of AR outpatients was significantly more among subjects with a past smoking habit (OR; 1.19, 95% CI; 1.08-1.31, P < 0.001), insufficient sleep (OR; 2.93, 95% CI; 2.52-3.42, P < 0.001), psychological distress (OR; 1.71, 95% CI; 1.62-1.80, P < 0.001), high household expenditures (OR; 1.68, 95% CI; 1.56-1.80, P < 0.001), and asthma and atopic dermatitis outpatients (OR; 8.97, 95% CI; 8.13-9.89 P < 0.001 for asthma and OR; 7.61, 95% CI; 6.76-8.58 P < 0.001 for atopic dermatitis). We observed the same trend using the dataset of 2013 CLSC survey. CONCLUSION: This study revealed that smoking habit, psychological distress, insufficient sleep, high household expenditures and outpatients with other allergic diseases are the factors associated with AR outpatient visit.

ORMDL3/GSDMB genotype as a risk factor for early-onset adult asthma is linked to total serum IgE levels but not to allergic sensitization.

BACKGROUND: An orosomucoid-like 3 (ORMDL3)/gasdermin B (GSDMB) gene locus on chromosome 17q is consistently associated with childhood-onset asthma, which is highly atopic. As some evidence suggests the relationship between asthma and allergic sensitization reflects asthma patient susceptibility to augmented IgE responses driven by common environmental allergens rather than an increased asthma risk after allergen exposure, we aimed to determine any relationships between this locus region and childhood-onset adult asthma with regard to serum total IgE levels or allergic sensitization. METHODS: We conducted a case-control association study using three independent Japanese populations (3869 total adults) and analyzed the ORs for association of rs7216389, an expression quantitative trait locus for ORMDL3/GSDMB, with adult asthma according to onset age. Additionally, associations between the rs7216389 genotype and total serum IgE levels or allergic sensitization was examined. RESULTS: Rs7216389 was associated with both childhood-onset adult asthma (OR for asthmatic patients afflicted at the age of 10 years or younger = 1.61, p = 0.00021) and asthmatic patients with higher levels of total serum IgE (OR for asthmatic patients with IgE ≥1000IU/mL = 1.55, p = 0.0033). In both healthy controls and in the combined healthy and asthmatic individuals, rs7216389 was correlated with increased total serum IgE levels (p < 0.0005), but not allergic sensitization (p > 0.1). CONCLUSIONS: ORMDL3/GSDMB is an important susceptibility gene for childhood-onset adult asthma in Japanese populations and this association is linked to elevated total serum IgE levels but not to allergic sensitization.

Neoantigen prediction in human breast cancer using RNA sequencing data.

Neoantigens have attracted attention as biomarkers or therapeutic targets. However, accurate prediction of neoantigens is still challenging, especially in terms of its accuracy and cost. Variant detection using RNA sequencing (RNA-seq) data has been reported to be a low-accuracy but cost-effective tool, but the feasibility of RNA-seq data for neoantigen prediction has not been fully examined. In the present study, we used whole-exome sequencing (WES) and RNA-seq data of tumor and matched normal samples from six breast cancer patients to evaluate the utility of RNA-seq data instead of WES data in variant calling to detect neoantigen candidates. Somatic variants were called in three protocols using: (i) tumor and normal WES data (DNA method, Dm); (ii) tumor and normal RNA-seq data (RNA method, Rm); and (iii) combination of tumor RNA-seq and normal WES data (Combination method, Cm). We found that the Rm had both high false-positive and high false-negative rates because this method depended greatly on the expression status of normal transcripts. When we compared the results of Dm with those of Cm, only 14% of the neoantigen candidates detected in Dm were identified in Cm, but the majority of the missed candidates lacked coverage or variant allele reads in the tumor RNA. In contrast, about 70% of the neoepitope candidates with higher expression and rich mutant transcripts could be detected in Cm. Our results showed that Cm could be an efficient and a cost-effective approach to predict highly expressed neoantigens in tumor samples.

A Family with Gitelman Syndrome with Asymptomatic Phenotypes while Carrying Reported SLC12A3 Mutations.

Gitelman syndrome (GS) is an autosomal recessive disorder characterized by alkalosis, hypokalemia, and hypomagnesemia. Although hundreds of genetic variants associated with GS have been reported, many of them are categorized as of uncertain significance in ClinVar. Here, we describe a pediatric GS patient from a three-generation family whose mother and maternal grandmother were asymptomatic. The proband was a 16-year-old Japanese girl with muscle weakness and continuous hypokalemic metabolic alkalosis. The patient, her mother, and her maternal grandmother were compound heterozygous for, and each expressing a different combination of, previously reported SLC12A3variants in GS patients. The mother and the maternal grandmother had no symptoms related to GS, and blood gas tests showed that the blood potassium levels and venous pH were within normal limits; however, the venous blood HCO3- levels were slightly elevated. The phenotypic effect of missense mutations is difficult to evaluate, and accumulation of genotypic data with accurate phenotyping, including those of "healthy" and "asymptomatic" individuals in various ethnic populations, will improve the genetic diagnosis of GS.