[STATE-OF-THE-ART OF GLOBAL START-UP INVESTMENT FOR ALLERGY AND IMMUNOLOGY 2022].

BACKGROUND: In 2022, the "New Capitalism Grand Design and Implementation Plan" was adopted in Japan, emphasizing the promotion and environmental development of startups. Given this context, an investigation into the startup and investment landscape in the allergy sector, both domestically and internationally, becomes imperative. METHODS: We analyzed 156 allergy-related startups from Japan, the US, and Europe from 2010 to 2021. Data on corporate information and investment trends were extracted from databases and VC websites. RESULTS: The total investment reached approximately 7.2 billion USD, with a ratio of 20:6:1 for the US, Europe, and Japan, respectively. The US showed a decline post its peak from 2016-2018, while Europe and Japan experienced growth. Notably, the US primarily invested in biopharmaceuticals for atopic dermatitis and food allergies, Europe in asthma-related apps, and Japan in healthcare apps and cross-border startups. DISCUSSION AND CONCLUSION: While Japan's investment environment in the allergy sector remains in its nascent stages and has room for development, the US and Europe are evidently ahead. Considering the rise of startups and funding limitations in Japan, external funding from regions like the US becomes a potential avenue. These findings are anticipated to contribute to the strategic activation of startups in allergy research and development.

Updated mutational spectrum and genotype-phenotype correlations in ichthyosis patients with ABCA12 pathogenic variants.

Autosomal recessive congenital ichthyoses (ARCI) is a genetically heterogeneous condition that can be caused by pathogenic variants in at least 12 genes, including ABCA12. ARCI mainly consists of congenital ichthyosiform erythroderma (CIE), lamellar ichthyosis (LI) and harlequin ichthyosis (HI). The objective was to determine previously unreported pathogenic variants in ABCA12 and to update genotype-phenotype correlations for patients with pathogenic ABCA12 variants. Pathogenic variants in ABCA12 were detected using Sanger sequencing or a combination of Sanger sequencing and whole-exome sequencing. To verify the pathogenicity of a previously unreported large deletion and intron variant, cDNA analysis was performed using total RNA extracted from hair roots. Genetic analyses were performed on the patients with CIE, LI, HI and non-congenital ichthyosis with unusual phenotypes (NIUP), and 11 previously unreported ABCA12 variants were identified. Sequencing of cDNA confirmed the aberrant splicing of the variant ABCA12 in the patients with the previously unreported large deletion and intron variant. Our findings expand the phenotype spectrum of ichthyosis patients with ABCA12 pathogenic variants. The present missense variants in ABCA12 are considered to be heterogenous in pathogenicity, and they lead to varying disease severities in patients with ARCI and non-congenital ichthyosis with unusual phenotypes (NIUP).

Manufacturing processes of peanut (Arachis hypogaea) allergen powder-dnfp.

Background: Important components of drug safety, efficacy, and acceptability involve manufacturing and testing of the drug substance and drug product. Peanut flour sourcing/processing and manufacturing processes may affect final drug product allergen potency and contamination level, possibly impacting drug safety, quality, and efficacy. We describe key steps in the manufacturing processes of peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Palforzia®), a drug used in oral immunotherapy (OIT) for the treatment of peanut allergy. Methods: Established criteria for source material must be met for manufacturing PTAH drug product. Degree of roasting was determined with a Hunter colorimeter. Protein/allergen content, identity, potency, safety, and quality of each batch of PTAH drug substance were assessed with a combustion analyzer, allergen-specific Western blot (immunoblotting), ELISA, and HPLC. Contaminants (ie, aflatoxin) were measured by UPLC. Results: Roasting degree beyond "light roast" was associated with variable degrees of protein allergen degradation, or potentially aggregation. Relative potency and amounts of protein allergens showed variability due in part to seasonal/manufacturing variability. Proportion of lots not meeting aflatoxin limits has increased in recent years. Up to 60% of peanut flour source material failed to meet screening selection acceptance criteria for proceeding to drug substance testing, mostly because of failure to meet potency acceptance criteria. Other lots were rejected due to safety (ie, aflatoxin) and quality. Influence of potency variation, within specification parameters, on safety/tolerability observed in trials was considered low, in part due to stringent controls placed at each step of manufacturing. Conclusions: Extensive variability in allergen potency is a critical issue during immunotherapy, particularly during OIT initial dose escalation and up-dosing, as it may result in lack of efficacy or avoidable adverse allergic reactions. Based on EU and US regulatory requirements, the production of PTAH includes manufacturing controls to ensure drug product safety, potency, and quality. For example, although PTAH contains all peanut allergens, each lot has met strict criteria ensuring consistent allergenic potency of Ara h 1, Ara h 2, and Ara h 6. The rigor of PTAH's manufacturing process ensures reliable dose consistency and stability throughout its shelf life.

Evaluation of adrenaline auto-injector prescription profiles: A population-based, retrospective cohort study within the National Insurance Claims Database of Japan.

BACKGROUND: Adrenaline is the first-line medication for managing anaphylaxis. A better understanding of prescription trends for adrenaline auto-injectors (AAIs) is important to improving patient care as well as information on health education interventions and medical guidelines. However, it has been difficult to gather comprehensive data in a sustainable manner. Thus, we aimed to investigate trends in AAI prescriptions in Japan. METHODS: We searched the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB), a unique and comprehensive database of health insurance claims, and investigated prescriptions for AAIs for all ages (April 2017 to March 2018). We assessed the annual number of prescriptions per person as well as prescription rates per 100,000 population per year by age, sex, and geographic region. RESULTS: A total of 88,039 subjects (56,109 males, 31,930 female) and 116,758 devices (1.33 AAIs per patient per year) were prescribed AAIs at least once a year for all ages. The prescription rate for AAIs was 69.5 per 100,000 population-years. Patients aged 0-9 years were prescribed AAIs at the rate of 278.9 per 100,000 population-years. Patients aged 0-19 years were 6.4 times more likely to be prescribed AAIs than those over 20 years of age. Males were more frequently prescribed AAIs than females in all age groups, except for those aged 20-24 years. We also evaluated differences in prescription rates by geographic region. CONCLUSIONS: This comprehensive evaluation revealed trends in AAI prescriptions, thus helping develop preventive strategies with respect to anaphylaxis in Japan.

More than keratitis, ichthyosis, and deafness: Multisystem effects of lethal GJB2 mutations.

BACKGROUND: Infant death in keratitis-ichthyosis-deafness (KID) syndrome is recognized; its association with specific genotypes and pathophysiology is inadequately understood. OBJECTIVE: We sought to discover characteristics that account for poor outcomes in lethal KID syndrome. METHODS: We collected 4 new cases and 9 previously reported, genotyped cases of lethal KID syndrome. We performed new molecular modeling of the lethal mutants GJB2 p.A88V and GJB2 p.G45E. RESULTS: Infant death occurred in all patients with GJB2 p.G45E and p.A88V; it is unusual with other GJB2 mutations. Early death with those 2 "lethal" mutations is likely multifactorial: during life all had ≥1 serious infection; most had poor weight gain and severe respiratory difficulties; many had additional anatomic abnormalities. Structural modeling of GJB2 p.G45E identified no impact on the salt bridge previously predicted to account for abnormal central carbon dioxide sensing of GJB2 p.A88V. LIMITATIONS: This clinical review was retrospective. CONCLUSION: GJB2 p.G45E and p.A88V are the only KID syndrome mutations associated with uniform early lethality. Those electrophysiologically severe mutations in GJB2 reveal abnormalities in many organs in lethal KID syndrome. All patients with KID syndrome may have subtle abnormalities beyond the eyes, ears, and skin. Early genotyping of KID syndrome births will inform prognostic discussion.

Survey on patients with undiagnosed diseases in Japan: potential patient numbers benefiting from Japan's initiative on rare and undiagnosed diseases (IRUD).

BACKGROUND: There is now an international partnership to establish global programs for patients with rare and undiagnosed diseases, involving interdisciplinary expert panels and phenotype-driven genetic analyses utilizing next-generation sequencing and analytics. Whereas it is crucial to have data such as the actual number of undiagnosed patients, to help inform the implementation plan with such programs, there have been no systematic studies to quantitate the numbers of patients principally because of the inherent difficulty in most health systems to identify patients whose condition has not yet been diagnosed and coded. Our national experience with a rare disease program, Nan-Byo which was established in 1972, and the more recently expanded Initiative on Rare and Undiagnosed Diseases (IRUD), provided a unique opportunity to design a cross-sectional study to ascertain the undiagnosed patients in Japan based on the IRUD referral criteria. RESULTS: Two rounds of online surveys were performed: one survey targeting physicians affiliated with general hospitals (GH) and family clinics (FC) (the response rate: 30.6% (242/792)) and one nationwide survey targeting university hospitals (UH) in Japan (47.1% (839/1781)). A high percentage of doctors needing IRUD was seen in pediatrics at GH, FC, while there was a clear demand for IRUD in most departments at UH. We calculated the number of undiagnosed patients in Japan, as the "percentage of doctors needing IRUD" × "number of patients who would be referred to IRUD per doctor needing IRUD (cases/person)" × "total number of doctors in the relevant facilities in Japan (persons)", resulting in 3681 cases in pediatrics/pediatric surgery and 33,703 cases in other departments, for a total of 37,384 cases. CONCLUSIONS: Our study revealed the extant demand for IRUD in most departments and 37,000+ potential patients with undiagnosed diseases in the Japanese health system. These data inform the establishment of an equitable, sustainable, efficient and effective outpatient-based IRUD. These findings would serve as a valuable reference for undiagnosed diseases programs in different international jurisdictions and for countries and regions who also share vision(s) for societal implementation that help to advance international efforts to support patients with rare diseases who are direly waiting for diagnosis, subsequent treatment and care.

Biallelic Mutations in KDSR Disrupt Ceramide Synthesis and Result in a Spectrum of Keratinization Disorders Associated with Thrombocytopenia.

Mutations in ceramide biosynthesis pathways have been implicated in a few Mendelian disorders of keratinization, although ceramides are known to have key roles in several biological processes in skin and other tissues. Using whole-exome sequencing in four probands with undiagnosed skin hyperkeratosis/ichthyosis, we identified compound heterozygosity for mutations in KDSR, encoding an enzyme in the de novo synthesis pathway of ceramides. Two individuals had hyperkeratosis confined to palms, soles, and anogenital skin, whereas the other two had more severe, generalized harlequin ichthyosis-like skin. Thrombocytopenia was present in all patients. The mutations in KDSR were associated with reduced ceramide levels in skin and impaired platelet function. KDSR enzymatic activity was variably reduced in all patients, resulting in defective acylceramide synthesis. Mutations in KDSR have recently been reported in inherited recessive forms of progressive symmetric erythrokeratoderma, but our study shows that biallelic mutations in KDSR are implicated in an extended spectrum of disorders of keratinization in which thrombocytopenia is also part of the phenotype. Mutations in KDSR cause defective ceramide biosynthesis, underscoring the importance of ceramide and sphingosine synthesis pathways in skin and platelet biology.

Pityriasis Rubra Pilaris Type V as an Autoinflammatory Disease by CARD14 Mutations.

Importance: We found CARD14 mutations (2 de novo novel mutations and another previously reported mutation) in 3 of 3 patients with pityriasis rubra pilaris (PRP) type V, but not in patients with PRP of other types. Our findings, combined with the published literature, suggest that type V PRP, both familial and sporadic, can be caused by CARD14 mutations. Detailed clinical observation revealed that all 3 patients displayed unique patchy macular brown hyperpigmentation. Objective: To further determine how often patients with PRP have pathogenic mutations in CARD14 and to elucidate which clinical subtype of PRP is caused by CARD14 mutations. Design, Setting, and Participants: We sequenced the entire coding regions of CARD14 in genomic DNA from patients with 5 clinical subtypes of PRP. The detailed clinical features were analyzed in all the patients. The pathogenicity of each mutation was evaluated by several computational predictions. PRP was classified into 6 subgroups, types I to VI, based on clinical criteria. We categorized all the patients with PRP into the clinical subtypes using the classic PRP classification; 22 cases of PRP with varying subtypes were studied. Main Outcomes and Measures: The prevalence of CARD14 mutations in each subtype of PRP was evaluated. Clinical features and characteristics of patients with PRP with CARD14 mutations were analyzed. Results: Overall 22 patients with PRP were included in our study (12 men, 10 women; mean [SD] age, 26 [18] years). Among 3 patients with PRP type V, all were found to have CARD14 mutations: 2 de novo novel mutations (p.Cys127Ser and p.Gln136Leu), and another previously reported mutation (p.Gly117Ser). All were close to the reported pathogenic domains. In silico analysis of all 3 mutations suggested that they are functionally relevant to pathogenesis. All 3 patients displayed unique patchy macular brown hyperpigmentation additionally to other typical features of PRP. Patients with PRP type I and type IV, 1 patient each, had the rare variants in CARD14. Conclusions and Relevance: Pityriasis rubra pilaris type V is a distinct variant of PRP that is caused by CARD14 mutations. In addition, a rare variant of CARD14 might also be implicated in the pathophysiology of other forms of PRP.

Design and synthesis of a potent inhibitor of class 1 DYRK kinases as a suppressor of adipogenesis.

Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer's disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARγ and C/EBPα. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome.

Anti-PM/Scl antibodies are found in Japanese patients with various systemic autoimmune conditions besides myositis and scleroderma.

INTRODUCTION: Anti-PM/Scl antibodies are associated with polymyositis (PM)/systemic scleroderma (SSc) overlap syndromes and are also found in other systemic autoimmune diseases. Although anti-PM/Scl reactivity is found in 3-11% of PM or SSc patients and in approximately 25% of PM/SSc overlap patients, previous large studies of Japanese patients with scleroderma reported that anti-PM/Scl are not found in Japanese patients at all. The PM/Scl autoantigen complex comprises 11-16 different polypeptides; ELISA with PM1-α peptide, which is a major epitope of the PM/Scl complex, has frequently been used for the detection of these antibodies in recent studies. However, no ELISA kit is commercially available in Japan. METHODS: In this study, we developed an immunoassay for measuring antibodies against recombinant PM/Scl-100 and PM/Scl-75 polypeptides, which are the two major targets of the complex, and we investigated their presence in 600 Japanese patients with various systemic autoimmune conditions. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts were also applied to ELISA-positive sera. RESULTS: In ELISA, 11 patients were positive for anti-PM/Scl-100 antibodies and 7 of these 11 patients were also positive for anti-PM/Scl-75 antibodies. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts confirmed that 9 out of these 11 patients immunoprecipitated the typical sets of PM/Scl proteins. In total, 4/16 (25%) undifferentiated connective tissue disease (UCTD) patients, 3/126 (2.4%) dermatomyositis patients, 1/223 (0.4%) SSc patients, 1/88 (1.1%) Sjögren's syndrome patients, 0/123 patients with systemic lupus erythematosus, 0/17 patients with overlap syndrome and 0/7 patients with PM were judged to be positive for anti-PM/Scl antibodies. CONCLUSIONS: This is the first report of Japanese autoimmune patients with anti-PM/Scl antibodies. In Japanese patients, anti-PM/Scl antibodies are only very rarely found, and they are not always specific for dermatomyositis (DM) or SSc; they are also present in various autoimmune conditions with the highest prevalence being in UCTD. All anti-PM/Scl-positive DM cases are complicated with interstitial lung disease and/or cancer, while no life-threatening involvement was found in other anti-PM/Scl-positive cases. Further studies on larger cohorts are necessary to define the clinical significance of anti-PM/Scl antibodies in autoimmune diseases.

A palindromic motif in the -2084 to -2078 upstream region is essential for ABCA12 promoter function in cultured human keratinocytes.

ATP-binding cassette transporter family A member 12 (ABCA12) is a keratinocyte transmembrane lipid transporter that plays a critical role in preserving the skin permeability barrier. Biallelic loss of function of the ABCA12 gene is causative of some forms of recessive congenital ichthyosis, an intractable disease marked by dry, thickened and scaly skin on the whole body. Genetic diagnosis is essential, although the results may occasionally be inconclusive, because some patients with low ABCA12 expression have one mutant allele and one apparently intact allele. Aside from aberrant splicing or deletion mutations, one possible explanation for such discrepancy is loss of promoter function. This study aims to elucidate the promoter region of ABCA12 and to locate the essential elements therein, thus providing the necessary information for genetic diagnostic screening of congenital ichthyosis. Close examination of the 2980-bp upstream regions of the ABCA12 gene revealed that a palindromic motif (tgagtca) at -2084 to -2078 is essential for the promoter function, and a short fragment of -2200/-1934 alone has potent promoter activity. Identification of the key promoter element of ABCA12 in this study may provide relevant information for genetic diagnosis of recessive congenital ichthyosis.

Revertant mutation releases confined lethal mutation, opening Pandora's box: a novel genetic pathogenesis.

When two mutations, one dominant pathogenic and the other "confining" nonsense, coexist in the same allele, theoretically, reversion of the latter may elicit a disease, like the opening of Pandora's box. However, cases of this hypothetical pathogenic mechanism have never been reported. We describe a lethal form of keratitis-ichthyosis-deafness (KID) syndrome caused by the reversion of the GJB2 nonsense mutation p.Tyr136X that would otherwise have confined the effect of another dominant lethal mutation, p.Gly45Glu, in the same allele. The patient's mother had the identical misssense mutation which was confined by the nonsense mutation. The biological relationship between the parents and the child was confirmed by genotyping of 15 short tandem repeat loci. Haplotype analysis using 40 SNPs spanning the >39 kbp region surrounding the GJB2 gene and an extended SNP microarray analysis spanning 83,483 SNPs throughout chromosome 13 in the family showed that an allelic recombination event involving the maternal allele carrying the mutations generated the pathogenic allele unique to the patient, although the possibility of coincidental accumulation of spontaneous point mutations cannot be completely excluded. Previous reports and our mutation screening support that p.Gly45Glu is in complete linkage disequilibrium with p.Tyr136X in the Japanese population. Estimated from statisitics in the literature, there may be approximately 11,000 p.Gly45Glu carriers in the Japanese population who have this second-site confining mutation, which acts as natural genetic protection from the lethal disease. The reversion-triggered onset of the disesase shown in this study is a previously unreported genetic pathogenesis based on Mendelian inheritance.