小坂 仁

Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of PTSG2 and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.

Thermal airway injuries, usually accompanied by facial burns, require emergency management. We encountered a pediatric case of a late airway-scalding injury without any initial signs of scalding on the face or inside the oral cavity. A 16-month-old boy was accidentally exposed to boiling water from overhead and developed tachypnea and dyspnea at 8 h after the injury. When he visited our hospital at 12 h after the injury, there were no scalding-related findings on his face or inside his oral cavity; however, severe laryngeal edema was observed, which required emergency intubation. Thermal airway injuries can occur later, even if there is no evidence of facial or oral scalding immediately after the injury. Airway injuries should be considered when a patient has been exposed to hot water from overhead.

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.

BACKGROUND: Behavioral problems of foster children are an important issue for the maintenance of the foster care system, but they have not been adequately studied in Japan. We used the Eyberg Child Behavior Inventory (ECBI) to investigate behavioral problems among foster children and to examine associated factors. METHODS: Twenty-nine foster children and their foster parents and 479 non-foster children and parents were recruited for the foster and control groups, respectively. Both groups underwent statistical comparative analyses using data from their ECBI assessments. The ECBI has two scales: the Intensity Scale quantifies the severity of child behavioral problems, and the Problem Scale captures the caregiver's perceived difficulties handling each behavior. We conducted a retrospective investigation of the background of the foster parent-child pairs to explore potential causal relationships with behavioral problems. RESULTS: The mean intensity score for the foster group was significantly higher than that for the control group (p = 0.001). The mean problem scores for the foster group and the control group were not significantly different (p = 0.79). In the foster group, the retrospective investigation revealed two children with neurological or neurodevelopmental disorders, 17 with histories of abuse, and 10 with other issues. CONCLUSION: Intensity scores showed severe behavioral problems among foster children, perhaps caused by neurological disorders, abuse, parental mental health, or economic hardship. Problem scores showed no significant differences between groups. It can therefore be posited that foster parents might exhibit a more lenient parenting style when dealing with children who have a history of abuse by their biological parents.

BACKGROUND: Premature children are known to be at a high risk of developing behavioral problems. This study examined the effectiveness of parent-child interaction therapy (PCIT) in reducing behavioral problems in young children born premature. METHODS: The study included 18 child-parent pairs with children born at less than 35 weeks of gestation (range: 23-34 weeks, median: 31.0 weeks) and aged 27-52 months (median: 38.0 months). They were assigned to either the PCIT group (n = 7) or the non-PCIT group (n = 11) based on maternal desire for treatment. The study was designed to examine the effects of PCIT. Specifically, the Eyberg Child Behavior Inventory (ECBI) intensity score, ECBI problem score, and Parenting Stress Index Short Form (PSI-SF) scores were compared before treatment and after 6 months. RESULTS: In the PCIT group, the mean ECBI intensity score was 135.7 (SD = 13.5; T-score = 64) at baseline and 90.1 (SD = 15.5; T-score = 46) at post-assessment, the mean ECBI problem score was 9.8 (SD = 1.9; T-score = 54) at baseline and 4.4 (SD = 3.1; T-score = 44) at post-assessment, the mean PSI-SF total score was 60.1 (SD = 4.8; 95%tile) at baseline and 49.6 (SD = 5.6; 85%tile) at post-assessment, showing a significant improvement (ECBI intensity scores: p < 0.001, d = 2.03; ECBI problem scores: p < 0.001, d = 1.94; PSI-SF total scores: p = 0.004, d = 0.86). On the other hand, none of the scores showed significant change in the non-PCIT group. CONCLUSIONS: The PCIT can be considered as a potential treatment option for behavioral problems in young children born premature.

Mendelson症候群は胃酸の誤嚥による化学性肺炎であり,誤嚥時および誤嚥後数時間以降の二相性の呼吸障害が特徴的である。意識障害,喉頭反射低下,フルストマックなどの状況がリスク因子である。熱性けいれん時の誤嚥により発症した重症のMendelson症候群を報告する。症例は1歳男児。昼食後に強直間代性けいれんが出現した。止痙後に嘔吐し,嘔吐後から低酸素血症を認め気管挿管された。第2病日に急激な肺高血圧から心停止に至り静脈脱血・動脈送血体外式膜型人工肺(venoarterial extracorporeal membrane oxygenation,以下VA ECMO)を導入した。その後,一酸化窒素吸入療法を含めた集学的治療を行い心機能および肺高血圧の所見は徐々に改善し第9病日にECMOを離脱,第38病日に明らかな神経学的後遺症はなく退院した。食後に発症したけいれんでは胃酸誤嚥のリスクが高く,経時的な呼吸循環動態の評価が重要である。(著者抄録)

BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive inherited and neurodegenerative disorder. Approximately 10% of NPC patients have acute liver failure and sometimes need liver transplantation (LT), and 7% reportedly develop inflammatory bowel disease (IBD). We report the case of a girl with NPC who had a re- accumulation of cholesterol in the transplanted liver and NPC-related IBD. CASE REPORT: The patient underwent living donor liver transplantation (LDLT) due to severe acute liver failure caused by an unknown etiology inherited from her father. At 1 year and 6 months (1Y6M), she developed neurological delay, catalepsy, and vertical supranuclear gaze palsy. The foam cells were found in her skin, and fibroblast Filipin staining was positive; hence, she was diagnosed with NPC. It was identified that her father had NPC heterozygous pathogenic variant. At 2 years, she had anal fissure, skin tag and diarrhea. She was diagnosed with NPC-related IBD, using a gastrointestinal endoscopy. Three years after LT, liver biopsy revealed foam cells and numerous fatty droplets. At 8 years, broken hepatocytes and substantial fibrosis were observed. She died from circulation failure due to hypoalbuminemia at 8Y2M. CONCLUSIONS: In NPC, load of cholesterol metabolism is suggested to persist even after LT. LDLT from NPC heterozygous variant donor was insufficient to metabolize cholesterol overload. In NPC patients, the possibility of cholesterol re-accumulation should be considered when LT is performed. NPC-related IBD should be considered when NPC patients have anorectal lesions or diarrhea.

Corona virus infectious disease, emerged in 2019(COVID-19)は、流行の主体が感染性の高いオミクロン変異株に置き換わり小児の感染者数が増加し、けいれん、意識障害、脳炎・脳症例も増加し、重症化する症例や後遺症を残す症例も報告されている。われわれが経験した小児COVID-19の出血性ショック脳症症候群や、片側けいれん・片麻痺・てんかん症候群を含む3症例の急性脳症を提示する。小児COVID-19の急性脳症はインフルエンザ脳症と同様、さまざまな臨床病型・重症度、そして予後をとりうる。(著者抄録)

We discovered biallelic intragenic structural variations (SVs) inFGF12by applying long-read whole genome sequencing to an exome-negative patient with developmental and epileptic encephalopathy (DEE). We also found another DEE patient carrying a biallelic (homozygous) single-nucleotide variant (SNV) inFGF12that was detected by exome sequencing.FGF12heterozygous recurrent missense variants with gain-of-function or heterozygous entire duplication ofFGF12are known causes of epilepsy, but biallelic SNVs/SVs have never been described.FGF12encodes intracellular proteins interacting with the C-terminal domain of the alpha subunit of voltage-gated sodium channels 1.2, 1.5, and 1.6, promoting excitability by delaying fast inactivation of the channels. To validate the molecular pathomechanisms of these biallelicFGF12SVs/SNV, highly sensitive gene expression analyses using lymphoblastoid cells from the patient with biallelic SVs, structural considerations, andDrosophilain vivo functional analysis of the SNV were performed, confirming loss-of-function. Our study highlights the importance of small SVs in Mendelian disorders, which may be overlooked by exome sequencing but can be detected efficiently by long-read whole genome sequencing, providing new insights into the pathomechanisms of human diseases.

BACKGROUND: Enoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in the ECHS1 gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem. METHODS: Here, we constructed an assay system to verify VUS function for ECHS1 gene. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis. RESULTS: The functional validation of VUS identified novel variants causing loss of ECHS1 function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system. CONCLUSIONS: In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.

Autism spectrum disorder (ASD) is caused by combined genetic and environmental factors. Genetic heritability in ASD is estimated as 60-90%, and genetic investigations have revealed many monogenic factors. We analyzed 405 patients with ASD using family-based exome sequencing to detect disease-causing single-nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variations (CNVs) for molecular diagnoses. All candidate variants were validated by Sanger sequencing or quantitative polymerase chain reaction and were evaluated using the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines for molecular diagnosis. We identified 55 disease-causing SNVs/indels in 53 affected individuals and 13 disease-causing CNVs in 13 affected individuals, achieving a molecular diagnosis in 66 of 405 affected individuals (16.3%). Among the 55 disease-causing SNVs/indels, 51 occurred de novo, 2 were compound heterozygous (in one patient), and 2 were X-linked hemizygous variants inherited from unaffected mothers. The molecular diagnosis rate in females was significantly higher than that in males. We analyzed affected sibling cases of 24 quads and 2 quintets, but only one pair of siblings shared an identical pathogenic variant. Notably, there was a higher molecular diagnostic rate in simplex cases than in multiplex families. Our simulation indicated that the diagnostic yield is increasing by 0.63% (range 0-2.5%) per year. Based on our simple simulation, diagnostic yield is improving over time. Thus, periodical reevaluation of ES data should be strongly encouraged in undiagnosed ASD patients.

Coenzyme Q10 (CoQ10) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ10 receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ10 has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ10 or the ratio of reduced to total CoQ10 (reduced/total CoQ10) in patients with mitochondrial diseases. We measured reduced and oxidized CoQ10 in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ10 deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ10 ratio. In primary CoQ10 deficiency patients, total CoQ10 levels were significantly decreased, however, the reduced/total CoQ10 ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ10 deficiency patients, total CoQ10 levels did not decrease. However, the reduced/total CoQ10 ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency. Measurement of CoQ10 in fibroblasts proved useful for the diagnosis of primary CoQ10 deficiency. In addition, the reduced/total CoQ10 ratio may reflect the metabolic status of mitochondrial disease.

BACKGROUND: Eight waves of the coronavirus disease 2019 (COVID-19) epidemic have been observed in Japan. This retrospective study was conducted to clarify the clinical characteristics of pediatric COVID-19 patients. METHODS: We studied 121 patients admitted to the Jichi Children's Medical Center Tochigi between April 2020 and March 2023. Incidence of pediatric COVID-19 in Tochigi Prefecture was used to examine hospitalization and severe illness rates. RESULTS: The mean age of the patients was 3 years and 8 months. One hundred and eleven patients (91.7%) were hospitalized after January 2022 (after the 6th wave), when the Omicron strain became endemic in Japan. Convulsions occurred in 30 patients (24.8%), all of whom were admitted after the 6th wave. Twenty-three of the 30 patients had no underlying disease. Eleven patients (9.1%) were diagnosed with acute encephalopathy. One patient died due to hemorrhagic shock and encephalopathy syndrome and two had sequelae after the 6th wave. The patient who died due to encephalopathy had hypercytokinemia. In the Tochigi Prefecture, the number of pediatric COVID-19 patients increased after the 6th wave, but the hospitalization rate declined. The rate of severe illness did not change before the end of 5th and after the 6th wave. CONCLUSION: Although the rate of severe illness in patients with pediatric COVID-19 did not increase after the 6th wave, some patients had complicated critical illnesses. Systemic inflammatory reaction was considered to have been associated with the severe encephalopathy.

Abstract Static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration is a neurodegenerative disorder with brain iron accumulation caused by the variants of WDR45, a core autophagy-related gene that encodes WIPI4. However, the pathophysiology of the disease, particularly the function of WDR45/WIPI4 in iron metabolism, is largely unknown. As no other variants of core autophagy-related genes show abnormalities in iron metabolism, the relation between autophagy and iron metabolism remains to be elucidated. Since iron deposition in the brain is the hallmark of static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration, iron chelation therapy has been attempted, but it was found to worsen the symptoms; thus, the establishment of a curative treatment is essential. Here, we evaluated autophagy and iron metabolism in patient-derived cells. The expression of ferritin and ferric iron increased and that of ferrous iron decreased in the patient cells with WDR45 variants. In addition, the expression of nuclear receptor coactivator 4 was markedly reduced in patient-derived cells. Furthermore, divalent metal transporter 1, which takes in ferrous iron, was upregulated, while ferroportin, which exports ferrous iron, was downregulated in patient-derived cells. The transfer of WDR45 via an adeno-associated virus vector restored WIPI4 and nuclear receptor coactivator 4 expression, reduced ferritin levels, and improved other phenotypes observed in patient-derived cells. As nuclear receptor coactivator 4 mediates the ferritin-specific autophagy, i.e., ferritinophagy, its deficiency impaired ferritinophagy, leading to the accumulation of ferric iron‒containing ferritin and insufficiency of ferrous iron. Because ferrous iron is required for various essential biochemical reactions, the changes in divalent metal transporter 1 and ferroportin levels may indicate a compensatory response for maintaining the intracellular levels of ferrous iron. Our study revealed that the pathophysiology of static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration involves ferrous iron insufficiency via impaired ferritinophagy through nuclear receptor coactivator 4 expression reduction. Our findings could aid in developing a treatment strategy involving WDR45 manipulation, which may have clinical applications.

We report on a patient with a distal 16.4-Mb duplication at 2q36.3-qter, who presented with severe intellectual disability, microcephaly, brachycephaly, prominent forehead, hypertelorism, prominent eyes, thin upper lip, and progenia. Copy number analysis using whole exome data detected a distal 2q duplication. This is the first report describing a distal 2q duplication at the molecular level.

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.

PURPOSE: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects. METHODS: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated. RESULTS: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments. CONCLUSION: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families.

BACKGROUND: The delivery of adeno-associated virus (AAV) vectors via the cerebrospinal fluid (CSF) has emerged as a valuable method for widespread transduction in the central nervous system. While infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, the cisterna magna has been recognized as an alternative target for clinical studies, as it can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. METHODS: We evaluated the early distribution of fluorine-18-labeled AAV9 vectors infused into the lateral ventricle or cisterna magna of four non-human primates using positron emission tomography. The expression of the green fluorescent protein was immunohistochemically determined. RESULTS: In both approaches, the labeled vectors diffused into the broad arachnoid space around the brain stem and cervical spinal cord within 30 mins. Both infusion routes efficiently transduced neurons in the cervical spinal cord. CONCLUSIONS: For gene therapy that primarily targets the cervical spinal cord and brainstem, such as amyotrophic lateral sclerosis, cisterna magna infusion would be a feasible and effective administration method.

Hypertrophic cardiomyopathy is a common cardiac complication in mitochondrial disorders, and the morbidity rate in neonatal cases is up to 40%. The mortality rate within 3 months for neonatal-onset mitochondrial cardiomyopathy is known to be high because there is currently no established treatment.We report the case of a male infant with neonatal-onset mitochondrial disorder presenting lactic acidosis and hypertrophic cardiomyopathy. Genetic analysis of the patient revealed recurrent m.13513G>A, p.Asp393Asn in mitochondrially encoded NADH dehydrogenase 5 gene (MT-ND5). Low-dose propranolol was initially administered for cardiomyopathy; however, he developed hypertrophic obstructive cardiomyopathy (HOCM) at 3 months of age. To reduce the risk of hypoglycemia associated with high-dose propranolol, cibenzoline, a class Ia antiarrhythmic drug, was added at a dose of 2.5 mg/kg/day and increased weekly to 7.5 mg/kg/day with monitoring of the blood concentration of cibenzoline. Left ventricular outflow tract stenosis (LVOTS) dramatically improved from 5.4 to 1.3 m/second in LVOTS peak velocity after 6 weeks, without notable adverse effects. The plasma N-terminal pro-brain natriuretic peptide level decreased from 65,854 to 10,044 pg/mL. Furthermore, myocardial hypertrophy also improved, as the left ventricular mass index decreased from 173.1 to 108.9 g/m2 after 3 months of the treatment.The administration of cibenzoline, in conjunction with low-dose propranolol, may serve an effective treatment for HOCM in infantile patients with mitochondrial disorders.

Biotin-responsive basal ganglia disease (BBGD) with SLC19A3 mutation was first reported in 1998, and over 30 mutations have been reported. We report a neonatal BBGD case with sudden-onset feeding difficulty and impaired consciousness. Encephalopathy resolved after the initiation of biotin and thiamine treatment. Genetic testing revealed a novel heterozygous mutation [c.384_387del, p.Tyr128fs];[c.265 A > C, p.Ser89Arg] in SLC19A3. Early treatment for BBGD is essential, especially with onset in the neonatal or early infancy period.

Abstract Background Infants with trisomy 13 have a very high mortality rate. However, aggressive interventions for their complications, can improve their prognosis and may, thereby, increase the number of long‐term survivors with trisomy 13. To date, there is no study on the psychomotor developmental progress of patients with trisomy 13. We conducted this survey to clarify the prognostic factors, living circumstances, and developmental status of infants the trisomy 13. Methods Patients with trisomy 13 who were admitted to the Department of Pediatrics, Jichi Medical University Hospital were enrolled. Their clinical data were investigated retrospectively using clinical records. Results Nine patients with trisomy 13 were enrolled and divided into the early death (died at &lt;1 year) and long‐term survival (survived for &gt;1 year) groups. All the early death group patients had severe congenital heart disease. Heart failure at under 1 year of age was associated with early death. All the long‐term survival group patients underwent operations (e.g. tracheostomy or gastrostomy) and all used home nursing and/or a social care service. Three patients used home mechanical ventilation. None of the patients was able to stand alone or speak intelligible words. Two patients without severe brain anomalies were able to roll over, sit up, and smile by 3 years of age. Conclusions Long‐term survivors with trisomy 13 require extensive nursing and medical care. It is important to provide medical and welfare services to reduce the burden on families. In patients without severe brain anomalies, psychomotor development may be expected. However, no clear developmental prognostic factors were found.

OBJECTIVES: We aimed to validate a newly developed antigen-detecting rapid diagnostic test (Ag-RDT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using anterior nasal specimens. METHODS: Between February 12 and September 30, 2021, 16 patients (age range, <1 month-76 years) were enrolled, and samples were collected simultaneously from anterior nasal and nasopharyngeal sites continuously during hospitalization. The primary endpoints were the diagnostic accuracy of the Ag-RDT and utility of anterior nasal specimens. RESULTS: In total, 226 sets of paired samples were obtained. In 88.2% of specimens, the viral load was high at the nasopharyngeal site. The mean cycle threshold (Ct) values for the anterior nasal and nasopharyngeal sites were 32.4 and 29.9, respectively. Using the real-time polymerase chain reaction results as a reference, the Ag-RDT showed 100% sensitivity up to day 6 of the illness using specimens with moderate or high viral load (Ct <30) from either site. From day 7, the sensitivity was 70.4-90.6% and 83.9-84.6% for the anterior nasal and nasopharyngeal sites, respectively. The specificity remained at 100%. CONCLUSIONS: Our novel Ag-RDT meets the World Health Organization criteria and provides stable sensitivity and specificity and accurate results with anterior nasal specimens.

BACKGROUND: Actin, alpha, skeletal muscle 1 (ACTA1) is one of the causative genes of nemaline myopathy (NM) and congenital fiber-type disproportion (CFTD). CFTD is characterized by type 1 fiber atrophy and distinguished from NM in the absence of rods. Eight patients with CFTD, including one patient with dilated cardiomyopathy (DCM), have previously been reported. Herein, we report the case of a 10-year-old boy presenting with CFTD and DCM. METHODS: We performed exome sequencing and analyzed the effect of Met327Lys mutations on cultured C2C12 muscle cells compared with that seen in the wild type (WT, ACTA1) and previously identified Asp294Val mutations associated with a severe phenotype of CFTD without cardiomyopathy. RESULTS: Exome sequencing revealed a de novo mutation, c.980 T > A, p.(Met327Lys), in ACTA1 (NM_001100.4). C2C12 cells transfected with the WT plasmid expressed ACTA1 in the nucleus and cytoplasm. Cells with the Asp294Val mutant showed needle-like structures in the cytoplasm, whereas the expression of the Met327Lys mutant resulted in few aggregations but many apoptotic cells. CONCLUSION: Apoptosis induced in Met327Lys-transfected muscle cells supports the pathogenicity of the mutation and can be implicated as one of the histopathological features associated with CFTD, as in NM.

TUBB4A gene variants cause dystonia type 4 and hypomyelination with atrophy of the basal ganglia and cerebellum. We report the case of a child with delayed motor development, intellectual disability, and dystonia. Magnetic resonance imaging revealed hypomyelination and progressive cerebellar atrophy without atrophy of the basal ganglia. Whole-exome sequencing revealed a de novo heterozygous variant, c.1088T > C, p.(Met363Thr), in TUBB4A. The present case further supports the vulnerability of the cerebellum in patients with TUBB4A pathogenic variants.

症例は神経発達障害を呈した11歳の男児で、8歳時に撮像したMRI画像で、大脳白質の髄鞘形成不全が認められたが、大脳基底核や小脳の萎縮はみられなかった。また、10歳時には軽度の知的障害(IQ=52)と診断され、11歳時に撮像したMRI画像では小脳の髄鞘形成不全と萎縮が認められたが、大脳基底核の大きさは正常であった。これらの所見から、全エクソームシーケンシングによる遺伝子解析を行ったところ、TUBB4A遺伝子にde novoヘテロ接合性バリアントc.1088T>C, p.(Met363Thr)が検出された。本例から、TUBB4A遺伝子に病的バリアントを有する患者には小脳の脆弱性が認められるという仮説が支持された。

CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.