小坂 仁

Niemann–Pick disease type C1 (NPC1) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants of the NPC1 gene that encodes a protein essential for lysosomal cholesterol transport. A deficiency in NPC1 results in the accumulation of unesterified cholesterol and sphingolipids, leading to neurological, psychiatric, and hepatic manifestations from infancy to adulthood. The currently approved treatment is palliative. Although the efficacy of gene therapy has been demonstrated in murine models, reliable biomarkers for evaluating the treatment effects remain unknown. We evaluated adeno-associated virus (AAV) vector-mediated NPC1 gene therapy in Npc1 homo-knockout ( Npc1 ^−/− ) mice, focusing on blood-based biomarkers. An AAV vector carrying human NPC1 under a cytomegalovirus promoter (AAV- hNPC1 ) was administered intraperitoneally on days 6–8 after birth at varying vector doses and analyzed at multiple time points: 1.8 × 10 ¹¹ vector genomes/mouse analyzed at 7 weeks (Low/7w) and 1.0 × 10 ¹² vector genomes/mouse at 4 weeks (High/4w) and 9 weeks (High/9w). hNPC1 is expressed in the brain and liver, and a degree of neuronal cell survival is observed. High-dose AAV treatment improves body weight and rotarod performance. Plasma N -palmitoyl- O -phosphocholine-serine (PPCS) and lysosphingomyelin (lyso-SM) levels were significantly elevated in Npc1 ^−/− mice. PPCS increased with disease progression but was significantly decreased after later points of high-dose AAV treatment (saline-treated Npc1 ^−/− mice: 12.88 ± 3.53 ng/mL, AAV-treated Npc1 ^−/− mice: 7.87 ± 1.67 ng/mL, p = 0.0008). Lyso-SM and oxysterols showed limited changes after therapy. Vector genome analysis revealed higher and more sustained levels in the brain than in the liver, which is consistent with rapid hepatocyte proliferation-reducing vector persistence. These findings demonstrate that systemic AAV- hNPC1 therapy ameliorates motor and neurological deficits but has a limited impact on several cholesterol-related biomarkers. PPCS has been suggested as a sensitive biomarker of therapeutic response and warrants further evaluations in preclinical and clinical NPC1 gene therapy trials.

The accurate removal of intronic sequences from pre-mRNA by the spliceosome is essential for correct gene expression, with small nuclear RNAs (snRNAs) such as U4 playing structural and regulatory roles in catalyzing this. De novo variants in the highly constrained critical region including the T-loop region of RNU4-2 have been linked to ReNU syndrome, a neurodevelopmental disorder, but the broader mutational spectrum remains uncharacterized. Here, we show that, in a cohort of unresolved cases with neurodevelopmental disorder, monoallelic and biallelic RNU4-2 variants were identified in 16 affected individuals, expanding the genetic basis beyond the critical region: 12 with de novo T-loop variants and 4 from two families with compound heterozygous variants in non-critical regions (stem II, the k-turn, and the Sm-binding site). Previously reported functional mapping by saturation genome editing confirmed that most of the variant positions in this study are highly functionally constrained. Clinically, individuals with biallelic variants exhibited developmental delay and intellectual disability that were similar to but milder than those with monoallelic variants, notably lacking extracerebral organ involvement. These results suggest that pathogenic RNU4-2 variants act in both dominant and recessive manners, and that non-critical regions may also harbor disease-causing variants. More broadly, this study underscores the diagnostic importance of non-coding RNA genes in neurodevelopmental disorders and demonstrates the need to distinguish pathogenic variants from benign ones, together with their inheritance patterns.

INTRODUCTION: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare disorder caused by antibodies against platelet factor 4 (PF4) triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines using non-replicable adenoviral vectors. It emerged during the pandemic, with patients typically presenting with thrombosis at uncommon sites, thrombocytopenia, and elevated D-dimer levels. VITT antibodies and heparin-dependent antibodies bind to distinct PF4 epitopes. Recently, VITT-like clinical, laboratory, and anti-PF4 antibody features have also been observed in patients with adenoviral infections. Only four pediatric cases of cerebral venous sinus thrombosis (CVST) have been reported. CASE REPORT: The patient was a previously healthy 2-year-old girl with no history of heparin exposure or SARS-CoV-2 vaccination. She presented with fever and was diagnosed with adenovirus infection. The fever resolved by day 4, but by day 6 she became increasingly lethargic and experienced vomiting. On day 12, Laboratory data showed severe thrombocytopenia and elevated D-dimer levels. Computed tomography revealed CVST along with a secondary hemorrhage in the right temporal lobe. She underwent hematoma removal with external/internal decompression and was started on continuous intravenous unfractionated heparin, and she was switched to warfarin. The thrombus decreased, platelet count spontaneously increased. Platelet activation assays using acute-phase serum identified a PF4-dependent platelet-activating antibody. CONCLUSION: We report a case of CVST in a 2-year-old girl following adenovirus infection. Unlike heparin-induced thrombocytopenia, where heparin exacerbates the condition, it is effective here by competitively inhibiting anti-PF4 antibody binding. In patients with prior adenovirus infection presenting with CVST and thrombocytopenia, anti-PF4 disorders should be considered.

Creatine transporter deficiency (CTD) caused by mutations in SLC6A8 encoding the creatine transporter (CRT), leads to cerebral creatine deficiency syndromes; however, the cellular impact of CRT loss remains unclear. In this study, we investigated the consequences of the G561R mutation by examining fibroblasts using proteomics and functional assays. We observed severe intracellular creatine deficiency (> 90% reduction), leading to impaired energy metabolism (low ATP and high ADP/ATP). Proteomic analysis revealed significant alterations in the mitochondrial and extracellular vesicle pathways. Our investigation revealed impaired mitochondrial oxidative phosphorylation, reduced spare respiratory capacity, elevated oxidative stress, and significant alterations in amino acid transporter activity. Protein misfolding associated with G561R exacerbated these deficits compared to the deletion model. These findings elucidate the key pathological mechanisms induced by the CRT-G561R mutation-including energy metabolic reprogramming, mitochondrial dysfunction, and cellular stress-which significantly contribute to our understanding of the pathogenesis of creatine transporter deficiency and suggest potential therapeutic targets.

Pathogenic SNF2 related chromatin remodeling ATPase 1 (SMARCA1) variants have been reported in patients with X-linked intellectual disability (XLID) characterized by macrocephaly and variable neurological symptoms. Here, we report two unrelated male patients with XLID due to novel SMARCA1 variants detected by exome sequencing. Patient 1 showed macrocephaly, behavioral difficulty, and learning disability with a hemizygous SMARCA1 variant (NM_003069.5:c.1795 C > T p.[Gln599*]) leading to nonsense-mediated decay. Patient 2 had ataxia and speech delay with a hemizygous missense variant (NM_003069.5:c.1343 G > T p.[Arg448Leu]). Structural modeling suggested that the missense variant, p.(Arg448Leu) might destabilize interactions between SMARCA1 and nucleosomal DNA, thereby contributing to the abberant effect of mutant SMARCA1 protein. Both variants were inherited from their unaffected healthy mothers. This study suggests that hemizygous variants impairing SMARCA1 function can cause XLID with other variable features, such as macrocephaly and ataxia, in men.

The 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) is a mitochondria-localized protein involved in the biosynthesis of coenzyme Q10 in the electron transfer system, and its variants have been reported to cause progressive neurodegenerative diseases such as neonatal leukoencephalopathy and hereditary spastic paraplegia. In this case report, we present a case of a nine-year-old girl with exotropia with a novel HPDL variant who underwent strabismus surgery. She was referred to the ophthalmology department with exotropia and a history of progressive spastic paraplegia with gait disturbance. Brain MRI showed no remarkable findings. Whole-exome sequencing revealed a homozygous variant c.1040delC (p.Thr347Metfs*66) in the HPDL gene. Ophthalmic examination revealed a best-corrected visual acuity of 20/12 in both eyes. Fundoscopy showed retinal discoloration at the level of the retinal pigment epithelium in the right eye. As the patient had intermittent exotropia with good convergence, she was followed up conservatively. One year after the initial examination, the patient could not keep her eyes in a central position by convergence. The alternate prism cover test revealed exotropia of 80 prism diopters. We diagnosed that intermittent exotropia had deteriorated into constant exotropia. The patient's family requested a strabismus surgery, which was conducted under general anesthesia. Standard left lateral rectus recession, left medial rectus resection, and right lateral rectus recession were also performed. Postoperatively, her exotropia was reduced, and she achieved good convergence. The patient and her family were satisfied with the surgical outcome, and no recurrence was noted one year postoperatively. Our results provide important information for the associations of variant in HPDL with progressive spastic paraplegia, strabismus and retinal changes and broaden the genetic spectrum of HPDL-related disease. This is the first report to present a novel HPDL variant and document the performance of strabismus surgery for constant exotropia.

Seizures in patients with developmental and epileptic encephalopathies (DEEs) are often highly resistant to various antiseizure medications. Perampanel (PER) is a novel antiseizure medication that non-competitively inhibits the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and is expected to reduce seizure frequency not only for focal seizures and generalized tonic-clonic seizures (GTCS) but also for other seizure types. This study aimed to clarify the long-term therapeutic efficacy and tolerability of PER in patients with DEEs. We analyzed data regarding patients' background characteristics, medication retention, trends in seizure frequency, and adverse events obtained from 24 patients with DEEs who had been on PER treatment for 60 months. The retention rates were 62.5% and 46.9% at 12 and 60 months, respectively. At 60 months after PER initiation, the rate of patients with > 50% seizure reduction was 33.3%, 33.3%, 38.5%, 54.5%, 54.5%, and 36.4% among patients with atypical absence seizures, tonic seizures, focal seizures, GTCS, myoclonic seizures, and atonic seizures, respectively. The frequency of adverse events was 70.8%. PER showed long-term efficacy in various seizure types. PER is a promising treatment option for patients with DEEs.

We report a case of severe Aicardi-Goutières syndrome caused by a novel homozygous RNASEH2B intronic variant, NC_000013.10(NM_024570.4):c.65-13G > A p.Glu22Valfs*5. The patient was born with pseudo-TORCH symptoms, including intracranial calcification, cataracts, and hepatosplenomegaly. Furthermore, the patient exhibited profound intellectual impairment and died at 14 months due to aspiration pneumonia accompanied by interstitial lung abnormalities. The severity of the patient's symptoms underscores the critical role of the C-terminal region of RNase H2B.

It is difficult to differentiate between coronavirus disease 2019 (COVID-19) and influenza based on the symptoms. In the present study, a newly developed antigen rapid diagnostic test (Ag-RDT) called Panbio™ COVID-19/Flu A&B that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B virus was evaluated. Its accuracy was evaluated using 235 pairs of nasopharyngeal samples collected from patients with respiratory symptoms and fever (>37.5°C). Reverse transcription polymerase chain reaction was used as a reference method to evaluate the accuracy of the SARS-CoV-2 detection. We confirmed the accuracy of the developed Ag-RDT against the Omicron variant where the sensitivity and specificity were 94.8% and 100%, respectively. In addition, to identify the influenza A virus, a noninferiority test was conducted using a commercial Ag-RDT, which has a sensitivity and specificity in comparison with viral culture of 94.8% and 98.4%, respectively. The positive and negative predictive values for influenza A virus were 98.5% and 98.1%, respectively, for the Panbio COVID-19/Flu A&B test. The evaluation of this newly developed Ag-RDT using clinical samples suggests that it has a high efficacy in clinical settings.

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.

6歳男児。2歳過ぎより目立っていた尖足に対して、3歳2ヵ月時に当科へ紹介受診となった。所見では両下肢ともに尖足位で、独歩は可能であったが、膝関節を伸展させた状態で歩行していた。対処として両下肢の尖足に対して理学療法や下腿三頭筋へのボトックス注射が行われたが、5歳頃より連続歩行距離が短くなり、長距離の移動には車いすが必要となった。そこで、緩徐に進行する痙性対麻痺症状から遺伝子検査を行った結果、本症例は軽度知的障害を合併したp.Arg239Cysによる遺伝性痙性対麻痺と診断された。6歳7ヵ月時にアキレス腱延長術を行った結果、目下は連続歩行距離は500mで、疲労時や移動の際は車いすを使用している。

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.

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.

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.