小坂 仁

Structural variants (SVs), including duplications, deletions, inversions, translocations, and insertions, play major roles in human phenotypic diversity but remain difficult to detect because of variable size and structural complexity. Optical genome mapping (OGM) uses ultra-high molecular weight DNA (>150 kb) fluorescently labeled at a specific six-nucleotide sequence, enabling comprehensive SV detection by analyzing labeling patterns along long DNA molecules. This study aimed to fully characterize SVs using OGM. OGM was applied to 30 cases with exome sequencing-based copy number (CN) variants (16 CN losses, 7 CN gains, and 7 combined CN losses and gains). Additionally, targeted Oxford Nanopore long-read sequencing with adaptive sampling was used to determine breakpoints of SVs. This approach revealed undetected SVs in 14 cases (46.7%), and disclosed gene disruptions or CN alterations explaining clinical features in seven cases (23.3%). Even complex SVs involving numerous chromosomal segments and breakpoints were resolved efficiently, highlighting the power of combining OGM and long-read sequencing. Integrating OGM with long-read sequencing improves diagnostic resolution beyond sequencing alone and provides a robust framework for interpreting complex SVs. These findings highlight the potential clinical utility of combining OGM and long-read sequencing as a comprehensive diagnostic strategy for improved precision medicine in rare genetic diseases.

BACKGROUND: Primary coenzyme Q10 (CoQ10) deficiency is a group of mitochondrial disorders caused by pathogenic variants of genes involved in CoQ10 biosynthesis. Although some patients respond to oral CoQ10 supplementation, the pathophysiology remains poorly understood. Ferroptosis, a form of iron-dependent cell death driven by lipid peroxidation, is suppressed by reduced CoQ10via ferroptosis suppressor protein 1 (FSP1). However, its involvement in primary CoQ10 deficiency has not yet been studied using patient-derived cells. CASES AND RESULTS: We reported six patients from three families and investigated ferroptosis susceptibility in fibroblasts from three representative patients: one with COQ2 variants and two with COQ4 variants. Fibroblasts with COQ2 variants showed increased vulnerability to ferroptosis inducers, plasma membrane lipid peroxidation. In contrast, fibroblasts with COQ4 variants exhibited only mild changes. Notably, susceptibility to ferroptosis remained unchanged after increasing intracellular CoQ10 levels. Despite this persistent ferroptosis sensitivity in vitro, the COQ2 patient exhibited significant clinical improvement following CoQ10 supplementation. These findings suggest that ferroptosis may contribute to cellular vulnerability in primary CoQ10 deficiency but may not be the primary driver of renal and neurological symptoms. CONCLUSIONS: Our results highlight a complex interplay between CoQ10 biosynthesis, ferroptosis defense, and therapeutic response, warranting further investigation of subcellular CoQ10 distribution and ferroptosis-related mechanisms.

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.