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Abstract Bryant-Li-Bhoj syndrome (BLBS; OMIM # 619720, 619721), caused by germline H3F3A and H3F3B variants encoding histone H3.3, is characterized by mild to severe developmental delay, intellectual disability, failure to thrive, muscle tone abnormalities, and dysmorphic facial features. Here, we present a Japanese patient with a novel heterozygous p.A48G variant in H3F3A, displaying previously unrecognized symptoms of neonatal myoclonus. This case helps broaden the phenotypic spectrum of BLBS.

Abstract We report a case of a 61‐year‐old female with 22q11.2 deletion syndrome (22q11.2DS) and a novel heterozygous nonsense variant in MAP1A, identified through whole‐genome sequencing (WGS). The patient presented with intellectual developmental disorder, treatment‐resistant schizophrenia (SCZ), and multiple congenital anomalies. Despite aggressive pharmacotherapy, she experienced persistent auditory hallucinations and negative symptoms. WGS revealed a 3 Mb deletion at 22q11.2 and a nonsense variant in MAP1A (c.4652T>G, p.Leu1551*). MAP1A, encoding microtubule‐associated protein 1A, is crucial for axon and dendrite development and has been implicated in autism spectrum disorder and SCZ. The MAP1A variant may contribute to the severe psychiatric phenotype, as it is thought to influence synaptic plasticity, a process also affected by 22q11.2 deletion. This case highlights the importance of WGS in identifying additional pathogenic variants that may explain phenotypic variability in 22q11.2DS. Thus, WGS can lead to a better understanding of the genetic architecture of 22q11.2DS. However, further studies are needed to elucidate the role of secondary genetic contributors in the diverse clinical presentations of 22q11.2DS.

Abstract Nuclear clearance and cytoplasmic aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and its pathogenic mechanism is mediated by both loss-of-function and gain-of-toxicity of TDP-43. However, the role of TDP-43 gain-of-toxicity in oligodendrocytes remains unclear. To investigate the impact of excess TDP-43 on oligodendrocytes, we established transgenic mice overexpressing the ALS-linked mutant TDP-43M337V in oligodendrocytes through crossbreeding with Mbp-Cre mice. Two-step crossbreeding of floxed TDP-43M337V and Mbp -Cre mice resulted in the heterozygous low-level systemic expression of TDP-43M337V with (Cre-positive) or without (Cre-negative) oligodendrocyte-specific overexpression of TDP-43M337V. Although Cre-negative mice also exhibit subtle motor dysfunction, TDP-43M337V overexpression in oligodendrocytes aggravated clasping signs and gait disturbance accompanied by myelin pallor in the corpus callosum and white matter of the lumbar spinal cord in Cre-positive mice. RNA sequencing analysis of oligodendrocyte lineage cells isolated from whole brains of 12-month-old transgenic mice revealed downregulation of myelinating oligodendrocyte marker genes and cholesterol-related genes crucial for myelination, along with marked upregulation of apoptotic pathway genes. Immunofluorescence staining showed cleaved caspase 3–positive apoptotic oligodendrocytes surrounded by activated microglia and astrocytes in aged transgenic mice. Collectively, our findings demonstrate that an excess amount of ALS-linked mutant TDP-43 expression in oligodendrocytes exacerbates motor dysfunction in mice, likely through oligodendrocyte dysfunction and neuroinflammation. Therefore, targeting oligodendrocyte protection, particularly through ameliorating TDP-43 pathology, could represent a potential therapeutic approach for ALS.

Abstract Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.

Abstract Cardiospondylocarpofacial syndrome (CSCFS) is a congenital malformation characterized by growth retardation, facial features, short toes with carpal and tarsal fusion, extensive posterior neck vertebral fusion, congenital heart disease, and deafness. Here, we report a severe case of CSCFS with a novel variant, p.Thr187Ile, in MAP3K7. Thr187 is the main phosphorylation site for TGF-beta-activated kinase 1 encoded by MAP3K7, and this variant may cause significant abnormalities in downstream signaling.

Abstract CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.

Summary: Recent single-cell analyses have revealed the complexity of microglial heterogeneity in brain development, aging, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Disease-associated microglia (DAMs) have been identified in ALS mice model, but their role in ALS pathology remains unclear. The effect of genetic background variations on microglial heterogeneity and functions remains unknown. Herein, we established and analyzed two mice models of ALS with distinct genetic backgrounds of C57BL/6 and BALB/c. We observed that the change in genetic background from C57BL/6 to BALB/c affected microglial heterogeneity and ALS pathology and its progression, likely due to the defective induction of neurotrophic factor-secreting DAMs and impaired microglial survival. Single-cell analyses of ALS mice revealed new markers for each microglial subtype and a possible association between microglial heterogeneity and systemic immune environments. Thus, we highlighted the role of microglia in ALS pathology and importance of genetic background variations in modulating microglial functions.

The pervasive weak electromagnetic fields (EMF) inundate the industrialized society, but the biological effects of EMF as weak as 10 µT have been scarcely analyzed. Heat shock proteins (HSPs) are molecular chaperones that mediate a sequential stress response. HSP70 and HSP90 provide cells under undesirable situations with either assisting covalent folding of proteins or degrading improperly folded proteins in an ATP-dependent manner. Here we examined the effect of extremely low-frequency (ELF)-EMF on AML12 and HEK293 cells. Although the protein expression levels of HSP70 and HSP90 were reduced after an exposure to ELF-EMF for 3 h, acetylations of HSP70 and HSP90 were increased, which was followed by an enhanced binding affinities of HSP70 and HSP90 for HSP70/HSP90-organizing protein (HOP/STIP1). After 3 h exposure to ELF-EMF, the amount of mitochondria was reduced but the ATP level and the maximal mitochondrial oxygen consumption were increased, which was followed by the reduced protein aggregates and the increased cell viability. Thus, ELF-EMF exposure for 3 h activated acetylation of HSPs to enhance protein folding, which was returned to the basal level at 12 h. The proteostatic effects of ELF-EMF will be able to be applied to treat pathological states in humans.

Profiling of human DNA polymerase Polε and Polα demonstrates their roles in leading and lagging strand DNA synthesis, and their independent measures allowed accurate predictions of replication dynamics and effects of transcription.

Abstract Objective Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy in patients older than 50 years of age. sIBM is hardly responds to any immunosuppressing theraphies, and its pathophysiology remains elusive. This study aims to explore pathogenic pathways underlying sIBM and identify novel therapeutic targets using metabolomic and transcriptomic analyses. Methods In this retrospective observational study, we analyzed biopsied muscle samples from 14 sIBM patients and six non‐diseased subjects to identify metabolic profiles. Frozen muscle samples were used to measure metabolites with cation and anion modes of capillary electrophoresis time of flight mass spectrometry. We validated the metabolic pathway altered in muscles of sIBM patients through RNA sequencing and histopathological studies. Results A total of 198 metabolites were identified. Metabolomic and transcriptomic analyses identified specific metabolite changes in sIBM muscle samples. The pathways of histamine biosynthesis and certain glycosaminoglycan biosynthesis were upregulated in sIBM patients, whereas those of carnitine metabolism and creatine metabolism were downregulated. Histopathological examination showed infiltration of mast cells and deposition of chondroitin sulfate in skeletal muscle samples, supporting the results of metabolomic and transcriptomic analyses. Interpretation We identified alterations of several metabolic pathways in muscle samples of sIBM patients. These results suggest that mast cells, chondroitin sulfate biosynthesis, carnitine, and creatine play roles in sIBM pathophysiology.

Abstract Recently, whole-exome sequencing (WES) has been used for genetic diagnoses of patients who remain otherwise undiagnosed. WES was performed in 177 Japanese patients with undiagnosed conditions who were referred to the Tokai regional branch of the Initiative on Rare and Undiagnosed Diseases (IRUD) (TOKAI-IRUD). This study included only patients who had not previously received genome-wide testing. Review meetings with specialists in various medical fields were held to evaluate the genetic diagnosis in each case, which was based on the guidelines of the American College of Medical Genetics and Genomics. WES identified diagnostic single-nucleotide variants in 66 patients and copy number variants (CNVs) in 11 patients. Additionally, a patient was diagnosed with Angelman syndrome with a complex clinical phenotype upon detection of a paternally derived uniparental disomy (UPD) [upd(15)pat] wherein the patient carried a homozygous DUOX2 p.E520D variant in the UPD region. Functional analysis confirmed that this DUOX2 variant was a loss-of-function missense substitution and the primary cause of congenital hypothyroidism. A significantly higher proportion of genetic diagnoses was achieved compared to previous reports (44%, 78/177 vs. 24–35%, respectively), probably due to detailed discussions and the higher rate of CNV detection.

Abstract SLC26A4 is a known iodide transporter, and is localized at the apical membrane of thyrocytes. Previously, we reported that SLC26A7 is also involved in iodide transport and that Slc26a7 is a novel causative gene for congenital hypothyroidism. However, its detailed role in vivo remains to be elucidated. We generated mice that were deficient in Slc26a7 and Slc26a4 to delineate differences and associations in their roles in iodide transport. Slc26a7 −/− mice showed goitrous congenital hypothyroidism and mild growth failure on a normal diet. Slc26a7 −/− mice with a low iodine environment showed marked growth failure. In contrast, Slc26a4 −/− mice showed no growth failure and hypothyroidism in the same low iodine environment. Double-deficient mice showed more severe growth failure than Slc26a7 −/− mice. RNA-seq analysis revealed that the number of differentially expressed genes (DEGs) in Slc26a7 −/− mice was significantly higher than that in Slc26a4 −/− mice. These indicate that SLC26A7 is more strongly involved in iodide transport and the maintenance of thyroid function than SLC26A4.

Abstract Autism spectrum disorder (ASD) is a highly heritable, complex disorder in which rare variants contribute significantly to disease risk. Although many genes have been associated with ASD, there have been few genetic studies of ASD in the Japanese population. In whole exomes from a Japanese ASD sample of 309 cases and 299 controls, rare variants were associated with ASD within specific neurodevelopmental gene sets, including highly constrained genes, fragile X mental retardation protein target genes, and genes involved in synaptic function, with the strongest enrichment in trans-synaptic signaling (p = 4.4 × 10−4, Q-value = 0.06). In particular, we strengthen the evidence regarding the role of ABCA13, a synaptic function-related gene, in Japanese ASD. The overall results of this case-control exome study showed that rare variants related to synaptic function are associated with ASD susceptibility in the Japanese population.

RNA viruses are the most frequent pathogens responsible for respiratory infections, particularly in pediatric patients. Next-generation sequencing, represented by Illumina sequencing, is one of the most comprehensive methods for identifying pathogens. Nanopore sequencing has been used to identify and analyze pathogens with a shorter sequencing time. In this study, we evaluated the utility of nanopore sequencing for the detection of RNA viruses in bronchoalveolar lavage fluid (BALF) of pediatric patients with respiratory failure. Using the seven BALF samples, we first compared the nanopore and Illumina sequencing results. The nanopore sequencing detected the same RNA viruses as the Illumina sequencing. Subsequently, BALF samples from 24 additional pediatric patients with respiratory failure were analyzed by nanopore sequencing, and RNA viral pathogens were detected in 10 out of 24 patients. Among these 10 patients, nanopore sequencing identified the same viral pathogens as detected by the PCR and viral antigen tests in five patients. Furthermore, additional RNA viral pathogens were detected by nanopore sequencing with high genome coverage in five patients that were not detected by PCR and viral antigen tests. In conclusion, nanopore sequencing could comprehensively detect RNA viral pathogens in BALF samples with equivalent sensitivity and genome coverage as Illumina sequencing. This rapid sequencing platform may be more beneficial for detecting RNA viruses in clinical settings.

Abstract Background Congenital human cytomegalovirus (cCMV) infection can cause sensorineural hearing loss and neurodevelopmental disabilities in children. Ganciclovir and valganciclovir (GCV/VGCV) improve long-term audiologic and neurodevelopmental outcomes for patients with cCMV infection; however, antiviral drug resistance has been documented in some cases. Long-read sequencing can be used for the detection of drug resistance mutations. The objective of this study was to develop full-length analysis of UL97 and UL54, target genes with mutations that confer GCV/VGCV resistance using long-read sequencing, and investigate drug resistance mutation in patients with cCMV infection. Methods Drug resistance mutation analysis was retrospectively performed in 11 patients with cCMV infection treated with GCV/VGCV. UL97 and UL54 genes were amplified using blood DNA. The amplicons were sequenced using a long-read sequencer and aligned with the reference gene. Single nucleotide variants were detected and replaced with the reference sequence. The replaced sequence was submitted to a mutation resistance analyzer, which is an open platform for drug resistance mutations. Results Two drug resistance mutations (UL54 V823A and UL97 A594V) were found in one patient. Both mutations emerged after 6 months of therapy, where viral load increased. Mutation rates subsided after cessation of GCV/VGCV treatment. Conclusions Antiviral drug resistance can emerge in patients with cCMV receiving long-term therapy. Full-length analysis of UL97 and UL54 via long-read sequencing enabled the rapid and comprehensive detection of drug resistance mutations.

Self-healing collodion baby (SHCB), also called “self-improving collodion baby”, is a rare mild variant of autosomal recessive congenital ichthyosis and is defined as a collodion baby who shows the nearly complete resolution of scaling within the first 3 months to 1 year of life. However, during the neonatal period, it is not easy to distinguish SHCB from other inflammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report a case study of two Japanese SHCB patients with compound heterozygous mutations, c.235G>T (p.(Glu79∗))/ c.1189C>T (p.(Arg397Cys)) and c.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs∗3)), in CYP4F22, which encodes cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically, inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α was seen in the skin at birth. CYP4F22 is an ultra-long-chain FA ω-hydroxylase responsible for ω-O-acylceramide (acylceramide) production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal permeability barrier function. We found that the levels of ceramides with ω-hydroxy FAs including acylceramides and the levels of protein-bound ceramides were much lower in stratum corneum samples obtained by tape stripping from SHCB patients than in those from their unaffected parents and individuals without SHCB. Additionally, our cell-based enzyme assay revealed that two mutants, p.(Glu79∗) and p.(Arg397Cys), had no enzyme activity. Our findings suggest that genetic testing coupled with noninvasive ceramide analyses using tape-stripped stratum corneum samples might be useful for the early and precise diagnosis of congenital ichthyoses, including SHCB.

Non-immune hydrops fetalis (NIHF) indicates the risk for stillbirth. Although the causes vary and most NIHFs have no identifiable cause, recent advances in exome sequencing have increased diagnostic rates.We report a case of NIHF that developed into a giant cystic hygroma complicated by maternal mirror syndrome. Trio-based exome sequencing showed a de novo heterozygous missense variant in the RIT1 (NM_006912: c.246 T > G [p.F82L]). The RIT1 variants are known causative variants of Noonan syndrome (NS; OMIM #163950). The location of the RIT1 variants in the previously reported NS cases with NIHF or/and maternal mirror syndrome was mainly in the switch II region, including the present case.While a further accumulation of cases is needed, exome sequencing, which can identify the variant type in detail, might help predict the phenotype and severity of NIHF.

Hypophosphatasia (HPP), a genetic disorder characterized by decreased tissue-nonspecific alkaline phosphatase (TNSALP) activity, is caused by loss-of-function mutations in the ALPL gene, which encodes TNSALP. The most frequent pathogenic variant in Japanese patients with HPP is a frameshift mutation in the ALPL gene, c.1559delT, and its carrier frequency is reported to be one in 480 in the Japanese population. We report the cases of two Japanese children with HPP who had a heterozygous c.1559delT variant in the ALPL gene. One case (involving a neonate) exhibited respiratory insufficiency associated with vitamin B6 dependent convulsions, significant defective mineralization similar to the severe form of HPP, and extremely low ALP activity. Enzyme replacement therapy (ERT) using asfotase alfa promptly improved her respiratory insufficiency, bone mineralization, and maintained her motor development during infancy. The second case involved a 10-year-old boy who demonstrated diffuse musculoskeletal pain and weakness that progressively disturbed mobility. Although he showed no bony lesions, the clinical symptoms and biochemical abnormalities were compatible with childhood HPP. ERT successfully relieved the severe generalized pain and significantly improved motor function.

Aicardi–Goutières syndrome (AGS) is a rare genetic disorder characterised by progressive encephalopathy, involving microcephaly, intracranial calcification, and cerebrospinal fluid lymphocytosis with increased interferon-α concentrations. The clinical features of AGS overlap with fetal cerebral anomalies caused by congenital infections, such as TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes), or with those of other genetic disorders showing neonatal microcephaly, including Cockayne syndrome (CS) with transcription-coupled DNA repair deficiency, and Seckel syndrome (SS) showing aberrant cell-cycle checkpoint signaling. Therefore, a differential diagnosis to confirm the genetic cause or a proof of infection should be considered. In this report, we describe an individual who showed primordial dwarfism and encephalopathy, and whose initial diagnosis was CS. First, we conducted conventional DNA repair proficiency tests for the patient derived fibroblast cells. Transcription-coupled nucleotide excision repair (TC-NER) activity, which is mostly compromised in CS cases, was slightly reduced in the patient's cells. However, unscheduled DNA synthesis (UDS) was significantly diminished. These cellular traits were inconsistent with the diagnosis of CS. We further performed whole exome sequencing for the case and identified a compound heterozygous loss-of-function variants in the SAMHD1 gene, mutations in which are known to cause AGS. As SAMHD1 encodes deoxyribonucleoside triphosphate triphosphohydrolase, we reasoned that the deoxyribonucleoside triphosphate (dNTP) pool size in the patient's cells was elevated, and the labeling efficiency of UDS-test was hindered due to the reduced concentration of phosphorylated ethynyl deoxyuridine (EdU), a nucleoside analogue used for the assay. In conclusion, UDS assay may be a useful diagnostic tool to distinguish between AGS with SAMHD1 mutations and other related diseases.

Abstract Background Group B Streptococcus (GBS) is an important cause of invasive infection in neonates and infants. Cerebrospinal fluid (CSF) findings and culture may not show evidence of infection early in GBS meningitis. Next-generation sequencing (NGS) has the potential to detect microbial genetic material in patients with infectious diseases. We report two cases of infantile sepsis of GBS meningitis with negative results for CSF culture tests, but positive results for NGS analysis. Case presentation Patient 1 was a 22-day-old male infant diagnosed with sepsis and meningitis. His CSF findings showed pleocytosis, decreased glucose, and increased protein levels. However, CSF and blood culture results at admission were negative. He received a total of 3 weeks of treatment with ampicillin and cefotaxime, and showed clinical improvement. GBS was detected through NGS analysis of CSF collected at admission. Patient 2 was a 51-day-old male infant with sepsis. CSF findings on admission were normal, and blood and CSF cultures were also negative. Intravenous ampicillin and cefotaxime treatment were initiated. Treatment was de-escalated to ampicillin alone because Enterococcus faecalis was cultured from urine. He was discharged after a total of 1 week of antibiotic treatment. Six days after discharge, he was re-hospitalized for sepsis. Blood and CSF cultures were negative, and E. faecalis was again cultured from urine. He received a total of 3 weeks of ampicillin treatment for enterococcal-induced nephritis and did not relapse thereafter. NGS pathogen searches were retrospectively performed on both blood and CSF collected at the first and second admission. GBS was detected in the CSF collected at the first admission, but no significant pathogen was detected in the other samples. Inadequate treatment for GBS meningitis at the first admission may have caused the recurrence of the disease. Conclusion Infantile sepsis may present bacterial meningitis that is not diagnosed by either culture testing or CSF findings. NGS analysis for CSF may be useful for confirming the diagnosis of bacterial meningitis.

Abstract Background Immunosuppression during liver transplantation (LT) enables the prevention and treatment of organ rejection but poses a risk for severe infectious diseases. Immune modulation and antimicrobials affect the plasma microbiome. Thus, determining the impact of immunosuppression on the microbiome may be important to understand immunocompetence, elucidate the source of infection, and predict the risk of infection in LT recipients. We characterized the plasma microbiome of LT recipients at early post-LT and assessed the association between the microbiome and clinical events. Results In this study, 51 patients who received LT at Nagoya University Hospital from 2016 to 2018 were enrolled. Plasma samples were retrospectively collected at the following time points: 1) within a week after LT; 2) 4 ± 1 weeks after LT; 3) 8 ± 1 weeks after LT; and 4) within 2 days after a positive blood culture. A total of 111 plasma samples were analyzed using shotgun next-generation sequencing (NGS) with the PATHDET pipeline. Relative abundance of Anelloviridae, Nocardiaceae, Microbacteriaceae, and Enterobacteriaceae significantly changed during the postoperative period. Microbiome diversity was higher within a week after LT than that at 8 weeks after LT. Antimicrobials were significantly associated with the microbiome of LT recipients. In addition, the proportion of Enterobacteriaceae was significantly increased and the plasma microbiome diversity was significantly lower in patients with acute cellular rejection (ACR) than non-ACR patients. Sequencing reads of bacteria isolated from blood cultures were predominantly identified by NGS in 8 of 16 samples, and human herpesvirus 6 was detected as a causative pathogen in one recipient with severe clinical condition. Conclusions The metagenomic NGS technique has great potential in revealing the plasma microbiome and is useful as a comprehensive diagnostic procedure in clinical settings. Temporal dynamics of specific microorganisms may be used as indirect markers for the determination of immunocompetence and ACR in LT recipients.

Abstract Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer’s disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, App NL-G-F/NL-G-F with amyloid pathology, rTg4510 with tauopathy, and SOD1G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer’s change (n = 11) and control brain (n = 14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.

Abstract Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

Background Most sequencing studies of schizophrenia (SCZ) have focused on de novo genetic variants due to interpretability. However, investigating shared rare variants among patients in the same multiplex family is also important. Relatively large-scale analyses of SCZ multiplex families have been done in Caucasian populations, but whether detected variants are also pathogenic in the Japanese population is unclear because of ethnic differences in rare variants. Materials and methods We performed whole-exome sequencing (WES) of 14 Japanese SCZ multiplex families. After quality control and filtering, we identified rare variants shared among affected persons within the same family. A gene ontology (GO) analysis was performed to identify gene categories possibly affected by these candidate variants. Results We found 530 variants in 486 genes as potential candidate variants from the 14 SCZ multiplex families examined. The GO analysis demonstrated significant enrichment in calcium channel activity. Conclusion This study provides supporting evidence that calcium ion channel activity is involved in SCZ. WES of multiplex families is a potential means of identifying disease-associated rare variants for SCZ.

The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. pColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that “phosphatidylinositol signaling system” and “extracellular matrix receptor interaction” were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.

Abstract Background Achondroplasia (ACH), the most common form of short‐limbed skeletal dysplasia, is caused by gain‐of‐function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. More than 97% of patients result from a heterozygous p.G380R mutation in the FGFR3 gene. We present here a child who had two de novo variants in the FGFR3 on the same allele, a common p.G380R mutation and a novel p.S378N variant. Methods A 3‐year‐old Japanese girl born from non‐consanguineous healthy parents showed more severe clinical and radiological phenotypes than classic ACH, including severe short‐limbed short stature with marked ossification defects in the metaphysis and epiphysis, hydrocephalus and cervicomedullary compression due to foramen magnum stenosis, prolonged pulmonary hypoplasia, and significant delay in the gross motor development. Genomic DNA was extracted from the proband and whole‐exome sequencing was performed. The variants were subsequently confirmed by Sanger sequencing. Results Mutation analysis demonstrated that the proband had p.S378N (c.1133G>A) and p.G380R (c.1138G>A) variants in the FGFR3 gene. Both variants were not detected in her parents and therefore considered de novo. An allele‐specific PCR was developed in order to determine whether these mutations were on the same allele (cis) or on different alleles (trans). The c.1138G>A mutation was found in the PCR product generated with the primer for the mutant 1133A, but it was not detected in the product with the wild‐type 1133G, confirming that p.S378N and p.G380R variants were located on the same allele (cis). Conclusion This is the second case who had two FGFR3 variants in the transmembrane domain on the same allele. The p.S378N variant may provide an additive effect on the activating receptor with the p.G380R mutation and alter the protein function, which could be responsible for the severe phenotype of the present case.

Abstract Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

Repair of cyclobutane pyrimidine dimers requires access to DNA by the nucleotide excision repair machinery. Here the authors show that HBO1 facilitates accumulation of SNF2H and ACF1 to make chromatin more accessible after ultraviolet damage.

Nature Communications 8: Article number: 16102 (2017); Published online 18 July 2017; Updated 20 April 2018 The original version of this Article contained an error in Fig. 1b, in which the cells were incorrectly labelled as ‘HeLa’, rather than the correct ‘HEK293’ Also, the following was omitted from the end of the legend of Fig.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling.

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

Gamma-glutamyl carboxylase (GGCX) gene mutation causes GGCX syndrome (OMIM: 137167), which is characterized by pseudoxanthoma elasticum (PXE)-like symptoms and coagulation impairment. Here, we present a 55-year-old male with a novel homozygous deletion mutation, c.2,221delT, p.S741LfsX100, in the GGCX gene. Histopathological examination revealed calcium deposits in elastic fibers and vessel walls, and collagen accumulation in the mid-dermis. Studies of dermal fibroblasts from the patient (GGCX dermal fibroblasts) demonstrated that the mutated GGCX protein was larger, but its expression level and intracellular distribution were indistinguishable from those of the wild-type GGCX protein. Immunostaining and an enzyme-linked immunosorbent assay showed an increase in undercarboxylated matrix gamma-carboxyglutamic acid protein (ucMGP), a representative substrate of GGCX and a potent calcification inhibitor, indicating that mutated GGCX was enzymatically inactive. Under osteogenic conditions, calcium deposition was exclusively observed in GGCX dermal fibroblasts. Furthermore, GGCX dermal fibroblast cultures contained 23- and 7.7-fold more alkaline phosphatase (ALP)-positive cells than normal dermal fibroblast cultures (n = 3), without and with osteogenic induction, respectively. Expression and activity of ALP were higher in GGCX dermal fibroblasts than in normal dermal fibroblasts upon osteogenic induction. mRNA levels of other osteogenic markers were also higher in GGCX dermal fibroblasts than in normal dermal fibroblasts, which including bone morphogenetic protein 6, runt-related transcription factor 2, and periostin (POSTN) without osteogenic induction; and osterix, collagen type I alpha 2, and POSTN with osteogenic induction. Together, these data indicate that GGCX dermal fibroblasts trans-differentiate into the osteogenic lineage. This study proposes another mechanism underlying aberrant calcification in patients with GGCX syndrome.

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be defined.

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3' and 5' ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3' single-strand overhang at "clean" DSBs, thus initiating homologous recombination (HR)-dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3' single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP(S332A/-/-) cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP(+/-/-) cells. Finally, CtIP(S332A/-/-)BRCA1(-/-) and CtIP(+/-/-)BRCA1(-/-) showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

The organelle contact site of the endoplasmic reticulum and mitochondria, known as the mitochondria-associated membrane (MAM), is a multifunctional microdomain in cellular homeostasis. We previously reported that MAM disruption is a common pathological feature in amyotrophic lateral sclerosis (ALS); however, the precise role of MAM in ALS was uncovered. Here, we show that the MAM is essential for TANK-binding kinase 1 (TBK1) activation under proteostatic stress conditions. A MAM-specific E3 ubiquitin ligase, autocrine motility factor receptor, ubiquitinated nascent proteins to activate TBK1 at the MAM, which results in ribosomal protein degradation. MAM or TBK1 deficiency under proteostatic stress conditions resulted in increased cellular vulnerability in vitro and motor impairment in vivo. Thus, MAM disruption exacerbates proteostatic stress via TBK1 inactivation in ALS. Our study has revealed a proteostatic mechanism mediated by the MAM-TBK1 axis, highlighting the physiological importance of the organelle contact sites. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP. [ABSTRACT FROM AUTHOR]