👤 Hiroshi Imai

Advanced esophageal squamous cell carcinoma (ESCC) has a poor prognosis, and current treatments provide limited survival benefits. This study aimed to identify prognostic biomarkers and therapeutic targets by genomic profiling of advanced ESCC using circulating tumor DNA (ctDNA). The SCRUM-MONSTAR GOZILA study is a nationwide, plasma-based molecular profiling project using Guardant360, involving 31 core cancer institutions in Japan. We evaluated the genomic landscape of advanced ESCC and investigated associations between specific alterations and overall survival (OS). The correlation between blood tumor mutation burden (bTMB) and clinical outcomes in patients with PD-1 inhibitors was also assessed using multiple cutoff values (2, 4, 6, 8, and 10 mutations/Mb). Among 313 patients, alterations predominantly consisted of single nucleotide variants (SNVs, 68.9%) and copy number alterations (20.7%). ctDNA analysis identified key genomic alterations linked to poor outcomes in advanced ESCC, revealing potential prognostic biomarkers and therapeutic targets. In contrast, bTMB did not show predictive value for the efficacy of PD-1 inhibitors in this study. Show less

Epstein-Barr virus (EBV) is an enveloped, double-stranded DNA virus that selectively infects primates. Periodontitis, a common inflammatory disease characterized by alveolar bone destruction, affects more than half of the global adult population. While EBV has been linked to periodontitis due to its pro-inflammatory effects and presence in the human periodontium, its effects on bone metabolism, particularly alveolar bone resorption, remain unclear. This study demonstrated that EBV infection in humanized mice induced osteoclast differentiation and alveolar bone resorption, resulting in sparse trabecular bone patterns and increased lacunae resorption. Extracellular vesicles (EVs) from EBV-infected cells contained M-CSF, essential for osteoclast differentiation, and increased CTSK and RANKL expression in osteoclast precursor cells after uptake. EBV infection increased the expression of group IIA-secreted phospholipase A Show less

X-linked adrenoleukodystrophy (X-ALD) is a congenital metabolic disorder characterized mainly by inflammatory demyelination and adrenal insufficiency. Newborn screening using hexacosanoyl lysophosphatidylcholine (C26:0-LPC) in dried blood spots as a diagnostic marker can successfully identify potential patients with X-ALD and prevent disease onset. C26:0-LPC accumulates in patients with X-ALD, although the machinery synthesizing it has remained unclear. In this study, we focused on phosphatidylcholine (PC) with C26:0 moiety as a precursor of C26:0-LPC. We identified that lysophospholipid (LPL) acyltransferase 10 (LPLAT10)/LPCAT4/LPEAT2/AGPAT7 (1-acylglycerol-3-phosphate O-acyltransferase 7) is the responsible LPL acyltransferase that produces PC with C26:0 moiety by transferring C26:0-CoA into 2-acyl-LPC. We also found that LPLAT10 deficiency decreased the amount of C26:0-LPC in fibroblasts from X-ALD patients. Mechanistically, LPLAT10 introduced saturated fatty acid-CoA of various chain lengths as substrates into the sn-1 position of LPC but did not transfer C26:0-CoA to other LPL classes, such as lysophosphatidylethanolamine. Structural analysis revealed that a trimethylamine group of PC was placed between two tryptophan residues (W242 and W244), forming a W-X-W motif, possibly through cation-π interaction. Finally, it was shown that exogenously administered C26:0 FFA-d Show less

Gingival crevicular fluid (GCF) reflects both local periodontal inflammation and systemic conditions. This review highlights the role of oxidative stress, oxidised low-density lipoprotein (oxLDL), and apolipoprotein B (apoB) as molecular links between periodontitis and metabolic disorders. Elevated GCF levels of oxLDL and apoB indicate enhanced vascular permeability and local oxidative modification, particularly in diabetes. Furthermore, oxLDL promotes the formation of neutrophil extracellular trap (NET) via connecting oxidative stress with immune-mediated tissue injury. These insights establish GCF as a valuable, non-invasive biomarker for understanding the interplay between periodontal and systemic diseases. Show less

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder characterized by progressive fibrofatty replacement of the myocardium. In the Japanese population, variants of the desmoglein-2 ( A 6-year-old asymptomatic girl was diagnosed with ACM based on abnormal electrocardiogram findings, including epsilon waves, and T-wave inversions in leads V This case illustrates the potential for severe pediatric ACM associated with compound heterozygous This case underscores the genetic heterogeneity and phenotypic variability in inherited cardiomyopathies. It emphasizes the importance of comprehensive genetic testing and close monitoring of affected individuals and their families. Show less

Prenatal maternal depression is known to affect the neurodevelopment of offspring. This study aimed to investigate the profile of umbilical cord serum in mothers with major depressive disorder (MDD). Liquid chromatography-tandem mass spectrometry (LC-MS) was conducted using umbilical cord serum from mothers with MDD (n = 5) and controls (control, n = 5). The levels of several differentially expressed proteins in umbilical cord serum were compared between the MDD (n = 10) and control groups (n = 10) by enzyme-linked immunosorbent assay. The proteomic profiles in the umbilical cord serum were different between the MDD and control groups, including the pathways of regulation of plasma lipoprotein particle levels, and synapse organization. Only apolipoprotein A4 (APOA4) was significantly higher in the cord blood of MDD group. APOA4 levels in maternal serum were also significantly higher in the MDD group than those in the control group. The APOA4 levels in the umbilical cord serum were higher than that in the maternal serum. The levels of APOA4, a biomarker of depression, in the umbilical cord serum at birth were elevated in the neonates of MDD mothers. It is, therefore, likely that fetuses of MDD mothers were exposed to higher APOA4 levels in utero and this could have developmental and mental health implications for the offspring. Show less

Fibroblast growth factor receptor (FGFR) alterations drive oncogenesis in multiple tumor types. Here we studied pemigatinib, a selective, potent, oral FGFR1-FGFR3 inhibitor, in the phase 2 FIGHT-207 basket study of FGFR-altered advanced solid tumors. Primary end points were objective response rate (ORR) in cohorts A (fusions/rearrangements, n = 49) and B (activating non-kinase domain mutations, n = 32). Secondary end points were progression-free survival, duration of response and overall survival in cohorts A and B, and safety. Exploratory end points included ORR of cohort C (kinase domain mutations, potentially pathogenic variants of unknown significance, n = 26) and analysis of co-alterations associated with resistance and response. ORRs for cohorts A, B and C were 26.5% (13/49), 9.4% (3/32) and 3.8% (1/26), respectively. Tumors with no approved FGFR inhibitors or those with alterations not previously confirmed to be sensitive to FGFR inhibition had objective responses. In cohorts A and B, the median progression-free survival was 4.5 and 3.7 months, median duration of response was 7.8 and 6.9 months and median overall survival was 17.5 and 11.4 months, respectively. Safety was consistent with previous reports. The most common any-grade treatment-emergent adverse events were hyperphosphatemia (84%) and stomatitis (53%). TP53 co-mutations were associated with lack of response and BAP1 alterations with higher response rates. FGFR1-FGFR3 gatekeeper and molecular brake mutations led to acquired resistance. New therapeutic areas for FGFR inhibition and drug failure mechanisms were identified across tumor types. ClinicalTrials.gov identifier: NCT03822117 . Show less

Renal interstitial fibrosis (RIF) is a common pathological manifestation of chronic kidney diseases. Epithelial-mesenchymal transition (EMT) of tubular epithelial cells is considered a major cause of RIF. Although long non-coding RNAs (lncRNAs) are reportedly involved in various pathophysiological processes, the roles and underlying molecular mechanisms of lncRNAs in the progression of RIF are poorly understood. In this study, we investigated the function of lncRNAs in RIF. Microarray assays showed that expression of the lncRNA lnc-CHAF1B-3 (also called claudin 14 antisense RNA 1) was significantly upregulated in human renal proximal tubular cells by both transforming growth factor-β1 (TGF-β1) and hypoxic stimulation, accompanied with increased expression of EMT-related genes. Knockdown of lnc-CHAF1B-3 significantly suppressed TGF-β1-induced upregulated expression of collagen type I alpha 1, cadherin-2, plasminogen activator inhibitor-1, snail family transcriptional repressor I (SNAI1) and SNAI2. Quantitative reverse transcriptase PCR analyses of paraffin-embedded kidney biopsy samples from IgA nephropathy patients revealed lnc-CHAF1B-3 expression was correlated positively with urinary protein levels and correlated negatively with estimated glomerular filtration rate. Show less

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous neuromuscular disorder characterized by muscle weakness and caused by mutations in more than 35 different genes. This condition should not be overlooked as a subset of patients with CMS are treatable. However, the diagnosis of CMS is often difficult due to the broad variability in disease severity and course. A five-year-old boy without remarkable family history was born with marked general muscle hypotonia and weakness, respiratory insufficiency, anomalies, and multiple joint contractures. Congenital myopathy was suspected based upon type 1 fiber predominance on muscle biopsy. However, he was diagnosed with CMS at age 4 years when his ptosis and ophthalmoplegia were found to be improved by edrophonium chloride and repetitive nerve stimulation showed attenuation of compound muscle action potentials. An exome sequencing identified a compound heterozygous missense variant of c.737C > T (p.A246V) and a novel intronic insertion c.1166 + 4₁₁₆₆ + 5insAAGCCCACCAC in RAPSN. RT-PCR analysis which showed the skipping of exon 7 in a skeletal muscle sample confirmed that the intronic insertion was pathogenic. His myasthenic symptoms were remarkably improved by pyridostigmine. The patient's diagnosis of CMS was confirmed by exome sequencing, and RT-PCR revealed that the skipping of exon 7 in RAPSN was caused by a novel intronic insertion. The genetic information uncovered in this case should therefore be added to the collection of tools for diagnosing and treating CMS. Show less

Hepatocyte growth factor (HGF) is an endogenously induced bioactive molecule that has strong anti-apoptotic and tissue repair activities. In this research, we identified APOA4 as a novel pharmacodynamic (PD) marker of the recombinant human HGF (rh-HGF), E3112. rh-HGF was administered to mice, and their livers were investigated for the PD marker. Candidates were identified from soluble proteins and validated by using human hepatocytes in vitro and an animal disease model in vivo, in which its c-Met dependency was also ensured. Among the genes induced or highly enhanced after rh-HGF exposure in vivo, a soluble apolipoprotein, APOA4 was identified as a soluble PD marker of rh-HGF with c-Met dependency. It should be worthwhile to clinically validate its utility through clinical trials with healthy subjects and ALF patients. Show less

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants. Show less

Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A. Show less

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins. Show less

Valproic acid, which is widely used to treat various types of epilepsy, may cause severe hyperammonemia. However, the mechanism responsible for this side effect is not readily apparent. Polymorphisms in the genes encoding carbamoyl-phosphate synthase 1 (CPS1) and N-acetylglutamate synthase (NAGS) were recently reported to be risk factors for the development of hyperammonemia during valproic acid-based therapy. This study aimed to examine the influence of patient characteristics, including polymorphisms in CPS1 4217C>A and NAGS -3064C>A, on the development of hyperammonemia in Japanese pediatric epilepsy patients. The study included 177 pediatric epilepsy patients. The presence of a 4217C>A polymorphism in CPS1 was determined using an allele-specific polymerase chain reaction (PCR)-based method, and the presence of a -3064C>A polymorphism in NAGS was determined using a PCR-based restriction fragment length polymorphism method. Hyperammonemia was defined as a plasma ammonia level exceeding 200 μg/dL. We observed a significant difference between the combination of valproic acid with phenytoin and the development of hyperammonemia in both univariate and multivariate analyses. With regard to the CPS1 4217C>A polymorphism, we did not observe a significant association with the development of hyperammonemia. In conclusion, CPS1 4217C>A polymorphism may not be associated with the development of hyperammonemia in Japanese population. Show less

Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by multistep interactions between lymphocytes and high endothelial venules (HEVs). Heparan sulfate (HS) has been implicated in the presentation of chemokines on the surface of HEVs during this process. However, it remains unclear whether this cell surface presentation is a prerequisite for lymphocyte homing. In this study, we generated conditional knockout (cKO) mice lacking Ext1, which encodes a glycosyltransferase essential for HS synthesis, by crossing Ext1(flox/flox) mice with GlcNAc6ST-2-Cre transgenic mice expressing Cre recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated in PLN HEVs but retained in other blood vessels in the cKO mice. The accumulation of a major secondary lymphoid tissue chemokine, CCL21, on HEVs was also abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated that lymphocyte homing to PLNs was diminished in the cKO mice by 30-40%. Consistent with this result, contact hypersensitivity responses were also diminished in the cKO mice. The residual lymphocyte homing to PLNs in the cKO mice was dependent on pertussis toxin-sensitive Gi protein signaling, in which lysophosphatidic acid-mediated signaling was partly involved. These results suggest that chemokine presentation by HS on the surface of HEVs facilitates but is not absolutely required for lymphocyte homing. Show less

Hypospadias is a common congenital anomaly caused by incomplete fusion of urethral folds. Development of the urethra and external genital system in the male fetus is an androgen-dependent process. In this regard, enzymes 17 β-hydroxysteroid dehydrogenase type 3 (17 β HSD3, encoded by HSD17B3) and steroid 5 α-reductase type 2 (encoded by SRD5A2) play crucial roles. To investigate the possible associations between common polymorphisms in HSD17B3 as well as well-known V89L polymorphism in SRD5A2 and risk of hypospadias. A case-control study was performed between 1999 and 2005. There were 89 Japanese boys with hypospadias and 291 newborn controls. We genotyped HSD17B3-1999T>C, +10A>G, +20A>G, +139G>A (V31I), +913G>A (G289S), and SRD5A2+336G>C (V89L) polymorphisms by allelic discrimination assay. We measured mRNA expression of the wildtype G289 allele and the mutant S289 allele of the HSD17B3 gene in the transfected human fetal kidney HEK293 cells. Assessment of hypospadias including its severity and HSD17B3 and SRD5A2 genes using DNA blood samples: allele and genotype distribution of single nucleotide polymorphisms in these two genes in cases and controls. In our study, the risk of hypospadias was significantly higher in subjects carrying homozygous HSD17B3+913A (289S) alleles (odds ratio [OR]: 3.06; 95% confidence interval [CI]: 1.38-6.76). The risk of severe hypospadias was much higher in these subjects (OR: 3.93; 95% CI: 1.34-11.49). The mRNA expression levels of HSD17B3 G289 were higher than those of HSD17B3 S289 mutant (P < 0.001). In addition, the risk of severe hypospadias increased in boys carrying the SRD5A2+336C (89L) allele (OR: 3.19; 95% CI: 1.09-9.36). These results suggest that the HSD17B3 G289S polymorphism may be a potential risk modifier for hypospadias. Our findings provide evidence that a certain genotype related to androgen production may potentiate risk of hypospadias. Show less

Activation of the Wnt signaling pathway is frequently observed in hepatocellular carcinoma (HCC), though mutation of three of its components, CTNNB1, AXIN1, and AXIN2, is observed substantially less often. We examined the relationship between Wnt signaling and epigenetic alteration of secreted frizzled-related protein (SFRP) genes in HCC. We frequently detected the active form of beta-catenin and accumulation of nuclear beta-catenin in liver cancer cell lines. We detected methylation of SFRP family genes in liver cancer cell lines (SFRP1, 9/12, 75%; SFRP2, 7/12, 58%; SFRP4, 3/12, 25%; SFRP5, 7/12, 58%) and primary HCCs (SFRP1, 9/19, 47%; SFRP2, 12/19, 63%; SFRP5, 8/19, 42%), though methylation of SFRP4 was not found in primary HCCs. SFRP methylation also was detected in hepatitis B or C virus-associated chronic hepatitis (SFRP1, 6/37, 16%; SFRP2, 14/37, 38%; SFRP5, 5/37, 14%) and liver cirrhosis (SFRP1, 10/28, 36%; SFRP2, 9/28, 32%; SFRP5, 3/28, 11%), suggesting that methylation of these genes is an early event in liver carcinogenesis. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcriptional activity in liver cancer cells, while overexpression of a beta-catenin mutant and depletion of SFRP1 using siRNA synergistically upregulated TCF/LEF transcriptional activity. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in HCC, and suggest that their loss of function contributes to activation of Wnt signaling during hepatocarcinogenesis. Show less

Although mutations of APC, CTNNB1 (beta-catenin) and AXIN1 are rare in oral squamous cell carcinoma (OSCC), activation of the Wnt signaling pathway is thought to play an important role in oral carcinogenesis. In the present study, we examined the relationship between Wnt signaling and epigenetic alteration of the secreted frizzled-related protein (SFRP) genes in OSCC. We frequently detected loss of membrane localization of beta-catenin and its cytoplasmic or nuclear accumulation in OSCC cell lines, although these cell lines showed no APC or CTNNB1 (beta-catenin) mutations and no methylation of CDH1 (E-cadherin). By contrast, we frequently detected methylation of SFRP1 (7/17, 41%) SFRP2 (16/17, 94%) and SFRP5 (14/17, 82%) in a panel of OSCC cell lines, as well as in specimens of primary tumors collected from 44 OSCC patients (SFRP1, 10/42, 24%; SFRP2, 16/44, 36%; SFRP5, 7/43, 16%). We also observed that OSCC cell lines express various Wnt ligands, and that ectopic expression of SFRPs inhibited cancer cell proliferation. Our results confirm the frequent methylation and silencing of SFRP genes in OSCC, and suggest that their loss of function contributes to activation of Wnt signaling that leads to cell proliferation during oral carcinogenesis. Show less

We have previously shown that MUPP1, which has an MRE domain and 13 PDZ domains, is expressed in epithelial cells and localize at tight junctions (TJs) and apical membranes. Using yeast two-hybrid screening, we found here that MUPP1 interacts with angiomotin (Amot), JEAP/Amot-like 1 and MASCOT/Amot-like 2, which we refer to as Amot/JEAP family proteins. PDZ2 and -3 were responsible for MUPP1's interaction with Amot and MASCOT, whereas only PDZ3 was responsible for its interaction with JEAP. All the Amot/JEAP family proteins also interacted with Patj, a close relative of MUPP1. The C-terminal PDZ-binding motives of the Amot/JEAP family were required for these interactions. We successfully generated specific antibodies for these proteins and analyzed the endogenous molecular properties of the family in parallel. Immunofluorescence microscopy of cultured epithelial cells showed that in subcellular distribution, the Amot/JEAP family proteins were indistinguishable; they were apparent at TJs as well as apical membranes, and mostly co-localized with MUPP1. They were also located at TJs in several mouse tissues, but each protein showed a distinct tissue distribution. In biochemical fractionation assays, the Amot/JEAP family behaved not as transmembrane but as peripheral membrane proteins. Unexpectedly, the PDZ-binding motives were not necessarily required for their localization to TJs, and dominant negative MUPP1 or Patj did not affect the localization of Amot/JEAP family proteins, suggesting that the interaction with MUPP1/Patj is not necessarily responsible for their proper subcellular distribution. Show less

The rare fragile site is a specific point on a chromosome that is expressed as an isochromatid gap or break under certain conditions of cell culture and is inherited in a Mendelian codominant fashion. Five folate-sensitive fragile sites were cloned, and the molecular basis of fragile site mutation was shown to be a new class of mutation, called dynamic mutation, resulting from an allelic expansion of (CCG)n repeats. The mechanism responsible for other types of rare fragile sites, i.e., distamycin A-inducible and BrdU-requiring, is unknown, although cytogenetic studies suggested that these fragile sites play a mechanistic role in breakage and recombination and may also be integration and modification sites of foreign viral DNA genomes. A distamycin A-inducible fragile site, FRA8E, is mapped to 8q24.1 in which various loci implicated in genomic instability are located. Here we identified a YAC clone spanning both FRA8E and the hereditary multiple exostosis (EXT1) gene, using fluorescence in situ hybridization (FISH) analysis of a yeast artificial chromosome (YAC) contig. By using P1 clones as probes, the FRA8E locus was further localized to a 400-kb region including the EXT1 gene. Furthermore, the integration and amplification site of human papillomavirus 16 DNA in the ASCC (argyrophil small cell carcinoma) cells were shown not to coincide with FRA8E, but to be involved in an extensively broad genomic region of 8q24.1, including the c-myc gene. Show less