Serum MYCN as a predictive biomarker of prognosis and therapeutic response in the prevention of hepatocellular carcinoma recurrence.

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment.

Metabolic Dysfunction-Associated Steatotic Liver Disease in Japan: Prevalence Trends and Clinical Background in the 10 Years before the Coronavirus Disease 2019 Pandemic.

Background and Objectives: The trends in metabolic dysfunction-associated steatotic liver disease (MASLD) and related metabolic dysfunctions in Japan are unknown. Thus, we aimed to clarify these trends before the novel coronavirus disease 2019 pandemic in Japan. Materials and Methods: We included Japanese individuals aged 25-79 years who underwent health examinations at our center. We analyzed anthropometry, lifestyle-related disease, and nutritional intake in relation to MASLD trends from 2010-2019. Results: The prevalence of MASLD increased in all ages and body mass index (BMI) classes, reaching 30.3% in males and 16.1% in females, with MASLD accounting for 75% of steatotic liver cases and more than half of all type 2 diabetes mellitus (T2DM) and high waist circumference (HWC) cases. The increase in the prevalence of MASLD was thought to be largely attributable to an increase in that of the incidence of steatotic liver itself, and there was no increase in the prevalence of other factors, such as overweight, T2DM, hypertension, and dyslipidemia. The prevalence of glucose metabolic disorders (GMDs) and hypertension decreased. National nutritional data showed an increase in energy intake, total fat, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, which correlated with a decrease in GMDs. Salt intake also decreased, which correlated with hypertension. The MASLD group had a higher prevalence of all related metabolic factors than the non-MASLD group, especially HWC, T2DM, and hyperlipidemia. Conclusions: The prevalence of MASLD increased with that of steatotic liver, regardless of age or BMI. A relationship between increased dietary fat, increased steatotic liver, and decreased GMDs was suggested.

Determination of a Serum 25-Hydroxyvitamin D Reference Ranges in Japanese Adults Using Fully Automated Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Despite an increasing interest in vitamin D status, a reference range of the nutrient has not been fully established. This is partly due to a paucity of standardized measuring systems with high throughput. In addition, the range may vary by populations and may change with modernization of lifestyles. OBJECTIVES: This study aims to calculate the current reference concentration of 25-hydroxyvitamin D (25(OH)D) among healthy people living in an urban area in Japan. METHODS: A newly developed fully automated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) system was used to measure serum 25(OH)D concentrations. Reproducibility was assessed by measuring standardized samples. Accuracy was validated by comparing with commercially available immunoassays. Then, mass screening was conducted targeting participants who received medical checkups in Tokyo from April 2019 to March 2020, and the reference ranges were calculated. RESULTS: The coefficients of variations of interoperator and interday reproducibility were 4.1%-8.5% and 3.7%-8.0% for 25-hydroxyvitamin D2 (25(OH)D2) and 4.7%-7.0% and 4.0%-6.9% for 25-hydroxyvitamine D3, respectively. The measured total 25(OH)D concentrations correlated well with those measured by immunoassays. In total, 5518 participants were measured for 25(OH)D concentrations, among whom 98% showed inadequate concentrations (<30 ng/mL). The reference ranges of total 25(OH)D for female, male, and total participants were 7-30 ng/mL, 5-27 ng/mL, and 6-29 ng/mL, respectively. After excluding those with abnormal renal and liver function, the range was 6-30 ng/mL. CONCLUSIONS: The high prevalence of vitamin D insufficiency among seemingly healthy population may be attributed to lifestyle characteristics of people living in urban areas of Japan, including spending less time outdoors and lower intake of traditional foods. Longitudinal follow-up and mass screenings targeting different population will help elucidate reasons for discrepancies between official guidelines and the observed concentrations, to which the well-validated measurement system is essential.

A mild form of POC1B-associated retinal dystrophy with relatively preserved cone system function.

PURPOSE: Biallelic variants in POC1B are rare causes of autosomal recessive cone dystrophy associated with generalized cone system dysfunction. In this report, we describe the clinical characteristics of a Japanese male patient with POC1B-associated retinopathy with relatively preserved cone system function. METHODS: We performed whole-exome sequencing (WES) to identify the disease-causing variants and a comprehensive ophthalmic examination, including full-field and multifocal electroretinography (ffERG and mfERG). RESULTS: Our WES analysis identified novel compound heterozygous POC1B variants (p.Arg106Gln and p.Arg452Ter) in the patient. His unaffected mother carried the p.Arg452Ter variant heterozygously. The patient experienced decreased visual acuity in his 50s. At the age of 63, his corrected visual acuity was 20/22 in the right and 20/20 in the left eye. Fundus and fundus autofluorescence images for each eye showed no remarkable finding, except for a subtle hyperautofluorescent spot in the fovea of the left eye. Cross-sectional optical coherence tomography demonstrated blurred but a relatively preserved ellipsoid zone. The ffERG showed that amplitudes of rod and standard-flash responses were within the reference range, whereas the cone and light-adapted 30-Hz flicker amplitudes were close to, or slightly below, the reference range. The mfERG revealed substantially reduced responses with relative preservation of central function. CONCLUSIONS: We reported the case of an older patient with POC1B-associated retinopathy, demonstrating late-onset visual decrease, good visual acuity, and relatively preserved cone system function. The disease condition was much milder than previously reported in patients with POC1B-associated retinopathy.

SEB genotyping: SmartAmp-Eprimer binary code genotyping for complex, highly variable targets applied to HBV.

BACKGROUND: SmartAmp-Eprimer Binary code (SEB) Genotyping is a novel isothermal amplification method for rapid genotyping of any variable target of interest. METHODS: After in silico alignment of a large number of sequences and computational analysis to determine the smallest number of regions to be targeted by SEB Genotyping, SmartAmp primer sets were designed to obtain a binary code of On/Off fluorescence signals, each code corresponding to a unique genotype. RESULTS: Applied to HBV, we selected 4 targets for which fluorescence amplification signals produce a specific binary code unique to each of the 8 main genotypes (A-H) found in patients worldwide. CONCLUSIONS: We present here the proof of concept of a new genotyping method specifically designed for complex and highly variable targets. Applied here to HBV, SEB Genotyping can be adapted to any other pathogen or disease carrying multiple known mutations. Using simple preparation steps, SEB Genotyping provides accurate results quickly and will enable physicians to choose the best adapted treatment for each of their patients.

Prostaglandin E-major urinary metabolite diagnoses mucosal healing in patients with ulcerative colitis in remission phase.

BACKGROUND AND AIM: Ulcerative colitis (UC) is usually detected by clinical symptoms, such as bleeding and diarrhea; however, it is rather difficult to assess during asymptomatic clinical remission (CR). Hence, there is a need for a biomarker that can reliably detect UC during remission. We previously reported on the utility of the prostaglandin E-major urinary metabolite (PGE-MUM) as a biomarker reflecting UC activity. In this study, we evaluated the effectiveness of the PGE-MUM in the diagnosis of endoscopic, histological, and histo-endoscopic mucosal remission of UC, comparing with fecal tests. METHODS: This prospective study was conducted at the Jikei University Hospital between August 2017 and January 2021. Patients with UC in CR scheduled to undergo colonoscopy were included. The association between the PGE-MUM with endoscopic remission (ER), histological remission (HR), and complete mucosal healing (CMH, defined as histo-endoscopic remission) was analyzed. We also compared the area under the curve (AUC) for the receiver operating characteristic curves between PGE-MUM, fecal calprotectin (FC), and fecal immunochemical test (FIT). RESULTS: In total, 128 patients were analyzed. PGE-MUM differed significantly in ER versus non-ER (14.5 vs 16.7, P = 0.028), HR versus non-HR (14.2 vs 17.4, P = 0.004), and CMH versus non-CMH (14.3 vs 16.7, P = 0.021). There were no significant differences between the AUCs for PGE-MUM, FC, and FIT for ER, HR, or CMH. CONCLUSIONS: The PGE-MUM can determine CMH in UC even during CR, regardless of the disease phenotype, indicating its clinical benefit for non-invasive monitoring.

Clinical characterization of autosomal dominant retinitis pigmentosa with NRL mutation in a three-generation Japanese family.

PURPOSE: Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal disorders. NRL-associated autosomal dominant (AD)-RP is a rare form of AD-RP in the Japanese population. This study aimed to report a clinical characterization of NRL-associated retinopathy in a three-generation Japanese family. CASE PRESENTATION: A total of 4 patients from a Japanese family were referred to The Jikei University School of Medicine for clinical and genetic examination. The patients included a male proband (41 years old), his daughters (5 and 6 years old), and his mother (71 years old); they underwent ophthalmic examinations, and genetic testing was performed using whole exome sequencing analysis, revealing a known variant [c.152C > T (p.Pro51Leu)] heterozygously in exon 2 of the NRL gene. Fundus photograph showed that retinal degeneration expanded to the macular and peripheral retina in an age-dependent manner. Fundus autofluorescence imaging showed hyper-autofluorescence (AF) within the macular with slightly hypo-AF in younger patients and obvious hypo-AF in older patients. Optical coherence tomography showed that the length of the ellipsoid zone tended to be longer in younger patients than in older patients. Goldmann perimetry showed an age-dependent decrease in the visual field. Furthermore, full-field electroretinographic findings revealed non-recordable rod and cone function in older patients and non-recordable rod function with preserved cone function in younger patients. CONCLUSIONS: Our results indicated that retinal construction and function were aggravated in an age-dependent manner, and retinal degeneration, especially in the macular region, revealed milder findings than in previous cases with NRL-associated AD-RP.

The second Japanese family with Malattia Leventinese/Doyne honeycomb retinal dystrophy.

PURPOSE: To describe the clinical and genetic findings of patients in the second Japanese family with Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD). METHODS: Two patients (a 41-year-old male proband and his third son) underwent comprehensive ophthalmic examinations, including full-field and multifocal electroretinography (ERG). Sanger sequencing was performed to detect an EFEMP1 gene variant (p.Arg345Trp), which was identified as the only causative pathogenic variant. RESULTS: Genetic analysis revealed that both patients carried the heterozygous variant, but the other unaffected family members did not. Although the proband exhibited innumerable radially distributed drusen in both the posterior poles and good visual acuity at initial presentation, bilateral choroidal neovascularization (CNV) developed during the 15-year follow-up. The proband received 15 intravitreal anti-vascular endothelial growth factor (VEGF) injections in the left eye (LE) and two injections in the right eye (RE). At 56 years of age, his decimal best-corrected visual acuity was 0.1 and 1.2 in the LE and RE, respectively. Full-field ERG showed that while the rod and combined responses were within normal amplitudes, the cone and 30-Hz flicker responses had slightly decreased amplitudes. Multifocal ERG revealed attenuated central responses in the LE and decreased temporal responses in the RE. In the 20-year-old son, multifocal ERG showed normal responses in both eyes. CONCLUSION: This is the first report of ML/DHRD in a patient who developed bilateral CNV and received anti-VEGF treatment in both eyes. Although multifocal ERG exhibited worsening of macular function, the generalized photoreceptor function was preserved until middle age.

Design, synthesis, and target identification of new hypoxia-inducible factor 1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety.

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure-activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC50 = 1.2 µM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic ß subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase suppression.

Progress of macular atrophy during 30 months' follow-up in a patient with spinocerebellar ataxia type1 (SCA1).

PURPOSE: To report the 30-months' course of macular dystrophy in a patient with genetically confirmed spinocerebellar ataxia type1 (SCA1). METHODS: Detailed ophthalmological examinations including best-corrected visual acuity (BCVA), perimetry, multimodal fundus imaging, and electrophysiological recordings were performed on a 52-year-old woman with SCA1. The number of CAG sequence repeats of the candidate gene was verified. RESULTS: The baseline decimal BCVA was 0.2 OD and 0.3 OS. Goldman perimetry showed relative central scotomas and slight enlargements of Mariotte blind spot bilaterally. Ophthalmoscopy revealed no abnormalities in the macula and optic disk. Fundus autofluorescence (FAF) showed a circular hyperautofluorescence and round-shaped hypoautofluorescence in the macula. Optical coherence tomography (OCT) showed a loss of the interdigitation zone and ellipsoid zone (EZ) in the macula. Full-field scotopic and photopic Full-field electroretinograms (ERGs) were normal, and multifocal ERGs were decreased in the central area. After 30 months, the BCVA had not changed, but the FAF showed a spark-like hypoautofluorescence in the macula. The abnormal area of the EZ had expanded toward the periphery, and the rate of EZ loss was 199.7%/year OD and 206.8%/year OS. Genetic examinations revealed an increase in the number of heterozygous CAG repeats in the ATXN1 gene, and the CAG repeat number of the mutant allele ranged from 43 to 48. CONCLUSIONS: The full-field scotopic and photopic ERGs were normal. The mfERGs were significantly smaller in the central region. OCT demonstrated bilateral photoreceptor atrophy in the macula, and the rate of EZ loss was more rapid than in other macular dystrophies. Spark-like hypoautofluorescence appeared during the course of the disease process which might be a specific feature of SCA1-related retinopathy.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor.

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.

Novel biallelic splice-site BBS1 variants in Bardet-Biedle syndrome: a case report of the first Japanese patient.

PURPOSE: To report the clinical and genetic features of a 9-year-old female Japanese patient with Bardet-Biedl syndrome (BBS). METHODS: Genetic analysis using whole-exome sequencing (WES) was performed for the patient and her parents to identify disease-causing variants. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to investigate the impact of splice-site variants. Comprehensive ophthalmic and systemic examinations, including electroretinography (ERG), were performed. RESULTS: In the patient, WES identified novel compound heterozygous splice-site variants (c.124+2T>G and c.723+2T>G) in the BBS1 gene, and RT-PCR revealed skipping of exons 2 and 8 (p.N17AfsX56 and p.T198_K241del). Each parent had one of the variants. Ophthalmologically, the patient's decimal best-corrected visual acuity was 0.6 in the right eye and 0.4 in the left eye. Funduscopy revealed no apparent retinal degeneration or narrowed blood vessels in the periphery, but macular abnormalities were found on fundus autofluorescence imaging and optical coherence tomography images. Unexpectedly, non-recordable responses in rod ERG were found, with a non-recordable response of the right eye and an extremely reduced and delayed a-wave of the left eye in standard ERG, non-recordable responses in cone ERG, and extremely decreased responses in 30 Hz flicker ERG. Finally, the patient fulfilled four primary features of BBS diagnostic criteria: rod-cone dystrophy, polydactyly, central obesity, and learning disabilities, being diagnosed with BBS. CONCLUSIONS: This is the first report of a BBS patient with biallelic splice-site BBS1 variants in the Japanese population. Disparity between funduscopic and ERG findings may be a feature of BBS1-associated rod-cone dystrophy.

The prevalence and antimicrobial susceptibility of Streptococcus pneumoniae isolated from patients at Jikei University Hospitals after the implementation of the pneumococcal vaccination program in Japan.

Studies have shown that pneumococcal vaccination reduces the incidence of Streptococcus pneumoniae infections but does not change the prevalence of S. pneumoniae nasopharyngeal colonization. To comprehensively and longitudinally assess the epidemiology of S. pneumoniae after the introduction of pneumococcal vaccination, we monitored the prevalence and antimicrobial susceptibility of S. pneumoniae, irrespective of its serotypes or pathogenicity, by analyzing specimens collected from a large number of patients at Jikei University Hospitals from 2009 to 2017. A total of 5763 S. pneumoniae isolates were identified out of 375,435 specimens from various sources of patients in different age groups. The prevalence of S. pneumoniae isolated only from patients <5 years old was significantly reduced with the widespread use of pneumococcal vaccines, although this reduction differed by areas where patients resided. The incidence of pneumococcal infections, including bacteremia and otitis media, clearly decreased among patients <5 years old after the introduction of pneumococcal vaccination, while the prevalence of S. pneumoniae isolated from blood specimens of patients 15-64 years old increased, suggesting the involvement of non-vaccine serotypes in the incidence of invasive pneumococcal infections. The antimicrobial susceptibility of S. pneumoniae improved after the introduction of pneumococcal vaccination. Our results show that pneumococcal vaccination has a suppressive effect on the prevalence of S. pneumoniae and the incidence of pneumococcal infections, at least for children <5 years old, in association with an improvement in the antimicrobial susceptibility of S. pneumoniae. However, further measures will be needed to control invasive pneumococcal infections caused by non-vaccine serotypes.

Autosomal dominant optic atrophy with OPA1 gene mutations accompanied by auditory neuropathy and other systemic complications in a Japanese cohort.

Purpose: This study aimed to describe the genetic and clinical characteristics of four Japanese patients with autosomal dominant optic atrophy (DOA) accompanied by auditory neuropathy and other systemic complications (i.e., DOA-plus disease). Methods: Four patients from four independent families underwent comprehensive ophthalmic and auditory examinations and were diagnosed with DOA-plus disease. The disease-causing gene variants in the OPA1 gene were identified by direct sequencing. The genetic and clinical data of 48 DOA patients without systemic complications-that is, with simple DOA-were compared to those of DOA-plus patients. Results: DOA-plus patients noticed a decrease in vision before the age of 14 and hearing impairment 3 to 13 years after the development of visual symptoms. Two patients had progressive external ophthalmoplegia, and one patient had vestibular dysfunction and ataxia. The DOA-plus phenotypes accounted for 13.3% (4/30) of the families with the OPA1 gene mutations. Each DOA-plus patient harbored one of the monoallelic mutations in the OPA1 gene: c.1334G>A, p.R445H, c.1618A>C, p.T540P, and c.892A>C, p.S298R. Missense mutations accounted for 100% (4/4) of the DOA-plus families and only 11.5% (3/26) of the families with simple DOA. Conclusions: All the patients with the DOA-plus phenotype carried one of the missense mutations in the OPA1 gene. They all had typical ocular symptoms and signs of DOA in their first or second decade, and other systemic complications-such as auditory neuropathy, vestibular dysfunction, and ataxia-followed the ocular symptoms. We should consider the occurrence of extraocular complications in cases with DOA, especially when they carry the missense mutations in the OPA1 gene.

Novel biallelic loss-of-function KCNV2 variants in cone dystrophy with supernormal rod responses.

PURPOSE: To report clinical and genetic features including long-term full-field electroretinography (FF-ERG) findings of a patient with cone dystrophy with supernormal rod responses (CDSRR). METHODS: Ophthalmological medical records including FF-ERG were retrospectively reviewed. Genetic analysis using whole-exome sequencing (WES) was performed. Identified KCNV2 variants were confirmed by Sanger sequencing. RESULTS: A 30-year-old female patient was referred to our hospital for assessment of decreased vision from childhood. Funduscopy showed macular atrophy in both eyes. FF-ERG showed decreased amplitudes and delayed peak time of b-waves for dark-adapted (DA) 0.01 ERG, increased b/a-wave ratio with a slightly diminished a-wave for DA 3.0 and DA 25.7 ERG, residual a-waves and almost extinguished b-waves for light-adapted (LA) 3.0 ERG, and extremely diminished amplitudes in LA 30-Hz flicker responses. At 45 years of age, funduscopy showed progressive macular atrophy, whereas the responses for her FF-ERG remained unchanged compared to those observed at 30 years of age. WES identified the compound heterozygous KCNV2 variants (p.W67X and p.D174GfsX198) in the patient. These variants have previously been unreported as pathogenic variants. Each parent had one of the variants. Subsequently, the patient was finally diagnosed with CDSRR with the novel compound heterozygous KCNV2 variants. CONCLUSIONS: Biallelic loss-of-function KCNV2 variants (p.W67X and p.D174GfsX198) were identified as the cause of CDSRR. Long-term FF-ERG findings demonstrated there were no ERG changes during 15 years of observation, indicating that there was no evidence of progressive peripheral retinal dysfunction, in spite of worsening macular atrophy.

Comparative assessment of sarcopenia using the JSH, AWGS, and EWGSOP2 criteria and the relationship between sarcopenia, osteoporosis, and osteosarcopenia in patients with liver cirrhosis.

BACKGROUND: Sarcopenia and osteoporosis reduce life quality and worsen prognosis in patients with liver cirrhosis (LC). When these two complications coexist, a diagnosis of osteosarcopenia is made. We aimed to investigate the actual situations of sarcopenia, osteoporosis, osteosarcopenia, and vertebral fracture, and to clarify the relationship among these events in patients with LC. METHODS: We describe a cross-sectional study of 142 patients with LC. Sarcopenia was defined according to the Japan Society of Hepatology (JSH) criteria, Asian Working Group for Sarcopenia (AWGS) criteria, and European Working Group on Sarcopenia in Older People (EWGSOP2) criteria. The skeletal muscle mass index (SMI) and handgrip strength were assessed using bioelectrical impedance analysis and a digital grip strength dynamometer, respectively. Bone mineral density (BMD) was measured using dual energy X-ray absorptiometry, and vertebral fracture was evaluated using spinal lateral X-rays. The severity of LC was assessed using the Child-Pugh classification. RESULTS: Among the 142 patients, the prevalence of sarcopenia was 33.8% (48/142) according to the JSH and AWGS criteria and 28.2% (40/142) according to the EWGSOP2 criteria. The number of patients with osteoporosis, osteosarcopenia, and vertebral fracture was 49 (34.5%), 31 (21.8%), and 41 (28.9%), respectively. Multivariate analysis revealed a close association between sarcopenia and osteoporosis. Osteoporosis was independently associated with sarcopenia [odds ratio (OR) = 3.923, P = 0.010]. Conversely, sarcopenia was independently associated with osteoporosis (OR = 5.722, P < 0.001). Vertebral fracture occurred most frequently in patients with osteosarcopenia (19/31; 61.3%) and least frequently in those without both sarcopenia and osteoporosis (12/76; 15.8%). The SMI and handgrip strength values were significantly correlated with the BMD of the lumbar spine (r = 0.55 and 0.51, respectively; P < 0.001 for both), femoral neck, (r = 0.67 and 0.62, respectively; P < 0.001 for both), and total hip (r = 0.67 and 0.61, respectively; P < 0.001 for both). CONCLUSIONS: Sarcopenia, osteoporosis, osteosarcopenia, and vertebral fracture were highly prevalent and closely associated with one another in patients with LC. Specifically, patients with osteosarcopenia had the highest risk of vertebral fractures. Early diagnosis of these complications is essential for treatment intervention.

Critical role of CREBH-mediated induction of transforming growth factor ß2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells.

Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up-regulation of transforming growth factor ß (TGF-ß), especially TGF-ß2, which is mediated by activation of liver-enriched transcription factor cAMP-responsive element-binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV-infected cells with hepatic stellate cells (HSCs). Compared to TGF-ß1, expression of TGF-ß2 mRNA was induced faster and to a higher level upon HCV infection. Serum TGF-ß2 levels in hepatitis C patients were higher compared to those in healthy individuals and were positively correlated with hepatic fibrosis stages F0-F2. TGF-ß2 promoter activity was decreased and increased, respectively, by silencing and overexpression of CREBH. CREBH recognition sites were identified in the TGF-ß2 promoter. CREBH binding to the promoter and its increase in cells expressing HCV Core-NS2 were shown by gel mobility shift and chromatin immunoprecipitation, respectively. The active form of CREBH was detectable in HCV-infected chimeric mice with human livers and cells expressing HCV proteins. Involvement of CREBH in HCV-induced fibrogenic response was further demonstrated in the CREBH null-mutant mouse model. Fibrogenic phenotypes were assessed using co-cultures of HCV-infected cells and HSCs. Expressions of fibrogenic factors and TGF-ß1 increasing in the co-cultures was prevented by TGF-ß2- or CREBH silencing. CONCLUSION: CREBH was identified as a key positive regulator of TGF-ß2 transcription in HCV-infected cells. TGF-ß2 released from infected cells potentially contributes to cross-induction of TGF-ß in an autocrine manner through its own signaling pathway, leading to an increase in fibrogenic responses in adjacent HSCs. (Hepatology 2017;66:1430-1443).

Early onset flecked retinal dystrophy associated with new compound heterozygous RPE65 variants.

Purpose: To report genetic and clinical features of two unrelated Japanese patients with early onset flecked retinal dystrophy. Methods: Patients underwent comprehensive ophthalmic examinations that included electroretinography (ERG) after 30 min and 24 h of dark adaptation (DA). Disease-causing gene variants were identified with whole exome sequencing (WES), with identified candidates confirmed with direct sequencing. Results: WES identified compound heterozygous RPE65 variants in both patients. Variants in patient 1 included c.1543C>T (p.R515W) and c.683A>C (p.Q228P), while patient 2 exhibited c.1028T>A (p.L343*) and c.683A>C (p.Q228P). Although variants p.R515W and p.L343* have been previously reported as pathogenic, variant p.Q228P was reported as uncertain significance. Each unaffected parent carried the variant heterozygously. Both patients had similar ophthalmic findings, including decreased visual acuity with early onset night blindness, numerous dense white dots/flecks occurring mainly outside the vascular arcades, a diffuse and/or disrupted ellipsoid line as shown with optical coherence tomography, and non-recordable rod and combined responses along with decreased cone responses after 30 min of DA. After 24 h of DA, both patients exhibited marked or partial recovery of the combined responses. Conclusions: The results indicate that the recovery of combined or residual cone responses might be associated with a mild form of RPE65-related early onset flecked retinal dystrophy with new compound heterozygous variants.