Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

Mastocytosis in a Case of Noonan Syndrome Caused by a De Novo Pathogenic CBL Variant.

Noonan syndrome is an autosomal dominant disease characterized by multi-organ disorders caused by variants of genes involved in the RAS/MAPK signaling pathway. The nine causative genes including PTPN11 and CBL have been identified. Mastocytosis is a disease characterized by mast cell proliferation in skin, bone marrow, and other organs. To date, no previous cases of Noonan syndrome with mastocytosis caused by a pathogenic CBL variant have been reported. A boy was diagnosed with Noonan syndrome at 8 months of age with facial features and minor anomaly of his body. He presented with brown nodules of 5-10 mm on his body since the age of 2 months. The patient was diagnosed with mastocytosis by a biopsy specimen from brown nodules, which showed infiltration of mast cells. Whole-exome sequencing of the parent-patient trio revealed a de novo pathogenic CBL variant. The occurrence of mastocytosis may be a cue for the analysis of the CBL gene in Noonan syndrome. The CBL gene is involved in mastocytosis and various cancers. In the case of the pathogenic variant, long-term follow-up for the risk of cancers related to the CBL variant is necessary.

Association of IL-4 with pachychoroid neovasculopathy.

The purpose of this study was to identify the inflammatory cytokines that were associated with pachychoroid neovasculopathy (PNV). Seventy-five eyes of 75 patients with PNV, 145 eyes of 145 patients with neovascular age-related macular degeneration without pachyvessels, and 150 eyes of 150 normal subjects were examined for the levels of intraocular cytokines. In eyes with PNV, the levels of IL-1α, IL-1ß, IL-2, IL-4, IL-10, and VEGF were significantly higher than that of the controls. Logistic regression analysis showed that the highest association with the pachyvessels was found for IL-4, IL-2, and IL-1α. In eyes with PNV, the levels of IL-4, IL-2, IL-5, IL-13, IL-1α, and IL-1ß were significantly higher in eyes with both increased choroidal thickness and choroidal vessel diameter. The strongest correlation with the choroidal thickness and vessel diameter was observed for IL-4. In PNV eyes with polypoidal lesions, the levels of IL-4, IL-17, and TNFß were significantly correlated with the number of polypoidal lesions. Of these cytokines, IL-4 was especially associated with the thickness of the choroidal vessels and the formation of polypoidal lesions. We conclude that IL-4 is most likely involved in establishing the clinical characteristics of PNV and polypoidal vascular remodeling.

CSF P-Tau181 and Other Biomarkers in Patients With Neuronal Intranuclear Inclusion Disease.

BACKGROUND AND OBJECTIVES: CSF tau phosphorylated at threonine 181 (p-tau181) is a widely used biomarker for Alzheimer disease (AD) and has recently been regarded to reflect ß-amyloid and/or p-tau deposition in the AD brain. Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by intranuclear inclusions in neurons, glial cells, and other somatic cells. Symptoms include dementia, neuropathy, and others. CSF biomarkers were not reported. The objective of this study was to investigate whether CSF biomarkers including p-tau181 are altered in patients with NIID. METHODS: This was a retrospective observational study. CSF concentrations of p-tau181, total tau, amyloid-beta 1-42 (Aß42), monoamine metabolites homovanillic acid (HVA), and 5-hydroxyindole acetic acid (5-HIAA) were compared between 12 patients with NIID, 120 patients with Alzheimer clinical syndrome biologically confirmed based on CSF biomarker profiles, and patients clinically diagnosed with other neurocognitive disorders (dementia with Lewy bodies [DLB], 24; frontotemporal dementia [FTD], 13; progressive supranuclear palsy [PSP], 21; and corticobasal syndrome [CBS], 13). Amyloid PET using Pittsburgh compound B (PiB) was performed in 6 patients with NIID. RESULTS: The mean age of patients with NIID, AD, DLB, FTD, PSP, and CBS was 71.3, 74.6, 76.8, 70.2, 75.5, and 71.9 years, respectively. CSF p-tau181 was significantly higher in NIID (72.7 ± 24.8 pg/mL) compared with DLB, PSP, and CBS and was comparable between NIID and AD. CSF p-tau181 was above the cutoff value (50.0 pg/mL) in 11 of 12 patients with NIID (91.7%). Within these patients, only 2 patients showed decreased CSF Aß42, and these patients showed negative or mild local accumulation in PiB PET, respectively. PiB PET scans were negative in the remaining 4 patients tested. The proportion of patients with increased CSF p-tau181 and normal Aß42 (A-T+) was significantly higher in NIID (75%) compared with DLB, PSP, and CBS (4.2%, 4.8%, and 7.7%, respectively). CSF HVA and 5-HIAA concentrations were significantly higher in patients with NIID compared with disease controls. DISCUSSION: CSF p-tau181 was increased in patients with NIID without amyloid accumulation. Although the deposition of p-tau has not been reported in NIID brains, the molecular mechanism of tau phosphorylation or secretion of p-tau may be altered in NIID.

Clinical course of a Japanese patient with developmental delay linked to a small 6q16.1 deletion.

There is only one report of patients with developmental delay due to a 6q16.1 deletion that does not contain the SIM1 gene. A 3-year-old female showed strabismus, cleft soft palate, hypotonia at birth, and global developmental delay. Exome sequencing detected a de novo 6q16.1 deletion (chr6: 99282717-100062596) (hg19). The following genes were included in this region: POU3F2, FBXL4, FAXC, COQ3, PNISR, USP45, TSTD3, CCNC, and PRDM13.

Clinical course of epilepsy and white matter abnormality linked to a novel DYRK1A variant.

Epilepsy and white matter abnormality have been reported in DYRK1A-related intellectual disability syndrome; however, the clinical course has yet to be elucidated. Here, we report the clinical course of an 18-year-old male with a novel heterozygous DYRK1A variant (NM_001396.4: c.957C>G, p.Tyr319*); based on previous reports, epilepsy with this syndrome tends to be well controlled. Follow-up MRIs of the patient's lesion revealed slightly reduced signal intensity compared to the first image.

Online questionnaire on genetic testing for intractable diseases in Japan: response to and issues associated with the revised medical care act.

In Japan, most genetic testing for intractable diseases has been conducted in research laboratories in the past. However, since the Revised Medical Care Act came into effect on December 1, 2018, genetic testing in compliance with this act has become a major issue. To collect information on this topic, we conducted an online survey of members of the research groups for intractable diseases, which play a central role in medical care and research on intractable diseases with the support of the Ministry of Health, Labor and Welfare, five months after the enactment of the act. We separated the surveyed facilities into those that conducted genetic testing in their own laboratories ("testing facilities") and those that outsourced genetic testing ("outsourcing facilities"). Ninety-five and 66 responses regarding genetic testing were obtained from the testing and outsourcing facilities, respectively. Genetic analysis was the most commonly conducted genetic testing method, accounting for 60% or more of the tests. At the testing facilities that conducted comprehensive analysis with a next-generation sequencer, the number of target diseases for genetic testing was observed to be higher. In these testing facilities, more than 70% were research laboratories. In contrast, at the outsourcing facilities, testing was outsourced to registered clinical laboratories in many cases or to research laboratories. The proportion of genetic testing covered by public medical insurance at the outsourcing facilities was two times higher than that at the testing facilities. The importance of quality control for genetic testing was generally well acknowledged, but there was apprehension regarding the increased cost and burden on staff of quality control assurance, and many testing facilities viewed genetic testing as difficult. The research groups could handle the examination and interpretation of the genetic testing results, and many groups gathered and registered patient information. Within the intractable disease medical support network, there was a relatively large number of collaborations, with studies supported by the Japan Agency for Medical Research and Development (AMED) and Initiative on Rare and Undiagnosed Diseases (IRUD) projects. There were many requests for genetic testing to be covered by public medical insurance. In the future, the implementation of genetic testing using a next-generation sequencer at clinical laboratories with guaranteed quality control and the development of a system for collaboration with research groups will be necessary.

Clinical Characteristics of Fragile X Syndrome Patients in Japan.

BACKGROUND: Fragile X syndrome (FXS) is a well-known X-linked disorder clinically characterized by intellectual disability and autistic features. However, diagnosed Japanese FXS cases have been fewer than expected, and clinical features of Japanese FXS patients remain unknown. METHODS: We evaluated the clinical features of Japanese FXS patients using the results of a questionnaire-based survey. RESULTS: We presented the characteristics of seven patients aged 6 to 20 years. Long face and large ears were observed in five of seven patients. Macrocephaly was observed in four of five patients. The meaningful word was first seen at a certain time point between 18 and 72 months (median = 60 months). Developmental quotient or intellectual quotient ranged between 20 and 48 (median = 29). Behavioral disorders were seen in all patients (autistic spectrum disorder in six patients, hyperactivity in five patients). Five patients were diagnosed by polymerase chain reaction analysis, and two patients were diagnosed by the cytogenetic study. All physicians ordered FXS genetic testing for suspicious cases because of clinical manifestations. CONCLUSION: In the present study, a long face, large ears, macrocephaly, autistic spectrum disorder, and hyperactivity were observed in almost cases, and these characteristics might be common features in Japanese FXS patients. Our finding indicated the importance of clinical manifestations to diagnosis FXS. However, the sample size of the present study is small, and these features are also seen to patients with other disorders. We consider that genetic testing for FXS should be performed on a wider range of intellectually disabled cases.

Cord Blood from SGA Preterm Infants Exhibits Increased GLUT4 mRNA Expression.

BACKGROUND: Insulin and insulin-like growth factor (IGF) signaling plays an important role in prenatal and postnatal growth and glucose metabolism. Both small-for-gestational age (SGA) and preterm infants have abnormal growth and glucose metabolism. However, the underlying mechanism remains unknown. Recently, we showed that term SGA infants have abnormal insulin/IGF signaling in cord blood. In this study, we examined whether preterm infants show similar aberrations in cord blood insulin/IGF signaling. METHODS: A total of 41 preterm cord blood samples were collected. Blood glucose, insulin, IGF-1, and C-peptide concentrations were measured, and mRNA expression of IGF1R, INSR, IRS1, IRS2, and SLC2A4 (i.e., GLUT4) was analyzed by quantitative reverse-transcription PCR. RESULTS: This study included 34 appropriate-for-gestational age (AGA) and 7 SGA preterm neonates. No hyperinsulinemia or any differences in IGF1R or INSR mRNA expression were detected between the two groups. However, GLUT4 mRNA levels were increased in preterm SGA. Moreover, the expression level in hypoglycemic preterm SGA was significantly higher than that in hypoglycemic preterm AGA. IRS2 mRNA expression did not show a statistically significant difference between preterm SGA and AGA neonates. CONCLUSION: SGA preterm birth does not induce hyperinsulinemia; however, it modifies insulin/IGF signaling components such as GLUT4 in umbilical cord blood. Our study suggests that prematurity or adaptation to malnutrition alters the insulin/IGF signaling pathway.

CGG repeat RNA G-quadruplexes interact with FMRpolyG to cause neuronal dysfunction in fragile X-related tremor/ataxia syndrome.

Fragile X-related tremor/ataxia syndrome (FXTAS) is a neurodegenerative disease caused by CGG triplet repeat expansions in FMR1, which elicit repeat-associated non-AUG (RAN) translation and produce the toxic protein FMRpolyG. We show that FMRpolyG interacts with pathogenic CGG repeat-derived RNA G-quadruplexes (CGG-G4RNA), propagates cell to cell, and induces neuronal dysfunction. The FMRpolyG polyglycine domain has a prion-like property, preferentially binding to CGG-G4RNA. Treatment with 5-aminolevulinic acid, which is metabolized to protoporphyrin IX, inhibited RAN translation of FMRpolyG and CGG-G4RNA-induced FMRpolyG aggregation, ameliorating aberrant synaptic plasticity and behavior in FXTAS model mice. Thus, we present a novel therapeutic strategy to target G4RNA prionoids.

A case of infantile Tay-Sachs disease with late onset spasms.

BACKGROUND: Epilepsy is known to be associated with Tay-Sachs disease (TSD); however, no detailed reports are available. This case report aimed to present the clinical features of late onset spasms (LOS) in a patient with infantile TSD, and to elucidate the pathophysiology leading to LOS, using proton magnetic resonance spectroscopy (MRS). CASE PRESENTATION: At 11 months old, our patient had an afebrile seizure. At 14 months, he showed developmental stagnation and an increase in the frequency of epileptic seizures. Magnetic resonance imaging (T2-weighted images) showed high signal intensities in the thalamus bilaterally, and in the head of the caudate nucleus. Serum ß-hexosaminidase enzyme activity was reduced, and he was diagnosed with TSD with a homozygous pathogenic variant of the HEXA gene (c. 571-1 G > T [IVS5, -1 G > T]), confirmed using direct sequence analysis. At 20 months, epileptic spasms in series around times of drowsiness and waking were observed on long-term video-electroencephalogram monitoring, in which ictal findings were different from those of startle seizures and non-epileptic myoclonus. Therefore, the epilepsy was classified as LOS. Epileptic spasms stopped following adrenocorticotropic hormone therapy, after which his vitality and consciousness improved. Serial MRS results showed a progressive decline in N-acetyl aspartate, and an increase in myoinositol in the grey matter over time. DISCUSSION AND CONCLUSION: Our patient's MRS results suggested that cortical and subcortical axonal and neuronal degeneration with widespread gliosis in the cerebrum might lead to the development of LOS, and that LOS might be underestimated in patients with TSD.

Gallbladder cancer with ascites in a child with metachromatic leukodystrophy.

INTRODUCTION: Metachromatic leukodystrophy (MLD) refers to leukodystrophy caused by the accumulation of sulfatide from arylsulfatase A (ARSA) gene mutations. Sulfatide also accumulates in various organs, including the peripheral nerves, kidney, and gallbladder. Proliferative changes in the gallbladder have been reported in several patients, while gallbladder cancer is reported in only two adult MLD cases. We report what is likely the first pediatric case of MLD with gallbladder cancer. CASE REPORT: The patient was a 5-year-old girl diagnosed with MLD using head magnetic resonance imaging and detecting a homozygous mutation of c.302G>A (p.Gly101Asp) in ARSA. Abdominal bloating was observed at the age of 4 years; CT revealed a giant tumor in the gallbladder and massive ascites. Cholecystectomy was performed and pathological examination revealed adenocarcinoma. Measurement of serum sulfatide revealed increased levels compared to the average healthy range. DISCUSSION: Rapidly increased ascites and large polyps which are reported as risk factors for cancer were characteristic in our MLD case. When such lesions are detected, they should be removed immediately because of the possibility of cancer, even in a pediatric patient.

Establishment of FXS-A9 panel with a single human X chromosome from fragile X syndrome-associated individual.

Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5' untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.

Isolated cortical tuber in an infant with genetically confirmed tuberous sclerosis complex 1 presenting with symptomatic West syndrome.

Tuberous sclerosis complex (TSC) is an autosomal dominant hereditary disorder caused by mutations in either TSC1 on chromosome 16 or TSC2 on chromosome 9, clinically characterized mainly by facial angiofibroma, epilepsy, and intellectual disability. Cortical dysplasias, subependymal nodules, and subependymal giant cell astrocytoma are characteristic central nervous system lesions among 11 major features in the current clinical diagnostic criteria for TSC. We encountered an unusual case of genetically confirmed TSC1 presenting with symptomatic West syndrome due to an isolated cortical dysplasia in the left occipital lobe of a six-month-old male infant who did not meet the clinical diagnostic criteria for TSC. The patient underwent left occipital lesionectomy at age 11 months and has been seizure-free for nearly six years since then. Histological examination of the resection specimen revealed cortical neuronal dyslamination with abundant dysmorphic neurons and ballooned cells, consistent with focal cortical dysplasia (FCD) type IIb. However, the lesion was also accompanied by unusual features, including marked calcifications, dense fibrillary gliosis containing abundant Rosenthal fibers, CD34-positive glial cells with abundant long processes confined to the dysplastic cortex, and multiple nodular lesions occupying the underlying white matter, consisting exclusively of ballooned cell and/or balloon-like astrocytes with focal calcifications. Genetic testing for TSC1 and TSC2 using the patient's peripheral blood revealed a germline heterozygous mutation in exon 7 (NM_000368.5: c.526dupT, p.Tyr176fs) in TSC1. Isolated FCD with unusual features such as calcification, dense fibrillary gliosis, Rosenthal fibers and/or subependymal nodule-like lesions in the white matter may indicate the possibility of a cortical tuber even without a clinical diagnosis of TSC. Identification of such histopathological findings has significant implications for early and accurate diagnosis and treatment of TSC, and is likely to serve as an important supplementary feature for the current clinical diagnostic criteria for TSC.

Duchenne muscular dystrophy-like phenotype in an LGMD2I patient with novel FKRP gene variants.

A 32-year-old man initially received a diagnosis of Duchenne muscular dystrophy (DMD). Genetic analysis revealed two novel heterozygous FKRP variants: c.169G>A (p.Glu57Lys) and c.692G>A (p.Trp231*). These results indicated that the patient had limb-girdle muscular dystrophy type 2I (LGMD2I) caused by recessive FKRP variants. Patients with LGMD2I and DMD have many overlapping phenotypes. LGMD2I should be considered in patients who have a DMD phenotype but not a DMD pathogenic variant.

An autopsy case of GM1 gangliosidosis type II in a patient who survived a long duration with artificial respiratory support.

GM1 gangliosidosis is a storage disorder with autosomal recessive inheritance caused by deficiency of ß-galactosidase (GLB1), which is a lysosomal hydrolase, due to mutations in GLB1. We describe here an autopsy case of GM1 gangliosidosis in a female patient who survived for 38 years with a long period of artificial respiratory support (ARS). She was born after a normal pregnancy and delivery. Although development was normal until one year old, she was unable to walk at two years old and started having seizures by nine years old. At 21 years old, she became unable to communicate and was bed-ridden. At 36 years old, she suffered from pneumonia and required ARS. She died of pneumonia at 40 years old. Neuropathological examination revealed severe atrophy, predominantly found in the frontal lobes. Microscopically, severe gliosis and neuronal loss were observed in the cerebral cortex, putamen, cerebellum, the latter including Purkinje cell and granule cell layers. The hippocampus was relatively preserved. Severe neuronal swelling was observed in the limbic regions and stored a material in these neurons negative for periodic acid-Schiff (PAS). A PAS-positive granular storage material in neurons and macrophages was mainly observed in the brainstem and limbic regions. Exome analysis showed a known c.152T>C (p.I51T) variant that has been described in type III patients and a novel c.1348-2A>G variant in GLB1. Detailed analysis of reverse transcription-polymerase chain reaction products of GLB1 mRNA revealed that these variants were present in a compound heterozygous state. In our case, clinical features and neuropathological findings were most consistent with type II, although the entire course was longer than any previously reported cases. This may be explained by the residual enzyme activity in this patient whose severity lay between types II and III. Our finding of relative preservation of the limbic regions suggests that neuronal loss in GM1 gangliosidosis has regional selectivity.

[Successful treatment with enzyme replacement therapy for pelvic fragile fracture in an elderly case of type I Gaucher's disease].

A 76-year-old male with lower-limb weakness was admitted to our hospital where thrombocytopenia and anemia were noticed. CT showed massive splenomegaly and multiple nodules inside the spleen. Bone marrow examination showed an increase of macrophages with large cytoplasm. Suspected of splenic lymphoma, the patient underwent splenectomy. Spleen specimens were histologically analyzed and suggested the probability of Gaucher's disease (GD). Leukocyte glucocerebrosidase (GBA) enzyme activity had decreased to 1.25 nmol/mg, and mutation analysis of GBA revealed two missense variants, p.D448H (D409H), p.L483P (L444P), which confirmed the diagnosis of type I GD. Fourteen months after splenectomy, he developed right buttock pain, and pelvic magnetic resonance imaging showed a fragile right pubic and pelvic fracture. We initiated injection of imiglucerase as enzyme replacement therapy (ERT) and administered bisphosphonate. His symptoms gradually improved without surgical treatment. In addition, thrombocytopenia and anemia also improved, and angiotensin-converting enzyme levels decreased. Type I GD should be considered a differential diagnosis of giant splenomegaly and thrombocytopenia, even in the elderly. ERT or substrate reduction therapy should be administrated to GD patients, while paying attention to the development of bone lesions.

Comparison of Causative Variant Prioritization Tools Using Next-generation Sequencing Data in Japanese Patients with Mendelian Disorders.

BACKGROUND: During the investigation of causative variants of Mendelian disorders using next-generation sequencing, the enormous number of possible candidates makes the detection process complex, and the use of multidimensional methods is required. Although the utility of several variant prioritization tools has been reported, their effectiveness in Japanese patients remains largely unknown. METHODS: We selected 5 free variant prioritization tools (PhenIX, hiPHIVE, Phen-Gen, eXtasy-order statistics, and eXtasy-combined max) and assessed their effectiveness in Japanese patient populations. To compare these tools, we conducted 2 studies: one based on simulated data of 100 diseases and another based on the exome data of 20 in-house patients with Mendelian disorders. To this end we selected 100 pathogenic variants from the "Database of Pathogenic Variants (DPV)" and created 100 variant call format (VCF) files that each had pathogenic variants based on reference human genome data from the 1000 Genomes Project. The later "in-house" study used exome data from 20 Japanese patients with Mendelian disorders. In both studies, we utilized 1-5 terms of "Human Phenotype Ontology" as clinical information. RESULTS: In our analysis based on simulated disease data, the detection rate of the top 10 causative variants was 91% for hiPHIVE, and 88% for PhenIX, based on 100 sets of simulated disease VCF data. Also, both software packages detected 82% of the top 1 causative variants. When we used data from our in-house patients instead, we found that these two programs (PhenIX and hiPHIVE) produced higher detection rates than the other three systems in our study. The detection rate of the top 1 causative variant was 71.4% for PhenIX, 65.0% for hiPHIVE. CONCLUSION: The rates of detecting causative variants in two Exomizer software packages, hiPHIVE and PhenIX, were higher than for the other three software systems we analyzed, with respect to Japanese patients.

First isolation and characterization of vancomycin-resistant Enterococcus faecium harboring vanD5 gene cluster recovered from a 79-year-old female inpatient in Japan.

This study reports the first isolation and characterization of a vanD5 genotype vancomycin-resistant Enterococcus faecium strain (E. faecium IPHb306) recovered from a 79-year-old Japanese female inpatient. Species identification was determined by biochemical testing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and species-specific PCR. Susceptibility tests indicated that E. faecium IPHb306 was resistant to vancomycin but susceptible to teicoplanin. Southern hybridization analyses indicated that E. faecium IPHb306 harbored a vanD5 gene cluster on chromosomal DNA. Growth curve analyses showed that a vancomycin resistance phenotype could be inducible. Sequencing analyses of the vanD5 gene cluster and the ddlE. faecium gene demonstrated several point mutations were present. Because this strain belongs to ST203, a major hospital-adapted lineage, spread of the vanD5 genotype E. faecium ST203 is considered a clinical threat in Japan.