Publications by authors named "Kenichiro Hata"

190 Publications

Frequent FGFR3 and Ras Gene Mutations in Skin Tags/Acrochordons.

J Invest Dermatol 2021 Apr 30. Epub 2021 Apr 30.

Department of Dermatology, Keio University School of Medicine, Tokyo 160-8582, Japan; Keio-Maruho Laboratory of Skin Barriology, Keio University School of Medicine, Tokyo 160-8582, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2021.03.028DOI Listing
April 2021

Comparison of efficacy between anti-IL-6 receptor antibody and other biological disease-modifying antirheumatic drugs in the patients with rheumatoid arthritis who have knee joint involvement: the ANSWER cohort, retrospective study.

Rheumatol Int 2021 Apr 26. Epub 2021 Apr 26.

Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan.

Objective: We aimed to investigate the efficacy of anti-IL-6 receptor antibody (aIL-6) and other biologic disease-modifying antirheumatic drugs (bDMARDs), such as TNF inhibitor and CTLA4-Ig in the treatment of rheumatoid arthritis (RA) in patients with knee joint involvement.

Methods: We retrospectively analyzed 1059 treatment courses of patients with RA who visited our hospitals and were treated with bDMARDs. We categorized them into two groups, with or without knee joint involvement. We investigated the clinical disease activity index (CDAI) at baseline and 12 weeks after the initiation of bDMARDs. We compared the improvement of the markers between aIL-6 and other bDMARDs.

Results: Treatment with aIL-6 significantly increased ΔCDAI (n = 91, 15.4 ± 1.1; mean ± SEM) in patients with knee joint involvement, compared to other bDMARDs (n = 232, 11.0 ± 0.7) at 12 weeks (P = 0.006). Following the multivariate analysis adjusted by the CDAI levels at baseline, age, gender, concomitant use of methotrexate, and the first use of bDMARDs, ΔCDAI levels were significantly higher in aIL-6, compared to other bDMARDs (P = 0.02). However, there was no significant difference in ΔCDAI improvement between aIL-6 (n = 162, 5.9 ± 0.6) and other bDMARDs (n = 573, 6.2 ± 0.4) in patients without swollen knee joints. ΔCDAI levels were equally increased in patients with shoulder and elbow joint involvement.

Conclusion: aIL-6 was more effective in the patients with RA and knee joint involvement, compared to other bDMARDs.
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http://dx.doi.org/10.1007/s00296-021-04862-yDOI Listing
April 2021

Comparison of therapeutic effects of combination therapy with prednisolone and tacrolimus or azathioprine on progressive interstitial pneumonia with systemic sclerosis.

Mod Rheumatol 2021 Apr 25:1-17. Epub 2021 Apr 25.

Division of Rheumatology, Department of Internal Medicine IV, Osaka Medical College, Osaka, Japan.

Objectives: We retrospectively compared the therapeutic effects of combination therapy with prednisolone (PSL) and oral tacrolimus (TAC) or azathioprine (AZA) on progressive interstitial pneumonia with systemic sclerosis (SSc-PIP).

Methods: The effects of PSL (0.2-0.5 mg/kg/day) and TAC (3 mg/day) or AZA (1-2 mg/kg/day) therapies (n = 18) were evaluated for short (12 months) and long (36 months or more) periods.

Results: In the short period, IP improved in 6 and 5 patients and was stable in 12 and 13 patients in the TAC and AZA groups, respectively. In the long period, 11 patients were followed up in the TAC group and 12 in the AZA group. IP improved in 4 and 2 patients and was stable in seven and nine in the TAC and AZA groups, respectively. The rates of evolution of total fibrosis score, and those corrected by disease duration for the long period, in the TAC group were significantly lower than those in the AZA group ( = 0.017 and 0.025, respectively).

Conclusions: The inhibitory effect of PSL and TAC combination therapy on the progression of fibrosis in SSc-PIP may be superior to that of PSL and AZA in the long period.
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http://dx.doi.org/10.1080/14397595.2021.1918864DOI Listing
April 2021

Intestinal outcome of bone marrow transplantation for monogenic inflammatory bowel disease.

Pediatr Int 2021 Apr 21. Epub 2021 Apr 21.

Center for Pediatric Inflammatory Bowel Disease, Division of Gastroenterology, National Center for Child Health and Development, Tokyo, Japan.

Background: Some monogenic inflammatory bowel diseases (IBDs) are known to be refractory to conventional treatments. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become a curative therapeutic option for certain monogenic IBDs, its effectiveness regarding endoscopic improvements has not been clarified.

Methods: The clinical course and endoscopic findings of patients with monogenic IBDs, who were treated with allo-HSCT between December 2017 and November 2018 at the National Center for Child Health and Development, were retrospectively reviewed. The clinical disease activity was assessed using the weighted Pediatric Crohn's Disease Activity Index (wPCDAI) and the endoscopic finding was evaluated using the Simple Endoscopic Score for Crohn's Disease (SES-CD). Clinical remission was defined as a wPCDAI <10 and endoscopic remission was defined as an SES-CD of 2 or less.

Results: Four patients with severe monogenic IBDs, including three with X-linked inhibitor of apoptosis protein (XIAP) deficiency and one with interleukin-10 signaling defect, were treated with allo-HSCT with reduced-intensity conditioning. In four patients, the maximum scores of wPCDAI and SES-CD before allo-HSCT ranged from 67.5 to 120 and 20 to 34, respectively. After allo-HSCT, all four patients showed a significant improvement in intestinal inflammation and achieved both clinical and endoscopic remission. Although patients with XIAP deficiency presented with post-transplant hemophagocytic lymphohistiocytosis and a relatively late engraftment, all patients achieved prolonged clinical remission, and IBD medications were successfully discontinued in all patients.

Conclusion: Allo-HSCT for monogenic IBD resulted in complete clinical resolution with endoscopically confirmed mucosal healing.
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http://dx.doi.org/10.1111/ped.14750DOI Listing
April 2021

Deleterious fibronectin type III-related gene variants may induce a spinal extradural arachnoid cyst: an exome sequencing study of identical twin cases.

Childs Nerv Syst 2021 Mar 27. Epub 2021 Mar 27.

Department of Neurosurgery, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-Ku, Tokyo, 157-8535, Japan.

Purpose: Despite numerous studies, the etiology of spinal extradural arachnoid cyst (SEDAC), a lesion associated with neurological symptoms, remains unknown. In this genomic twin study, we investigated the genetic etiology of SEDACs.

Methods: The subjects were identical twins who developed notably similar SEDACs at the same vertebral level. Accordingly, we performed whole-exome sequencing analyses of genomic material from the twins and their parents using a next-generation sequencer. Additionally, we determined their detailed family history and analyzed the family pedigree.

Results: The pedigree analysis suggested the potential presence of SEDACs in certain family members, indicating a genetic disease. Sequenced data were analyzed and filtered using a purpose-built algorithm, leading to the identification of 155 novel single-nucleotide polymorphisms (SNPs), of which 118 encoded missense or nonsense variants. A functional analysis of the proteins encoded by these SNP alleles revealed strong enrichment for the fibronectin type III (FN3) protein domain (q = 0.00576). Specifically, the data indicated that a missense variant affecting the FN3 protein domain of fibronectin 1 (FN1, p.P969S) can be the causal mutation underlying the SEDACs.

Conclusion: The data suggest that deleterious mutations in fibronectin-related genes may cause SEDACs. In particular, it was suspected that a variant of FN1 may be the cause of the SEDACs in the twin cases studied herein. Detailed studies with a larger number of cases are needed.
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http://dx.doi.org/10.1007/s00381-021-05137-4DOI Listing
March 2021

Maintenance of mouse trophoblast stem cells in KSR-based medium allows conventional 3D culture.

J Reprod Dev 2021 Mar 20. Epub 2021 Mar 20.

Department of Animal Resource Sciences/Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOut Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.
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http://dx.doi.org/10.1262/jrd.2020-119DOI Listing
March 2021

The addition of iguratimod can reduce methotrexate dose in rheumatoid arthritis with clinical remission.

Mod Rheumatol 2021 Mar 16:1-6. Epub 2021 Mar 16.

Department of Internal Medicine, Osaka Medical College, Osaka, Japan.

Objectives: We prospectively evaluated whether the addition of iguratimod (IGU) could sustain clinical remission in rheumatoid arthritis (RA) patients after tapering of methotrexate (MTX).

Methods: The study included 47 patients; 25 patients in the MTX maintenance group, and 22 patients in the IGU addition group who were treated with additional IGU and tapering of MTX dose. Clinical efficacy and safety were evaluated at 12, 24, and 36 weeks.

Results: In the IGU addition group, the dose of MTX could be reduced from 8.6 ± 2.4 mg/week at baseline to 4.7 ± 2.2 mg/week at 36 weeks ( < .001). Clinical remission was maintained (disease activity score [DAS]28-ESR 1.48 ± 0.63 at baseline and 1.69 ± 0.76 at 36 weeks,  = .911), and disease activity remained low (clinical disease activity index [CDAI] 2.4 ± 1.5 at baseline and 3.1 ± 3.4 at 36 weeks,  = .825). The US-GLOSS score significantly decreased from 9.2 ± 5.3 at baseline to 6.4 ± 4.3 at 36 weeks ( = .034). In the IGU addition group, two patients discontinued IGU because of stomatitis and three patients relapsed during the follow-up period (flare rate: 15.0%). There was no significant difference in RA disease activity at 36 weeks between the two groups.

Conclusion: Additional use of IGU can effectively reduce the MTX dose required by patients during clinical remission without inducing a flare.
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http://dx.doi.org/10.1080/14397595.2021.1892945DOI Listing
March 2021

Effects of denosumab on rheumatic diseases and refractory glucocorticoid-induced osteoporosis: a prospective study.

Arch Osteoporos 2021 02 23;16(1):39. Epub 2021 Feb 23.

Department of Internal Medicine (IV), Osaka Medical College, 2-7 Daigaku-Machi, Takatsuki, Osaka, 569-8686, Japan.

This study evaluates the clinical efficacy of denosumab for glucocorticoid-induced osteoporosis (GIOP) refractory to previous osteoporosis treatment. Our results show that denosumab significantly increased BMD of the lumbar spine and bilateral hip over the 24-month study period. Denosumab demonstrates potential as a treatment for GIOP refractory to previous therapy.

Introduction: The aim of this study was to evaluate the clinical efficacy and safety of denosumab in patients with rheumatic diseases and glucocorticoid-induced osteoporosis (GIOP) refractory to previous osteoporosis treatment.

Methods: All patients were treated with 60 mg of denosumab subcutaneously every 6 months for 2 years after administration of bisphosphonates or rhPTH was stopped. We assessed bone mineral density (BMD) of the lumbar spine and bilateral hip at baseline, and at 6, 12, 18, and 24 months. We measured serum levels of bone alkaline acid phosphatase (BAP) and tartrate-resistant acid phosphatase (TRACP)-5b at baseline, and at 3, 6, 12, 18, and 24 months.

Results: Fifty-five patients with rheumatic diseases and GIOP were enrolled in this study. All patients were treated with bisphosphonates (n=40), recombinant human parathyroid hormone (n=4), or active vitamin D (n=11). Over the 24-month study period, denosumab significantly increased the mean BMD of the lumbar spine and bilateral hip (5.8 ± 0.7%, and 1.3 ± 0.4%, respectively). Additionally, denosumab also significantly reduced the serum levels of TRACP-5b and BAP over this same period (by -38.8 ± 3.5% and -16.3 ± 3.1%, respectively), although these changes in bone turnover markers were not predictive factors of an improvement in BMD values. While three patients developed fragility fractures during the study period, all three had several risk factors for fragility fractures in GIOP.

Conclusions: In conclusion, denosumab is a potential treatment for GIOP in rheumatic diseases, especially in patients refractory to previous therapy, including bisphosphonate therapy.
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http://dx.doi.org/10.1007/s11657-021-00899-5DOI Listing
February 2021

Identification of epigenetic memory candidates associated with gestational age at birth through analysis of methylome and transcriptional data.

Sci Rep 2021 Feb 9;11(1):3381. Epub 2021 Feb 9.

Department of Pediatrics, The University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Preterm birth is known to be associated with chronic disease risk in adulthood whereby epigenetic memory may play a mechanistic role in disease susceptibility. Gestational age (GA) is the most important prognostic factor for preterm infants, and numerous DNA methylation alterations associated with GA have been revealed by epigenome-wide association studies. However, in human preterm infants, whether the methylation changes relate to transcription in the fetal state and persist after birth remains to be elucidated. Here, we identified 461 transcripts associated with GA (range 23-41 weeks) and 2093 candidate CpG sites for GA-involved epigenetic memory through analysis of methylome (110 cord blood and 47 postnatal blood) and transcriptional data (55 cord blood). Moreover, we discovered the trends of chromatin state, such as polycomb-binding, among these candidate sites. Fifty-four memory candidate sites showed correlation between methylation and transcription, and the representative corresponding gene was UCN, which encodes urocortin.
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http://dx.doi.org/10.1038/s41598-021-83016-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873311PMC
February 2021

CCL2 Produced by CD68+/CD163+ Macrophages as a Promising Clinical Biomarker of Microscopic Polyanigiitis-Interstitial Lung Disease.

Rheumatology (Oxford) 2021 Jan 25. Epub 2021 Jan 25.

Department of Internal Medicine (IV), Osaka Medical College, Takatsuki, Osaka, Japan.

Objectives: Microscopic polyangiitis (MPA) is often complicated by interstitial lung disease (ILD); however, biomarkers that can be used to diagnose and predict the progression of MPA-ILD have not been identified. In this study we evaluated various serum biomarkers in MPA-ILD to assess their diagnostic and predictive performance.

Methods: We enrolled 49 patients with anti-neutrophil cytoplasmic antibody (ANCA)+ MPA and 10 healthy controls, with 32 of the MPA patients also presented ILD. The presence of ILD was assessed by high-resolution computed tomography and evaluated by ground-glass opacity and fibrosis score. We compared 16 biomarker profiles among MPA-ILD patients, those without ILD, and healthy controls and extracted biomarkers with higher levels in MPA-ILD groups to determine correlations with disease activity and other biomarkers. Three lung biopsies were examined by hematoxylin-eosin staining and immunostaining.

Results: Initial serum C-C motif chemokine ligand 2 (CCL2) levels were significantly higher in the MPA-ILD group than those of the MPA group, and were significantly higher in MPA-ILD patients 1 year after immunosuppressive therapy than those before treatment. Initial serum CCL2 levels positively correlated with an increased fibrosis score during the year after treatment and with initial serum platelet-derived growth factor levels. Immunohistochemical staining showed intense CCL2 signals in CD68+/CD163+ macrophages and metaplastic epithelial cells in MPA-ILD lungs.

Conclusion: CCL2 is associated with MPA-ILD pathogenesis and suggested its potential efficacy as a useful marker for diagnosing and predicting MPA-ILD progression. Therefore, targeting CCL2 in alveolar CD68+/CD163+ macrophages might represent a therapeutic intervention in ANCA+ MPA-ILD.
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http://dx.doi.org/10.1093/rheumatology/keab064DOI Listing
January 2021

Evaluation of poor prognostic factors of respiratory related death in microscopic polyangiitis complicated by interstitial lung disease.

Sci Rep 2021 Jan 15;11(1):1490. Epub 2021 Jan 15.

Department of Internal Medicine (IV), Osaka Medical College, Daigaku-Machi 2-7, Takatsuki, Osaka, 569-8686, Japan.

The prognosis of microscopic polyangiitis (MPA) with interstitial lung disease (ILD) is significantly worse than that of MPA without ILD. However, the clinical characteristics in MPA-ILD, especially poor prognostic factors, are not elucidated. We evaluated demographic, clinical, laboratory, and radiological findings, treatments, and outcomes of 80 patients with MPA, and investigated prognostic factors of respiratory-related death in patients with myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA) positive MPA-ILD. Ground-glass opacity and fibrosis were evaluated as scores on high-resolution computed tomography (HRCT). The presence of ILD was consistent with a high risk of respiratory-related death (hazard ratio, 4.8; P = 0.04). Multivariable logistic regression analyses using propensity scoring showed right or left lower lobe fibrosis score to be significantly associated with respiratory-related death (P = 0.0005 and 0.0045, respectively). A right or left lower lobe fibrosis score ≥ 2, indicating the presence of honeycombing at 1 cm above the diaphragm, was determined to be the best cut-off value indicating a poor prognosis. The 5-year survival rate was significantly lower in patients with right or left lower lobe fibrosis score ≥ 2 (survival rates: 37% and 19%, respectively) than those with a score < 2 (71% and 68%, respectively) (P = 0.002 and 0.0007, respectively). These findings suggest that the presence of honeycomb lesions in bilateral lower lobes on chest HRCT was associated with respiratory-related death in patients with MPO-ANCA positive MPA-ILD.
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http://dx.doi.org/10.1038/s41598-021-81311-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810976PMC
January 2021

Novel TNFAIP3 microdeletion in a girl with infantile-onset inflammatory bowel disease complicated by a severe perianal lesion.

Hum Genome Var 2021 Jan 14;8(1). Epub 2021 Jan 14.

Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, 157-8535, Japan.

A20 haploinsufficiency (HA20), a disease caused by loss-of-function TNFAIP3 mutations, manifests various autoinflammatory and/or autoimmune symptoms. Some cases of HA20 were initially diagnosed as very early onset inflammatory bowel disease (VEO-IBD). We performed whole-exome sequencing (WES) for a Japanese girl with infantile-onset IBD and a severe perianal lesion and detected a novel de novo 119 kb microdeletion containing only TNFAIP3 (arr[GRCh37] 6q23.3(138125829_138244816) × 1).
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http://dx.doi.org/10.1038/s41439-020-00128-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809258PMC
January 2021

Gestational arsenic exposure induces site-specific DNA hypomethylation in active retrotransposon subfamilies in offspring sperm in mice.

Epigenetics Chromatin 2020 12 2;13(1):53. Epub 2020 Dec 2.

Department of Maternal-Fetal Biology, National Center for Child Health and Development, Tokyo, 157-8535, Japan.

Background: Environmental impacts on a fetus can disrupt germ cell development leading to epimutations in mature germ cells. Paternal inheritance of adverse health effects through sperm epigenomes, including DNA methylomes, has been recognized in human and animal studies. However, the impacts of gestational exposure to a variety of environmental factors on the germ cell epigenomes are not fully investigated. Arsenic, a naturally occurring contaminant, is one of the most concerning environmental chemicals, that is causing serious health problems, including an increase in cancer, in highly contaminated areas worldwide. We previously showed that gestational arsenic exposure of pregnant C3H mice paternally induces hepatic tumor increase in the second generation (F2). In the present study, we have investigated the F1 sperm DNA methylomes genome-widely by one-base resolution analysis using a reduced representation bisulfite sequencing (RRBS) method.

Results: We have clarified that gestational arsenic exposure increases hypomethylated cytosines in all the chromosomes and they are significantly overrepresented in the retrotransposon LINEs and LTRs, predominantly in the intergenic regions. Closer analyses of detailed annotated DNA sequences showed that hypomethylated cytosines are especially accumulated in the promoter regions of the active full-length L1MdA subfamily in LINEs, and 5'LTRs of the active IAPE subfamily in LTRs. This is the first report that has identified the specific positions of methylomes altered in the retrotransposon elements by environmental exposure, by genome-wide methylome analysis.

Conclusion: Lowered DNA methylation potentially enhances L1MdA retrotransposition and cryptic promoter activity of 5'LTR for coding genes and non-coding RNAs. The present study has illuminated the environmental impacts on sperm DNA methylome establishment that can lead to augmented retrotransposon activities in germ cells and can cause harmful effects in the following generation.
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http://dx.doi.org/10.1186/s13072-020-00375-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709384PMC
December 2020

Premature aging syndrome showing random chromosome number instabilities with CDC20 mutation.

Aging Cell 2020 11 23;19(11):e13251. Epub 2020 Oct 23.

Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.
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http://dx.doi.org/10.1111/acel.13251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681047PMC
November 2020

Genome-wide methylation analysis in Silver-Russell syndrome, Temple syndrome, and Prader-Willi syndrome.

Clin Epigenetics 2020 10 22;12(1):159. Epub 2020 Oct 22.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan.

Background: Imprinting disorders (IDs) show overlapping phenotypes, particularly in Silver-Russell syndrome (SRS), Temple syndrome (TS14), and Prader-Willi syndrome (PWS). These three IDs include fetal and postnatal growth failure, feeding difficulty, and muscular hypotonia as major clinical features. However, the mechanism that causes overlapping phenotypes has not been clarified. To investigate the presence or absence of methylation signatures associated with overlapping phenotypes, we performed genome-wide methylation analysis (GWMA).

Results: GWMA was carried out on 36 patients with three IDs (SRS [n = 16], TS14 [n = 7], PWS [n = 13]) and 11 child controls using HumanMethylation450 BeadChip including 475,000 CpG sites across the human genome. To reveal an aberrantly methylated region shared by SRS, TS14, and PWS groups, we compared genome-wide methylation data of the three groups with those of control subjects. All the identified regions were known as SRS-, TS14-, and PWS-related imprinting-associated differentially methylated regions (iDMRs), and there was no hypermethylated or hypomethylated region shared by different ID groups. To examine the methylation pattern shared by SRS, TS14, and PWS groups, we performed clustering analysis based on GWMA data. The result focusing on 620 probes at the 62 known iDMRs (except for SRS-, TS14-, and PWS-related iDMRs) classified patients into two categories: (1) category A, grossly normal methylation patterns mainly consisting of SRS group patients; and (2) category B, broad and mild hypermethylation patterns mainly consisting of TS14 and PWS group patients. However, we found no obvious relationship between these methylation patterns and phenotypes of patients.

Conclusions: GWMA in three IDs found no methylation signatures shared by SRS, TS14, and PWS groups. Although clustering analysis showed similar mild hypermethylation patterns in TS14 and PWS groups, further study is needed to clarify the effect of methylation patterns on the overlapping phenotypes.
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http://dx.doi.org/10.1186/s13148-020-00949-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583213PMC
October 2020

Comprehensive genetic analysis of pediatric germ cell tumors identifies potential drug targets.

Commun Biol 2020 09 30;3(1):544. Epub 2020 Sep 30.

Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

To elucidate the molecular pathogenesis of pediatric germ cell tumors (GCTs), we performed DNA methylation array analysis, whole transcriptome sequencing, targeted capture sequencing, and single-nucleotide polymorphism array analysis using 51 GCT samples (25 female, 26 male), including 6 germinomas, 2 embryonal carcinomas, 4 immature teratomas, 3 mature teratomas, 30 yolk sac tumors, and 6 mixed germ cell tumors. Among the 51 samples, 11 were from infants, 23 were from young children, and 17 were from those aged ≥10 years. Sixteen of the 51 samples developed in the extragonadal regions. Germinomas showed upregulation of pluripotent genes and global hypomethylation. Pluripotent genes were also highly expressed in embryonal carcinomas. These genes may play essential roles in embryonal carcinomas given that their binding sites are hypomethylated. Yolk sac tumors exhibited overexpression of endodermal genes, such as GATA6 and FOXA2, the binding sites of which were hypomethylated. Interestingly, infant yolk sac tumors had different DNA methylation patterns from those observed in older children. Teratomas had higher expression of ectodermal genes, suggesting a tridermal nature. Based on our results, we suggest that KIT, TNFRSF8, and ERBB4 may be suitable targets for the treatment of germinoma, embryonal carcinomas, and yolk sac tumors, respectively.
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http://dx.doi.org/10.1038/s42003-020-01267-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528104PMC
September 2020

Simplified disease activity index and clinical disease activity index before and during pregnancy correlate with those at postpartum in patients with rheumatoid arthritis.

Mod Rheumatol 2020 Oct 20:1-8. Epub 2020 Oct 20.

Division of Rheumatology, Department of Internal Medicine, Osaka Medical College, Osaka, Japan.

Objectives: We explored rheumatoid arthritis (RA) disease activity before, during, and after pregnancy in patients treated with tight control and investigated the association between disease activity in the postpartum period and those before and during pregnancy.

Methods: We retrospectively reviewed disease activity and medications of 27 patients before pregnancy, at every trimester, and in the postpartum period.

Results: Prednisolone was administered to 33% of patients with a median dose of 0 (0-2.5) mg/day and biologic agents was 78% in the third trimester. The median remission rates during all periods were the Disease Activity Score-28-C-reactive Protein assessed with three variables (DAS28-CRP-3) 85%, Simplified Disease Activity Index (SDAI) 55%, and Clinical Disease Activity Index (CDAI) 54%. Although SDAI and CDAI decreased significantly from before pregnancy to the first trimester and increased from the third trimester to the postpartum period, DAS28-CRP-3 did not change during all periods. Although SDAI and CDAI before and during pregnancy were significantly correlated with those in the postpartum period, DAS28-CRP-3 was not.

Conclusions: Tight control before pregnancy suppressed RA disease activity during pregnancy and in the postpartum period. SDAI/CDAI before and during pregnancy were predictive for disease activity in the postpartum period.
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http://dx.doi.org/10.1080/14397595.2020.1829342DOI Listing
October 2020

Integrated multiomics analysis of hepatoblastoma unravels its heterogeneity and provides novel druggable targets.

NPJ Precis Oncol 2020 7;4:20. Epub 2020 Jul 7.

Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Although hepatoblastoma is the most common pediatric liver cancer, its genetic heterogeneity and therapeutic targets are not well elucidated. Therefore, we conducted a multiomics analysis, including mutatome, DNA methylome, and transcriptome analyses, of 59 hepatoblastoma samples. Based on DNA methylation patterns, hepatoblastoma was classified into three clusters exhibiting remarkable correlation with clinical, histological, and genetic features. Cluster F was largely composed of cases with fetal histology and good outcomes, whereas clusters E1 and E2 corresponded primarily to embryonal/combined histology and poor outcomes. E1 and E2, albeit distinguishable by different patient age distributions, were genetically characterized by hypermethylation of the HNF4A/CEBPA-binding regions, fetal liver-like expression patterns, upregulation of the cell cycle pathway, and overexpression of and . Inhibition of and in hepatoblastoma cells induced chemosensitization and growth suppression, respectively. Our results provide a comprehensive description of the molecular basis of hepatoblastoma and rational therapeutic strategies for high-risk cases.
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http://dx.doi.org/10.1038/s41698-020-0125-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341754PMC
July 2020

Monochorionic diamniotic twins of discordant external genitalia with 45,X/46,XY mosaicism.

Mol Genet Genomic Med 2020 09 25;8(9):e1382. Epub 2020 Jun 25.

Center for Maternal-Fetal-Neonatal and Reproductive Medicine, National Center for Child Health and Development, Setagaya-ku, Tokyo, Japan.

Background: Monozygotic twins with 45,X/46,XY mosaicism, discordant for phenotypic sex, are extremely rare.

Methods: This report describes the clinical findings of a rare case of 45,X/46,XY mosaicism in monozygotic twins with different external genitalia. Single nucleotide polymorphism (SNP) array analysis, performed by collecting DNA from each umbilical cord, showed identical SNPs in the autosomal chromosomes of both fetuses.

Results: Chorionic villus sampling of a 37-year-old primigravida carrying monozygotic twins revealed a 45,X/46,XY karyotype. Autopsy of the aborted fetuses revealed a penis and testes on one fetus and a vagina, uterus, and ovaries in the other fetus--which also had severe cystic hygroma. Cell counting using fluorescence in situ hybridization with XY probes (XY-FISH) showed 20% and 80% abundance of 45,X cells in the internal genitalia, liver, heart, lung, adrenal gland, bone marrow, and spine of the male and female fetuses, respectively.

Conclusion: These results indicated that the fetuses were genetically monozygotic twins and their different degrees of mosaicism may have resulted in different genital phenotypes.
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http://dx.doi.org/10.1002/mgg3.1382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503087PMC
September 2020

Identification of distinct loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development.

Nat Commun 2020 06 24;11(1):3199. Epub 2020 Jun 24.

Division of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan.

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.
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http://dx.doi.org/10.1038/s41467-020-16989-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314859PMC
June 2020

Loss of imprinting of the human-specific imprinted gene causes prenatal growth retardation and dysmorphic features: implications for phenotypic overlap with Silver-Russell syndrome.

J Med Genet 2020 Jun 23. Epub 2020 Jun 23.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

Background: , encoding a zinc-finger protein, is the human-specific maternally expressed imprinted gene located on 16p13.3. The parent-of-origin expression of is regulated by the :TSS-DMR, of which only the paternal allele acquires methylation during postimplantation period. Overexpression of may contribute to some of the phenotypes associated with maternal uniparental disomy of chromosome 16 (UPD(16)mat), and some patients with UPD(16)mat presenting with Silver-Russell syndrome (SRS) phenotype have recently been reported.

Methods: A 6-year-old boy presented with prenatal growth restriction, macrocephaly at birth, forehead protrusion in infancy and clinodactyly of the fifth finger. Methylation, expression, microsatellite marker, single nucleotide polymorphism array and trio whole-exome sequencing analyses were conducted.

Results: Isolated hypomethylation of the :TSS-DMR and subsequent loss of imprinting and overexpression of were confirmed in the patient. Epigenetic alterations, such as UPD including UPD(16)mat and other methylation defects, were excluded. Pathogenic sequence or copy number variants affecting his phenotypes were not identified, indicating that primary epimutation occurred postzygotically.

Conclusion: We report the first case of isolated imprinting defect, showing phenotypic overlap with SRS despite not satisfying the clinical SRS criteria. A novel imprinting disorder entity involving the imprinted domain can be speculated.
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http://dx.doi.org/10.1136/jmedgenet-2020-107019DOI Listing
June 2020

X-linked inhibitor of apoptosis protein deficiency complicated with Crohn's disease-like enterocolitis and Takayasu arteritis: A case report.

Clin Immunol 2020 08 12;217:108495. Epub 2020 Jun 12.

Center for Pediatric Inflammatory Bowel Disease, Division of Gastroenterology, National Center for Child Health and Development, Tokyo, Japan.

X-linked inhibitor of apoptosis protein (XIAP) deficiency results in monogenic inflammatory bowel disease. To date, no vasculitis associated with XIAP deficiency has been reported. A 10-year-old boy was diagnosed with Crohn's disease and he responded poorly to conventional treatment for Crohn's disease. He was dependent on corticosteroids and parenteral nutrition. To manage severe colitis, he underwent ileostomy followed by ileocolectomy for an ileo-sigmoid fistula. At the age of 15 years, he developed IgA vasculitis and at the age of 17 years, he developed refractory Takayasu arteritis (TAK), which was resistant to corticosteroid and immunosuppressive therapy. Whole-exome sequencing revealed a novel mutation of the splice acceptor site in XIAP (c.1057-1G > A) at the age of 19 years. Allogeneic hematopoietic stem cell transplantation was successful with subsequent withdrawal of intensive immunosuppressive therapy and clinical remission of both enterocolitis and TAK. This case suggests that patients with XIAP deficiency could develop intractable inflammatory disease involving the intestinal tract and blood vessels.
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http://dx.doi.org/10.1016/j.clim.2020.108495DOI Listing
August 2020

Direct Assessment of Single-Cell DNA Using Crudely Purified Live Cells: A Proof of Concept for Noninvasive Prenatal Definitive Diagnosis.

J Mol Diagn 2020 02;22(2):132-140

Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan. Electronic address:

Noninvasive testing techniques are often used for fetal diagnosis of genetic abnormalities but are limited by certain characteristics, including noninformative results. Thus, novel methods of noninvasive definitive diagnosis of fetal genetic abnormalities are needed. The aim of this study was to develop a single-cell DNA analysis method with high sensitivity and specificity that enables direct extraction of genetic information from live fetal cells in a crude mixture for simultaneous evaluation. Genomic DNA from circulating fetal CD45CD14 cells, an extremely rare cell type, extracted from 10-mL samples of maternal peripheral blood, was extracted using a single-cell-based droplet digital (sc-dd) PCR system with a modified amount of polymerase. A hexachloro-6-carboxyfluorescein-labeled RPP30 probe was used as an internal control and a 6-carboxyfluorescein-labeled SRY probe as a target. The results indicated that no droplets generated with samples from pregnant women carrying female fetuses were positive for both probe signals, whereas droplets prepared with samples from pregnant women carrying male fetuses were positive for both probe signals. The latter was considered a direct assessment of genetic information from single circulating male fetal cells. Thus, the modified sc-ddPCR system allows the detection of genetic information from rare target cells in a crudely purified cell population. This research also serves as a proof of concept for noninvasive prenatal definitive diagnosis.
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http://dx.doi.org/10.1016/j.jmoldx.2019.10.006DOI Listing
February 2020

Epitranscriptomic profiling in human placenta: N6-methyladenosine modification at the 5'-untranslated region is related to fetal growth and preeclampsia.

FASEB J 2020 01 25;34(1):494-512. Epub 2019 Nov 25.

Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan.

Intracellular mRNA levels are not always proportional to their respective protein levels, especially in the placenta. This discrepancy may be attributed to various factors including post-transcriptional regulation, such as mRNA methylation (N6-methyladenosine: mA). Here, we conducted a comprehensive mA analysis of human placental tissue from neonates with various birth weights to clarify the involvement of mA in placental biology. The augmented mA levels at the 5'-untranslated region (UTR) in mRNAs of small-for-date placenta samples were dominant compared to reduction of mA levels, whereas a decrease in mA in the vicinity of stop codons was common in heavy-for-date placenta samples. Notably, most of these genes showed similar expression levels between the different birth weight categories. In particular, preeclampsia placenta samples showed consistently upregulated SMPD1 protein levels and increased mA at 5'-UTR but did not show increased mRNA levels. Mutagenesis of adenosines at 5'-UTR of SMPD1 mRNAs actually decreased protein levels in luciferase assay. Collectively, our findings suggest that mA both at the 5'-UTR and in the vicinity of stop codon in placental mRNA may play important roles in fetal growth and disease.
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http://dx.doi.org/10.1096/fj.201900619RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027905PMC
January 2020

Two unrelated pedigrees with achondrogenesis type 1b carrying a Japan-specific pathogenic variant in SLC26A2.

Am J Med Genet A 2020 04 27;182(4):735-739. Epub 2019 Dec 27.

Department of Obstetrics, Hokkaido University, Sapporo, Hokkaido, Japan.

We present two unrelated Japanese pedigrees with achondrogenesis type 1b (ACG1B), characterized by prenatally lethal fetal hydrops and severe micromelia. The affected members in these pedigrees carried a common homozygous missense point mutation in solute carrier family 26 member 2 (SLC26A2), a gene associated with ACG1B (NM_000112:c.1987G>A). This loss-of-function point mutation causes substitution of glycine 663 with arginine in a highly conserved loop domain of SLC26A2. Interestingly, only a few cases of this mutation have been registered in Japanese genomic databases, and there are no reports of this mutation in any major genomic databases outside Japan. Furthermore, we confirmed the presence of a homozygous stretch of approximately 75 kb surrounding the pathogenic variant. Our findings suggest that this missense point mutation in SLC26A2, which is likely the cause of the ACG1B phenotypes in these unrelated fetuses, is distributed exclusively in Japan.
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http://dx.doi.org/10.1002/ajmg.a.61469DOI Listing
April 2020

Tetrasomy 21 pter→q21.3 due to an extra +dic(21;21)mat in a severely psychomotor-retarded female patient without Down syndrome phenotype.

Eur J Med Genet 2020 Apr 9;63(4):103824. Epub 2019 Dec 9.

Department of Maternal-Fetal Biology, Research Institute, National Center for Child Health and Development, Tokyo, Japan.

Complete or partial tetrasomy 21 has been reported only in rare cases. We report a Japanese female patient with tetrasomy 21 due to an extra chromosome derived from chromosome 21 (Chr21). The patient had severe psychomotor retardation without Down syndrome (DS) phenotype; she showed short stature, microcephaly, round face, cleft lip and palate, and other dysmorphic features. The chromosome analyses for the patient detected an extra dicentric Chr21 consisting of two partial Chr21 copies fused together within their long arms. Her karyotype was revealed to be 47,XX,+dic(21;21). Allelic ratios of heterozygous SNPs observed in the patient indicated the maternal origin of the extra Chr21. Copy number and structural variant analyses using whole genome sequencing data indicated that the distal breakpoint of the dicentric Chr21 was located within 21q21.3 and that the extra Chr21 did not simply consist of inverted duplications of the pter→q21.3 region, but likely contained multiple partial deletions, duplications, and inversions within it. Fluorescence in situ hybridization results were consistent with the karyotype and genomic analyses. The patient's lack of DS phenotype turned out to be due to the normal copy number of the DS critical region (21q22.13-22.3). A possible molecular mechanism leading to the complex genomic rearrangements in the tetrasomic region consists mainly of breakage-fusion-bridge cycles with an unequal crossing-over event.
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http://dx.doi.org/10.1016/j.ejmg.2019.103824DOI Listing
April 2020