Publications by authors named "Kazuhiko Nakabayashi"

176 Publications

Growth Suppression of Cancer Spheroids With Mutated KRAS by Low-toxicity Compounds from Natural Products.

Anticancer Res 2021 Aug;41(8):4061-4070

Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan

Background/aim: Among compounds from natural products selectively suppressing the growth of cancer spheroids, which have mutant (mt) KRAS, NP910 was selected and its derivatives explored.

Materials And Methods: The area of HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were measured in three-dimensional floating (3DF) cultures treated with 18 NP910 derivatives. The 50% cell growth inhibition (GI50) was determined by long-term 3DF (LT3DF) culture and nude mice assay.

Results: We selected NP882 (named STAR3) as the most effective inhibitor of growth of HKe3-mtKRAS spheroids with the least toxicity among NP910 derivatives. GI50s of STAR3 in LT3DF and nude mice assay were 6 μM and 30.75 mg/kg, respectively. However, growth suppression by STAR3 was observed in 50% of cell lines independent of KRAS mutation, suggesting that the target of STAR3 was not directly associated with KRAS mutation and KRAS-related signals.

Conclusion: STAR3 is a low-toxicity compound that inhibits growth of certain tumour cells.
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http://dx.doi.org/10.21873/anticanres.15207DOI Listing
August 2021

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

Reprod Med Biol 2021 Jul 14;20(3):352-360. Epub 2021 Jun 14.

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

Purpose: Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT.

Methods: Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing.

Results: The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA.

Conclusions: We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.
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http://dx.doi.org/10.1002/rmb2.12392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254165PMC
July 2021

Characteristics of genetic alterations of peripheral T-cell lymphoma in childhood including identification of novel fusion genes: the Japan Children's Cancer Group (JCCG).

Br J Haematol 2021 Jul 13. Epub 2021 Jul 13.

Department of Hematology/Oncology for Children and Adolescents, Sapporo Hokuyu Hospital, Hokkaido, Japan.

Peripheral T-cell lymphoma (PTCL) is a group of heterogeneous non-Hodgkin lymphomas showing a mature T-cell or natural killer cell phenotype, but its molecular abnormalities in paediatric patients remain unclear. By employing next-generation sequencing and multiplex ligation-dependent probe amplification of tumour samples from 26 patients, we identified somatic alterations in paediatric PTCL including Epstein-Barr virus (EBV)-negative (EBV ) and EBV-positive (EBV ) patients. As recurrent mutational targets for PTCL, we identified several previously unreported genes, including TNS1, ZFHX3, LRP2, NCOA2 and HOXA1, as well as genes previously reported in adult patients, e.g. TET2, CDKN2A, STAT3 and TP53. However, for other reported mutations, VAV1-related abnormalities were absent and mutations of NRAS, GATA3 and JAK3 showed a low frequency in our cohort. Concerning the association of EBV infection, two novel fusion genes: STAG2-AFF2 and ITPR2-FSTL4, and deletion and alteration of CDKN2A/2B, LMO1 and HOXA1 were identified in EBV PTCL, but not in EBV PTCL. Conversely, alterations of PCDHGA4, ADAR, CUL9 and TP53 were identified only in EBV PTCL. Our observations suggest a clear difference in the molecular mechanism of onset between paediatric and adult PTCL and a difference in the characteristics of genetic alterations between EBV and EBV paediatric PTCL.
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http://dx.doi.org/10.1111/bjh.17639DOI Listing
July 2021

Methylation status of genes escaping from X-chromosome inactivation in patients with X-chromosome rearrangements.

Clin Epigenetics 2021 Jun 30;13(1):134. Epub 2021 Jun 30.

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

Background: X-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes. However, about 15% of genes on the inactivated X chromosome (Xi) escape from XCI. The methylation level of the promoter region of the escape gene is lower than that of the inactivated genes. Dxz4 and/or Firre have critical roles for forming the three-dimensional (3D) structure of Xi. In mice, disrupting the 3D structure of Xi by deleting both Dxz4 and Firre genes led to changing of the escape genes list. To estimate the impact for escape genes by X-chromosome rearrangements, including DXZ4 and FIRRE, we examined the methylation status of escape gene promoters in patients with various X-chromosome rearrangements.

Results: To detect the breakpoints, we first performed array-based comparative genomic hybridization and whole-genome sequencing in four patients with X-chromosome rearrangements. Subsequently, we conducted array-based methylation analysis and reduced representation bisulfite sequencing in the four patients with X-chromosome rearrangements and controls. Of genes reported as escape genes by gene expression analysis using human hybrid cells in a previous study, 32 genes showed hypomethylation of the promoter region in both male controls and female controls. Three patients with X-chromosome rearrangements had no escape genes with abnormal methylation of the promoter region. One of four patients with the most complicated rearrangements exhibited abnormal methylation in three escape genes. Furthermore, in the patient with the deletion of the FIRRE gene and the duplication of DXZ4, most escape genes remained hypomethylated.

Conclusion: X-chromosome rearrangements are unlikely to affect the methylation status of the promoter regions of escape genes, except for a specific case with highly complex rearrangements, including the deletion of the FIRRE gene and the duplication of DXZ4.
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http://dx.doi.org/10.1186/s13148-021-01121-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244138PMC
June 2021

ZNF445: a homozygous truncating variant in a patient with Temple syndrome and multilocus imprinting disturbance.

Clin Epigenetics 2021 May 26;13(1):119. Epub 2021 May 26.

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

Background: ZNF445, as well as ZFP57, is involved in the postfertilization methylation maintenance of multiple imprinting-associated differentially methylated regions (iDMRs). Thus, ZNF445 pathogenic variants are predicted to cause multilocus imprinting disturbances (MLIDs), as do ZFP57 pathogenic variants. In particular, the MEG3/DLK1:IG-DMR would be affected, because the postzygotic methylation imprint of the MEG3/DLK1:IG-DMR is maintained primarily by ZNF445, whereas that of most iDMRs is preserved by both ZFP57 and ZNF445 or primarily by ZFP57.

Results: We searched for a ZNF445 variant(s) in six patients with various imprinting disorders (IDs) caused by epimutations and MLIDs revealed by pyrosequencing for nine iDMRs, without a selection for the original IDs. Re-analysis of the previously obtained whole exome sequencing data identified a homozygous ZNF445 variant (NM_181489.6:c.2803C>T:p.(Gln935*)) producing a truncated protein missing two of 14 zinc finger domains in a patient with Temple syndrome and MLID. In this patient, array-based genomewide methylation analysis revealed severe hypomethylation of most CpGs at the MEG3:TSS-DMR, moderate hypomethylation of roughly two-thirds of CpGs at the H19/IGF2:IG-DMR, and mild-to-moderate hypomethylation of a few CpGs at the DIRAS3:TSS-DMR, MEST:alt-TSS-DMR, IGF2:Ex9-DMR, IGF2:alt-TSS, and GNAS-AS1:TSS-DMR. Furthermore, bisulfite sequencing analysis for the MEG3/DLK1:IG-DMR delineated a markedly hypomethylated segment (CG-A). The heterozygous parents were clinically normal and had virtually no aberrant methylation pattern.

Conclusions: We identified a ZNF445 pathogenic variant for the first time. Since ZNF445 binds to the MEG3/DLK1:IG-DMR and other iDMRs affected in this patient, the development of Temple syndrome and MLID would primarily be explained by the ZNF445 variant. Furthermore, CG-A may be the target site for ZNF445 within the MEG3/DLK1:IG-DMR.
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http://dx.doi.org/10.1186/s13148-021-01106-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157728PMC
May 2021

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

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 Jul 27;37(7):2329-2334. 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
July 2021

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

J Reprod Dev 2021 Jun 20;67(3):197-205. 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238679PMC
June 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

Primary hypertrophic osteoarthropathy with severe arthralgia identified by gene mutation of .

Mod Rheumatol Case Rep 2021 Jul 18;5(2):404-408. Epub 2021 Jan 18.

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

Male, 41 years old (yo) had been complaining of severe arthralgia. Past History indicated obstruction of intestinal tract at 12 yo and gastric ulcer at 13 yo. He had been suffered from polyarthralgia especially at PIP and MP joints of both hands from 38 yo. Finally, he complained severe arthralgia at PIP and MP joints with clubbed fingers without swelling. Biochemical finding indicated negative rheumatoid factor and anti-CCP antibody and normal MMP-3 level, but slightly increased CRP and ESR levels. Radiological finding indicated periostosis of long bone without bone erosion and osteoporosis. His facial appearance was acromegalic with cutaneous manifestation of pachydermia and cutis vertices gyrate without abnormal growth hormone response. Histological findings of skin indicated oedema and hyperplasia of sebaceous glands with infiltration of lymphocytes around small blood vessels compatible with pachydermoperiostosis. In this case mutation of SLCO2A1 gene, which coded prostaglandin transport protein, was identified. The mutation c.940 + 1G > A of gene results in deletion of exon 7 and truncation of PG transporter (p.Arg288Glyfs*7). We suggest that severe arthralgia was originated from over production of prostaglandin E2. Further studies will be required.
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http://dx.doi.org/10.1080/24725625.2020.1861744DOI Listing
July 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

Genomic and Transcriptomic Analyses of Bioluminescence Genes in the Enope Squid Watasenia scintillans.

Mar Biotechnol (NY) 2020 Dec 24;22(6):760-771. Epub 2020 Oct 24.

Department of Genomics and Evolutionary Biology, National Institute of Genetics, Mishima, Japan.

Watasenia scintillans, a sparkling enope squid, has bioluminescence organs to illuminate its body with its own luciferase activity. To clarify the molecular mechanism underlying its scintillation, we analysed high-throughput sequencing data acquired previously and obtained draft genome sequences accomplished with comparative genomic data among the cephalopods. The genome mapped by transcriptome data showed that (1) RNA editing contributed to transcriptome variation of lineage specific genes, such as W. scintillans luciferase, and (2) two types of luciferase enzymes were characterized with reasonable 3D models docked to a luciferin molecule. We report two different types of luciferase in one organism and possibly related to variety of colour types in the W. scintillans fluorescent organs.
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http://dx.doi.org/10.1007/s10126-020-10001-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708342PMC
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

ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B-H4K8ac-BRD4 axis.

Nucleic Acids Res 2020 11;48(19):10848-10866

Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

Centromeres are genomic regions essential for faithful chromosome segregation. Transcription of noncoding RNA (ncRNA) at centromeres is important for their formation and functions. Here, we report the molecular mechanism by which the transcriptional regulator ZFAT controls the centromeric ncRNA transcription in human and mouse cells. Chromatin immunoprecipitation with high-throughput sequencing analysis shows that ZFAT binds to centromere regions at every chromosome. We find a specific 8-bp DNA sequence for the ZFAT-binding motif that is highly conserved and widely distributed at whole centromere regions of every chromosome. Overexpression of ZFAT increases the centromeric ncRNA levels at specific chromosomes, whereas its silencing reduces them, indicating crucial roles of ZFAT in centromeric transcription. Overexpression of ZFAT increases the centromeric levels of both the histone acetyltransferase KAT2B and the acetylation at the lysine 8 in histone H4 (H4K8ac). siRNA-mediated knockdown of KAT2B inhibits the overexpressed ZFAT-induced increase in centromeric H4K8ac levels, suggesting that ZFAT recruits KAT2B to centromeres to induce H4K8ac. Furthermore, overexpressed ZFAT recruits the bromodomain-containing protein BRD4 to centromeres through KAT2B-mediated H4K8ac, leading to RNA polymerase II-dependent ncRNA transcription. Thus, ZFAT binds to centromeres to control ncRNA transcription through the KAT2B-H4K8ac-BRD4 axis.
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http://dx.doi.org/10.1093/nar/gkaa815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641738PMC
November 2020

Rare variant of the epigenetic regulator SMCHD1 in a patient with pituitary hormone deficiency.

Sci Rep 2020 07 3;10(1):10985. Epub 2020 Jul 3.

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

Isolated hypogonadotropic hypogonadism (IHH), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) constitute a disease spectrum whose etiology remains largely unknown. This study aimed to clarify whether mutations in SMCHD1, an epigenetic regulator gene, might underlie this disease spectrum. SMCHD1 is a causative gene for Bosma arhinia microphthalmia syndrome characterized by arhinia, microphthalmia and IHH. We performed mutation screening of SMCHD1 in patients with etiology-unknown IHH (n = 31) or CPHD (n = 43, 19 of whom also satisfied the SOD diagnostic criteria). Rare variants were subjected to in silico analyses and classified according to the American College of Medical Genetics and Genomics guidelines. Consequently, a rare likely pathogenic variant, p.Asp398Asn, was identified in one patient. The patient with p.Asp398Asn exhibited CPHD, optic nerve hypoplasia, and a thin retinal nerve fiber layer, and therefore satisfied the criteria of SOD. This patient showed a relatively low DNA methylation level of the 52 SMCHD1-target CpG sites at the D4Z4 locus. Exome sequencing for the patient excluded additional variants in other IHH/CPHD-causative genes. In vitro assays suggested functional impairment of the p.Asp398Asn variant. These results provide the first indication that SMCHD1 mutations represent a rare genetic cause of the HH-related disease spectrum.
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http://dx.doi.org/10.1038/s41598-020-67715-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335161PMC
July 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 2021 Jun 23;58(6):427-432. 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142457PMC
June 2021

Contribution of gene mutations to Silver-Russell syndrome phenotype: multigene sequencing analysis in 92 etiology-unknown patients.

Clin Epigenetics 2020 06 16;12(1):86. Epub 2020 Jun 16.

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

Background: Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features. Major (epi)genetic causes of SRS are loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). However, IGF2, CDKN1C, HMGA2, and PLAG1 mutations infrequently cause SRS. In addition, other imprinting disturbances, pathogenic copy number variations (PCNVs), and monogenic disorders sometimes lead to SRS phenotype. This study aimed to clarify the frequency and clinical features of the patients with gene mutations among etiology-unknown patients with SRS phenotype.

Results: Multigene sequencing was performed in 92 out of 336 patients referred to us for genetic testing for SRS. The clinical features of the patients were evaluated based on the Netchine-Harbison clinical scoring system. None of the patients showed 11p15 LOM, upd(7)mat, abnormal methylation levels for six differentially methylated regions (DMRs), namely, PLAGL1:alt-TSS-DMR on chromosome 6, KCNQ1OT1:TSS-DMR on chromosome 11, MEG3/DLK1:IG-DMR on chromosome 14, MEG3:TSS-DMR on chromosome 14, SNURF:TSS-DMR on chromosome 15, and GNAS A/B:TSS-DMR on chromosome 20, PCNVs, or maternal uniparental disomy of chromosome 16. Using next-generation sequencing and Sanger sequencing, we screened four SRS-causative genes and 406 genes related to growth failure and/or skeletal dysplasia. We identified four pathogenic or likely pathogenic variants in responsible genes for SRS (4.3%: IGF2 in two patients, CDKN1C, and PLAG1), and five pathogenic variants in causative genes for known genetic syndromes presenting with growth failure (5.4%: IGF1R abnormality (IGF1R), SHORT syndrome (PIK3R1), Floating-Harbor syndrome (SRCAP), Pitt-Hopkins syndrome (TCF4), and Noonan syndrome (PTPN11)). Functional analysis indicated the pathogenicity of the CDKN1C variant. The variants we detected in CDKN1C and PLAG1 were the second and third variants leading to SRS, respectively. Our patients with CDKN1C and PLAG1 variants showed similar phenotypes to previously reported patients. Furthermore, our data confirmed IGF1R abnormality, SHORT syndrome, and Floating-Harbor syndrome are differential diagnoses of SRS because of the shared phenotypes among these syndromes and SRS. On the other hand, the patients with pathogenic variants in causative genes for Pitt-Hopkins syndrome and Noonan syndrome were atypical of these syndromes and showed partial clinical features of SRS.

Conclusions: We identified nine patients (9.8%) with pathogenic or likely pathogenic variants out of 92 etiology-unknown patients with SRS phenotype. This study expands the molecular spectrum of SRS phenotype.
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http://dx.doi.org/10.1186/s13148-020-00865-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298762PMC
June 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

Evolution of imprinting via lineage-specific insertion of retroviral promoters.

Nat Commun 2019 12 12;10(1):5674. Epub 2019 Dec 12.

Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

Imprinted genes are expressed from a single parental allele, with the other allele often silenced by DNA methylation (DNAme) established in the germline. While species-specific imprinted orthologues have been documented, the molecular mechanisms underlying the evolutionary switch from biallelic to imprinted expression are unknown. During mouse oogenesis, gametic differentially methylated regions (gDMRs) acquire DNAme in a transcription-guided manner. Here we show that oocyte transcription initiating in lineage-specific endogenous retroviruses (ERVs) is likely responsible for DNAme establishment at 4/6 mouse-specific and 17/110 human-specific imprinted gDMRs. The latter are divided into Catarrhini- or Hominoidea-specific gDMRs embedded within transcripts initiating in ERVs specific to these primate lineages. Strikingly, imprinting of the maternally methylated genes Impact and Slc38a4 was lost in the offspring of female mice harboring deletions of the relevant murine-specific ERVs upstream of these genes. Our work reveals an evolutionary mechanism whereby maternally silenced genes arise from biallelically expressed progenitors.
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http://dx.doi.org/10.1038/s41467-019-13662-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908575PMC
December 2019

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

Correction to: Gene expression and DNA methylation changes in BeWo cells dependent on tumor necrosis factor-α and insulin-like growth factor-I.

Hum Cell 2020 01;33(1):294

Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo, 181-8611, Japan.

In the original publication of the article, the author names were abbreviated in the author group. The corrected list of author names is presented above.
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http://dx.doi.org/10.1007/s13577-019-00310-zDOI Listing
January 2020

Exploring trophoblast-specific Tead4 enhancers through chromatin conformation capture assays followed by functional screening.

Nucleic Acids Res 2020 01;48(1):278-289

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

Tead4 is critical for blastocyst development and trophoblast differentiation. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. Using luciferase reporter assays with ES and TS cells for 34 candidate enhancer regions, we identified five genomic fragments that increased Tead4 promoter activity in a TS-specific manner. The five loci consisted of three intra- and two inter-chromosomal loci relative to Tead4 on chromosome 6. We established five mouse lines with one of the five enhancer elements deleted and evaluated the effect of each deletion on Tead4 expression in blastocysts. By quantitative RT-PCR, we measured a 42% decrease in Tead4 expression in the blastocysts with a homozygous deletion with a 1.5 kb genomic interval on chromosome 19 (n = 14) than in wild-type blastocysts. By conducting RNA-seq analysis, we confirmed the trans effect of this enhancer deletion on Tead4 without significant cis effects on its neighbor genes at least within a 1.7 Mb distance. Our results demonstrated that the genomic interval on chromosome 19 is required for the appropriate level of Tead4 expression in blastocysts and suggested that an inter-chromosomal enhancer-promoter interaction may be the underlying mechanism.
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http://dx.doi.org/10.1093/nar/gkz1034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943130PMC
January 2020

Gene expression and DNA methylation changes in BeWo cells dependent on tumor necrosis factor-α and insulin-like growth factor-I.

Hum Cell 2020 Jan 13;33(1):37-46. Epub 2019 Nov 13.

Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo, 181-8611, Japan.

Pregnant women with increased insulin resistance, characterized by elevated levels of tumor necrosis factor-alpha (TNF-α) and insulin-like growth factor-I (IGF-I), are at high risk of preeclampsia. We hypothesized that TNF-α and IGF-I affect the placentas and cause pathological changes leading to preeclampsia. To understand the genetic and epigenetic effects of TNF-α and IGF-I on trophoblast cells, gene expression microarray and DNA methylation array of BeWo cells stimulated by TNF-α (100 pg/ml, 100 ng/ml) and IGF-I (100 ng/ml) were conducted. Microarray analysis revealed the differential gene expression patterns in BeWo cells co-stimulated by TNF-α and IGF-I. Enrichment analysis identified the terms associated with NF-kappa B signaling pathways and arachidonic acid cascades such as PTGS2 and PTGER2. DNA methylation array revealed the distinct CpG methylation pattern in BeWo cells stimulated by high-TNF-α and IGF-I, while neither of them showed independent effects. Enrichment analysis identified the terms associated with major histocompatibility complex proteins. Integration of transcriptome and DNA methylome analyses identified three differentially expressed genes with significant DNA methylation change: C3, GP1BA, and NFKBIE, which are all possibly associated with pathogenesis of preeclampsia. In conclusion, co-stimulation of TNF-α and IGF-I induced the genetic and epigenetic changes associated with preeclampsia in BeWo cells. The results suggested that BeWo cells stimulated by TNF-α and IGF-I is a good in vitro model of preeclamptic placenta in pregnancy with increased insulin resistance.
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http://dx.doi.org/10.1007/s13577-019-00299-5DOI Listing
January 2020

Amplicon Sequencing-Based Noninvasive Fetal Genotyping for -Positive D Antigen-Negative Alleles.

Clin Chem 2019 10 5;65(10):1307-1316. Epub 2019 Sep 5.

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

Background: To avoid hemolytic disease of the fetus and newborn resulting from maternal alloantibodies against fetal Rh antigens, anti-D immunoglobulin is routinely administered to RhD-negative pregnant women in Japan. Fetal genotyping using cell-free DNA may prevent unnecessary antibody administration; however, current PCR-based methods, which detect deletion, do not address the higher rates of -positive D antigen-negative alleles in nonwhite populations without additional inspections.

Methods: We developed an amplicon-sequencing method that could estimate the type of paternally inherited fetal allele from 4 major alleles in the Japanese population: the D antigen-positive allele (*, 92.9%) and 3 D antigen-negative alleles (*, 6.6%; *, 0.3%; *, 0.1%) using cell-free DNA obtained from the blood plasma of pregnant women.

Results: The method correctly determined the fetal RhD type even when RhD-negative pregnant women possessed an -positive D antigen-negative allele: * or *.

Conclusions: This method is a reliable noninvasive fetal genotyping method for Japanese and other East Asian populations. The genotyping principle of amplifying 2 different regions using the same primer pair and distinguishing them by their sequence difference during the subsequent mapping procedure is also theoretically applicable to -positive D antigen-negative alleles prevalent in Africans. Therefore, this method offers an opportunity to consider targeted administration of anti-D immunoglobulin to RhD-negative pregnant women in East Asian and African countries and to increase the specificity of the fetal genotyping implemented nationwide in several European countries.
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http://dx.doi.org/10.1373/clinchem.2019.307074DOI Listing
October 2019
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