Publications by authors named "Yoichi Matsubara"

150 Publications

A novel missense variant of the GNAI3 gene and recognisable morphological characteristics of the mandibula in ARCND1.

J Hum Genet 2021 Mar 15. Epub 2021 Mar 15.

Department of Genome Medicine, National Center for Child Health and Development, Setagaya, Tokyo, Japan.

Auriculocondylar syndrome (ARCND) is an autosomal monogenic disorder characterised by external ear abnormalities and micrognathia due to hypoplasia of the mandibular rami, condyle and coronoid process. Genetically, three subtypes of ARCND (ARCND1, ARCND2 and ARCND3) have been reported. To date, five pathogenic variants of GNAI3 have been reported in ARCND1 patients. Here, we report a novel variant of GNAI3 (NM_006496:c.807C>A:p.(Asn269Lys)) in a Japanese girl with micrognathia using trio-based whole exome sequencing analysis. The GNAI3 gene encodes a heterotrimeric guanine nucleotide-binding protein. The novel variant locates the guanine nucleotide-binding site, and the substitution was predicted to interfere with guanine nucleotide-binding by in silico structural analysis. Three-dimensional computer tomography scan, or cephalogram, displayed severely hypoplastic mandibular rami and fusion to the medial and lateral pterygoid plates, which have been recognised in other ARCND1 patients, but have not been described in ARCND2 and ARCND3, suggesting that these may be distinguishable features in ARCND1.
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http://dx.doi.org/10.1038/s10038-021-00915-zDOI Listing
March 2021

Guide for diagnosis and treatment of hyperphenylalaninemia.

Pediatr Int 2021 Jan 10;63(1):8-12. Epub 2021 Jan 10.

Department of Pediatrics, The Jikei University, Tokyo, Japan.

Importance: Sapropterin hydrochloride, a natural coenzyme (6R-tetrahydrobiopterin) of phenylalanine hydroxylase, was first approved as a treatment for tetrahydrobiopterin deficiency in 1992 in Japan, and was then approved as a treatment for a tetrahydrobiopterin-responsive hyperphenylalaninemia in 2007 and 2008, in the USA and Japan, respectively. Guidelines are required on the proper use of sapropterin hydrochloride for tetrahydrobiopterin-responsive hyperphenylalaninemia.

Observations: It is recommended that tetrahydrobiopterin-responsive hyperphenylalaninemia should be diagnosed in all cases of hyperphenylalaninemia, including phenylketonuria, by tetrahydrobiopterin administration tests rather than by phenotype or blood phenylalanine levels.

Conclusions And Relevance: If tetrahydrobiopterin-responsive hyperphenylalaninemia is diagnosed, all ages can be treated with sapropterin hydrochloride. Although there are reports that sapropterin hydrochloride is effective and safe for the prevention of maternal phenylketonuria, further investigation is required.
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http://dx.doi.org/10.1111/ped.14399DOI Listing
January 2021

Research and Development Strategy for Future Embryonic Stem Cell-Based Therapy in Japan.

JMA J 2020 Oct 23;3(4):287-294. Epub 2020 Sep 23.

National Center for Child Health and Development, Tokyo, Japan.

Herewith, we review an updated progress of regenerative medical products using human embryonic stem cells (ESCs) in Japan. Two groups from Kyoto University and the National Center for Child Health and Development (NCCHD) established a novel derivation/cultivation system of ESCs for potential application in translational and clinical research. At the first stage of ESC derivation, murine feeder cells have been used in line with Japanese guidelines on public health associated with the implementation of the xenograft. To avoid exposure of ESCs to animal products in culture media, a xeno-free cultivating system has been established. Twelve ESCs (KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SEES-1, SEES-2, SEES-3, SEES-4, SEES-5, SEES-6, and SEES-7) are now available under a clinically relevant platform for industrially and clinically applicable regenerative medical products. NCCHD submitted an investigative new drug application to the Pharmaceuticals and Medical Devices Agency (PMDA) for using ESC-based products in patients with hyperammonemia due to genetic defects on March 2018 under the Pharmaceutical Affairs Law (now revised to the Pharmaceuticals, Medical Devices, and Other Therapeutic Products Act). Currently, up to ten ESC-based products are being prepared for intractable and rare disorders in Japan.
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http://dx.doi.org/10.31662/jmaj.2018-0029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676987PMC
October 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

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

Long-term efficacy and safety of two doses of Norditropin (somatropin) in Noonan syndrome: a 4-year randomized, double-blind, multicenter trial in Japanese patients.

Endocr J 2020 Aug 9;67(8):803-818. Epub 2020 May 9.

Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.

This 4-year randomized, double-blind, multicenter trial (NCT01927861) investigated the long-term efficacy and safety of Norditropin (NN-220; somatropin) in Japanese children with short stature due to Noonan syndrome. Pre-pubertal children with Noonan syndrome were randomized 1:1 to receive 0.033 mg/kg/day (n = 25, mean age 6.57 years) or 0.066 mg/kg/day (n = 26, mean age 6.06 years) GH. Height standard deviation score (SDS) change after 208 weeks from baseline was evaluated using an analysis of covariance model. Height SDS improved from -3.24 at baseline with a significantly greater increase (estimated mean [95% confidence interval]) with 0.066 vs. 0.033 mg/kg/day GH (1.84 [1.58; 2.10] vs. 0.85 [0.59; 1.12]; estimated mean difference 0.99 [0.62; 1.36]; p < 0.0001). The majority of treatment-emergent adverse events (TEAEs) were non-serious, mild and assessed as unlikely treatment-related. TEAE rates and frequencies of serious TEAEs were similar between groups. Three patients receiving 0.066 mg/kg/day were withdrawn; two due to TEAEs at days 1,041 and 1,289. Mean insulin-like growth factor-I SDS increased from -1.71 to -0.75 (0.033 mg/kg/day) and 0.57 (0.066 mg/kg/day) (statistically significant difference). In both groups, there were only minor glycosylated hemoglobin changes, similar oral glucose tolerance test insulin response increases and no clinically relevant changes in oral glucose tolerance test blood glucose, vital signs, electrocardiogram or transthoracic echocardiography. In conclusion, treatment with 0.033 and 0.066 mg/kg/day GH for 208 weeks improved height SDS in Japanese children with short stature due to Noonan syndrome with a significantly greater increase with 0.066 vs. 0.033 mg/kg/day GH and was well tolerated, with no new safety concerns.
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http://dx.doi.org/10.1507/endocrj.EJ19-0371DOI Listing
August 2020

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-Onset Chronic Pancreatitis.

Gastroenterology 2020 05 10;158(6):1626-1641.e8. Epub 2020 Jan 10.

Department of Gastroenterology and Hepatology, Tokyo Medical University, Tokyo, Japan.

Background & Aims: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca-selective ion channel, in an international cohort of patients and in mice.

Methods: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca imaging assays. CP was induced by repeated injections of cerulein in TRPV6 mice.

Results: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10). TRPV6 mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis.

Conclusions: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca balance in pancreatic cells. TRPV6 regulates Ca homeostasis and pancreatic inflammation.
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http://dx.doi.org/10.1053/j.gastro.2020.01.005DOI Listing
May 2020

Entire FGF12 duplication by complex chromosomal rearrangements associated with West syndrome.

J Hum Genet 2019 Oct 16;64(10):1005-1014. Epub 2019 Jul 16.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Complex rearrangements of chromosomes 3 and 9 were found in a patient presenting with severe epilepsy, developmental delay, dysmorphic facial features, and skeletal abnormalities. Molecular cytogenetic analysis revealed 46,XX.ish der(9)(3qter→3q28::9p21.1→9p22.3::9p22.3→9qter)(RP11-368G14+,RP11-299O8-,RP11-905L2++,RP11-775E6++). Her dysmorphic features are consistent with 3q29 microduplication syndrome and inv dup del(9p). Trio-based WES of the patient revealed no pathogenic single nucleotide variants causing epilepsy, but confirmed a 3q28q29 duplication involving FGF12, which encodes fibroblast growth factor 12. FGF12 positively regulates the activity of voltage-gated sodium channels. Recently, only one recurrent gain-of-function variant [NM_021032.4:c.341G>A:p.(Arg114His)] in FGF12 was found in a total of 10 patients with severe early-onset epilepsy. We propose that the patient's entire FGF12 duplication may be analogous to the gain-of-function variant in FGF12 in the epileptic phenotype of this patient.
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http://dx.doi.org/10.1038/s10038-019-0641-1DOI Listing
October 2019

Clonal Expansion of Second-Hit Cells with Somatic Recombinations or C>T Transitions Form Porokeratosis in MVD or MVK Mutant Heterozygotes.

J Invest Dermatol 2019 12 15;139(12):2458-2466.e9. Epub 2019 Jun 15.

Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; Keio-Maruho Laboratory of Skin Barriology, Keio University School of Medicine, Tokyo, Japan.

Patients with disseminated superficial actinic porokeratosis (DSAP) and linear porokeratosis (LP) exhibit monoallelic germline mutations in genes encoding mevalonate pathway enzymes, such as MVD or MVK. Here, we showed that each skin lesion of DSAP exhibited an individual second hit genetic change in the wild-type allele of the corresponding gene specifically in the epidermis, indicating that a postnatal second hit triggering biallelic deficiency of the gene is required for porokeratosis to develop. Most skin lesions exhibited one of two principal second hits, either somatic homologous recombinations rendering the monoallelic mutation biallelic or C>T transition mutations in the wild-type allele. The second hits differed among DSAP lesions but were identical in those of congenital LP, suggesting that DSAP is attributable to sporadic postnatal second hits and congenital LP to a single second hit in the embryonic period. In the characteristic annular skin lesions of DSAP, the central epidermis featured mostly second hit keratinocytes, and that of the annular ring featured a mixture of such cells and naïve keratinocytes, implying that each lesion reflects the clonal expansion of single second hit keratinocytes. DSAP is therefore a benign intraepidermal neoplasia, which can be included in the genetic tumor disorders explicable by Knudson's two-hit hypothesis.
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http://dx.doi.org/10.1016/j.jid.2019.05.020DOI Listing
December 2019

Transient multifocal genomic crisis creating chromothriptic and non-chromothriptic rearrangements in prezygotic testicular germ cells.

BMC Med Genomics 2019 05 28;12(1):77. Epub 2019 May 28.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, 157-8535, Japan.

Background: The co-occurrence of multiple de novo copy number variations (CNVs) is a rare phenomenon in the human genome. Recently, an "organismal CNV mutator phenotype" has been reported to result in transient genomic instability introducing multiple de novo CNVs in primary oocytes and early-stage zygotes. These findings opened a new area of human genome research.

Methods: We performed genome-wide copy number analysis for ~ 2100 individuals with various congenital defects. Furthermore, extensive molecular analyses, including synthetic long-read whole-genome sequencing and haplotype-phasing, were carried out for an individual with multiple de novo CNVs.

Results: A boy was found to have de novo rearrangements on five chromosomes. The rearrangements comprised simple duplication and inversion as well as chaotic changes, all of which affected paternally derived chromosomes. Postzygotic genomic instability was ruled out. The duplicated regions on 6q and 13q contained both diallelic and triallelic loci, indicating that the genomic rearrangements were initially created during premeiotic mitosis and subsequently modified by physiological cross-over during meiosis I. Breakpoints of the rearrangements were indicative of non-homologous end joining, replication-based errors, and/or chromothripsis. The mutagenic event was independent of specific local DNA motifs or de novo point mutations, but may be driven by spermatogenesis-specific factors.

Conclusions: These results indicate that during spermatogenesis, a transient multifocal genomic crisis can introduce several chromothriptic and non-chromothriptic changes into the genome. These findings broaden the concept of the "organismal CNV mutator phenotype". This study provides insights into mechanisms for altering the global chromosomal architecture of human embryos.
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http://dx.doi.org/10.1186/s12920-019-0526-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540402PMC
May 2019

KLF11 variant in a family clinically diagnosed with early childhood-onset type 1B diabetes.

Pediatr Diabetes 2019 09 3;20(6):712-719. Epub 2019 Jun 3.

Department of Pediatrics, Osaka City University School of Medicine, Osaka, Japan.

KLF11 is the causative gene for maturity-onset diabetes of the young 7 (MODY7). KLF11 regulates insulin gene expression through binding to the GC box in the promoter. To date, only two KLF11 mutations have been identified in three families with early-onset type 2 diabetes. Here, we report a novel KLF11 variant associated with early childhood-onset type 1B diabetes. The proband and his younger sister exhibited hyperglycemia at age 1 year, and their mother developed diabetes at age 4 years. These three individuals required insulin injection from the initial phase of the disease. Being negative for islet cell autoantibodies, they were diagnosed with type 1B diabetes. Mutation screening for 30 diabetes-associated genes identified a heterozygous KLF11 variant (p.His418Gln) in the proband and his sister. The variant was also detected in the affected mother, as well as in the allegedly unaffected maternal grandmother. In silico analyses indicated that this variant involves a highly conserved histidine residue in the first C H zinc finger domain which ligates a zinc ion. In vitro analyses showed that expression levels and intracellular localization of His418Gln-KLF11 were comparable to those of wildtype (WT)-KLF11. Luciferase assays demonstrated that while WT-KLF11 suppressed the activity of a 6 × GC box-containing reporter, His418Gln-KLF11 lacked the suppressive effect. Notably, His418Gln-KLF11 canceled the suppressive effect of co-transfected WT-KLF11. Such a dominant-negative effect was absent in the previously reported Ala347Ser-KLF11 variant. These results indicate that specific variants of KLF11 (MODY7) with a dominant-negative effect underlie early childhood-onset type 1B diabetes with incomplete penetrance. This study documents a novel monogenic mutation associated with diabetes in children.
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http://dx.doi.org/10.1111/pedi.12868DOI Listing
September 2019

Germline-Derived Gain-of-Function Variants of Gs-Coding Gene Identified in Nephrogenic Syndrome of Inappropriate Antidiuresis.

J Am Soc Nephrol 2019 05 8;30(5):877-889. Epub 2019 Apr 8.

Departments of Molecular Endocrinology,

Background: The stimulatory G-protein -subunit encoded by exons 1-13 (-Gs) mediates signal transduction of multiple G protein-coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function -Gs variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function -Gs variants have been detected in McCune-Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality.

Methods: We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of and functional studies for identified variants.

Results: Whole-exome sequencing revealed two -Gs candidate variants for NSIAD: -Gs p.(F68_G70del) in one family and -Gs p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, -Gs alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function-compatible conformational property for p.M255V-Gs, although such assessment was not possible for p.F68_G70del-Gs. Both variants had gain-of-function effects that were significantly milder than those of McCune-Albright syndrome-specific somatic Gs variants. Model mice for p.F68_G70del-Gs showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gs exhibited severe failure to thrive.

Conclusions: This study shows that germline-derived gain-of-function rare variants of -Gs exist and cause NSIAD as a novel Gs-mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to -Gs's gain-of-function effects.
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http://dx.doi.org/10.1681/ASN.2018121268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493982PMC
May 2019

Recurrent de novo MAPK8IP3 variants cause neurological phenotypes.

Ann Neurol 2019 06 25;85(6):927-933. Epub 2019 Apr 25.

Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan.

c-Jun-amino-terminal kinase-interacting protein 3 (JIP3), encoded by MAPK8IP3, is an adaptor protein of the kinesin-1 complex and essential for axonal transport in neurons. However, an association between MAPK8IP3 variants and human disease has not been established. We identified 5 individuals from four families with recurrent de novo variants c.1732C>T (p.Arg578Cys) and c.3436C>T (p.Arg1146Cys) in MAPK8IP3. The core phenotype includes spastic diplegia, intellectual disability, cerebral atrophy, and corpus callosum hypoplasia. Zebrafish embryos overexpressing human mutant JIP3 showed axon varicosities of the posterior lateral line nerve, suggesting an adverse effect on the developing axons. Our results suggest that MAPK8IP3 variants cause a neurodevelopmental disease. ANN NEUROL 2019;85:927-933.
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http://dx.doi.org/10.1002/ana.25481DOI Listing
June 2019

The desmosome is a mesoscale lipid raft-like membrane domain.

Mol Biol Cell 2019 06 3;30(12):1390-1405. Epub 2019 Apr 3.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.

Desmogleins (Dsgs) are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting syndrome. Molecular modeling predicts that this G-to-R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography indicates that the lipid bilayer within the desmosome is ∼10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via the establishment of a specialized mesoscale lipid raft-like membrane domain.
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http://dx.doi.org/10.1091/mbc.E18-10-0649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724694PMC
June 2019

New Noonan syndrome model mice with RIT1 mutation exhibit cardiac hypertrophy and susceptibility to β-adrenergic stimulation-induced cardiac fibrosis.

EBioMedicine 2019 Apr 18;42:43-53. Epub 2019 Mar 18.

Department of Medical Genetics, Tohoku University Graduate School of Medicine, Sendai, Japan. Electronic address:

Background: Noonan syndrome (NS) is a genetic disorder characterized by short stature, a distinctive facial appearance, and heart defects. We recently discovered a novel NS gene, RIT1, which is a member of the RAS subfamily of small GTPases. NS patients with RIT1 mutations have a high incidence of hypertrophic cardiomyopathy and edematous phenotype, but the specific role of RIT1 remains unclear.

Methods: To investigate how germline RIT1 mutations cause NS, we generated knock-in mice that carried a NS-associated Rit1 A57G mutation (Rit1). We investigated the phenotypes of Rit1 mice in fetal and adult stages as well as the effects of isoproterenol on cardiac function in Rit1 mice.

Findings: Rit1 embryos exhibited decreased viability, edema, subcutaneous hemorrhage and AKT activation. Surviving Rit1 mice had a short stature, craniofacial abnormalities and splenomegaly. Cardiac hypertrophy and cardiac fibrosis with increased expression of S100A4, vimentin and periostin were observed in Rit1 mice compared to Rit1 mice. Upon isoproterenol stimulation, cardiac fibrosis was drastically increased in Rit1 mice. Phosphorylated (at Thr308) AKT levels were also elevated in isoproterenol-treated Rit1 hearts.

Interpretation: The A57G mutation in Rit1 causes cardiac hypertrophy, fibrosis and other NS-associated features. Biochemical analysis indicates that the AKT signaling pathway might be related to downstream signaling in the RIT1 A57G mutant at a developmental stage and under β-adrenergic stimulation in the heart. FUND: The Grants-in-Aid were provided by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science KAKENHI Grant.
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http://dx.doi.org/10.1016/j.ebiom.2019.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491386PMC
April 2019

Dihydrotestosterone induces minor transcriptional alterations in genital skin fibroblasts of children with and without androgen insensitivity.

Endocr J 2019 Apr 19;66(4):387-393. Epub 2019 Feb 19.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.

Endogenous and exogenous androgens induce masculinization of external genitalia through binding to the androgen receptor (AR). The target genes of androgens in external genitalia remain to be determined, although previous studies have shown that the apolipoprotein D gene (APOD) was significantly upregulated by dihydrotestosterone (DHT), the most potent androgen in humans. In the present study, we performed microarray analysis for genital skin fibroblasts obtained from four boys with buried penis (the control individuals) and a patient with partial androgen insensitivity syndrome (PAIS) due to a hypomorphic mutation in AR (the PAIS patient). We identified 24 transcripts that were upregulated or downregulated by DHT in all samples of control individuals and, to a lesser extent, in the sample of the PAIS patient. Differences between DHT-treated and -untreated samples were small; the results of 24 transcripts did not reach statistical significance. The 24 transcripts included CYP1B1, a gene possibly involved in the development of genital tubercle in mice, and APOD, as well as several genes that have been reported as androgen targets in prostate or other tissues. The results of this study indicate that androgen-mediated masculinization of external genitalia is unlikely to depend on massive transcriptional changes in specific AR target genes. Rather, minor transcriptional changes of several genes, and/or a complex molecular network may play a major role in penile development. Importantly, our data suggest the possible involvement of CYP1B1 in human genital development and confirm the clinical importance of APOD as a biomarker for AR function.
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http://dx.doi.org/10.1507/endocrj.EJ18-0494DOI Listing
April 2019

Delineation of LZTR1 mutation-positive patients with Noonan syndrome and identification of LZTR1 binding to RAF1-PPP1CB complexes.

Hum Genet 2019 Jan 27;138(1):21-35. Epub 2018 Oct 27.

Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryomachi, Aobaku, Sendai, 980-8574, Japan.

RASopathies are a group of developmental disorders caused by mutations in genes that regulate the RAS/MAPK pathway and include Noonan syndrome (NS), Costello syndrome, cardiofaciocutaneous syndrome and other related disorders. Whole exome sequencing studies recently identified LZTR1, PPP1CB and MRAS as new causative genes in RASopathies. However, information on the phenotypes of LZTR1 mutation-positive patients and functional properties of the mutations are limited. To identify variants of LZTR1, PPP1CB, and MRAS, we performed a targeted next-generation sequencing and reexamined previously analyzed exome data in 166 patients with suspected RASopathies. We identified eight LZTR1 variants, including a de novo variant, in seven probands who were suspicious for NS and one known de novo PPP1CB variant in a patient with NS. One of the seven probands had two compound heterozygous LZTR1 variants, suggesting autosomal recessive inheritance. All probands with LZTR1 variants had cardiac defects, including hypertrophic cardiomyopathy and atrial septal defect. Five of the seven probands had short stature or intellectual disabilities. Immunoprecipitation of endogenous LZTR1 followed by western blotting showed that LZTR1 bound to the RAF1-PPP1CB complex. Cells transfected with a small interfering RNA against LZTR1 exhibited decreased levels of RAF1 phosphorylated at Ser259. These are the first results to demonstrate LZTR1 in association with the RAF1-PPP1CB complex as a component of the RAS/MAPK pathway.
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http://dx.doi.org/10.1007/s00439-018-1951-7DOI Listing
January 2019

Present State and Future Prospects of Pediatric Liver Transplantations.

JMA J 2018 Sep;1(1):50-56

National Center for Child Health and Development, Tokyo, Japan.

Regarding liver transplantations in Japan, with no progress having been made in deceased donor liver transplantations, a living donor liver transplantation performed on a boy with end-stage liver cirrhosis caused by biliary atresia by Nagasue et al. at Shimane University in November 1989 was the first case of its kind. Unlike deceased donor liver transplantations, living donor liver transplantations have two major advantages. First, because organs are donated from healthy adults, it is possible to transplant organs with better viability compared to deceased donor organs, which have been preserved in cold storage for a long time. Second, depending on the recipient's condition, it is possible to conduct elective surgery at the optimal time. In Japan, the number of annual liver transplantation cases is approximately 400, with the number of annual pediatric liver transplantation cases stable at approximately 120 cases. The patient survival rate of pediatric liver transplantation cases is relatively good at 89.4% over the course of 1 year, 86.8% over 5 years, 84.4% over 10 years, and 80.9% over 20 years. The liver transplantation program was initiated at the National Center for Child Health and Development, Tokyo, Japan, in November 2005, providing liver transplantation treatment to 510 pediatric patients with end-stage liver disease to date. This article outlines the history of liver transplantations in Japan along with the present state of liver transplantations at the National Center for Child Health and Development.
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http://dx.doi.org/10.31662/jmaj.2018-0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969907PMC
September 2018

Clinical and molecular characteristics of fusion-positive B-cell precursor acute lymphoblastic leukemia in childhood, including a novel translocation resulting in gene fusion.

Haematologica 2019 01 31;104(1):128-137. Epub 2018 Aug 31.

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

Fusion genes involving have recently been identified in precursor B-cell acute lymphoblastic leukemia, mutually exclusive of the common risk stratifying genetic abnormalities, although their true incidence and associated clinical characteristics remain unknown. We identified 16 cases of acute lymphoblastic leukemia and 1 of lymphoma harboring fusions, including (n=10), (n=6), and one novel fusion. The incidence of fusions overall was 2.4% among consecutive precursor B-cell acute lymphoblastic leukemia patients enrolled onto a single clinical trial. They frequently showed a cytoplasmic μ chain-positive pre-B immunophenotype, and often expressed an aberrant CD5 antigen. Besides up- and down-regulation of and , elevated expression was also a characteristic feature of fusion-positive patients. Mutations of , recurrent in T-cell acute lymphoblastic leukemia, also showed an unexpectedly high frequency (50%) in these patients. fusion-positive patients were older (median age 9 years) with elevated WBC counts (median: 27,300/ml) at presentation and, as a result, were mostly classified as NCI high risk. Although they responded well to steroid treatment, fusion-positive patients showed a significantly worse outcome, with 53.3% relapse and subsequent death. Stem cell transplantation was ineffective as salvage therapy. Interestingly, relapse was frequently associated with the presence of gene deletions. Our observations indicate that fusions comprise a distinct subgroup of precursor B-cell acute lymphoblastic leukemia with a characteristic immunophenotype and gene expression signature, associated with distinct clinical features.
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http://dx.doi.org/10.3324/haematol.2017.186320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312004PMC
January 2019

Correction: Nationwide survey for current clinical status of amniocentesis and maternal serum marker test in Japan.

J Hum Genet 2018 11 23;63(11):1195. Epub 2018 Aug 23.

Department of Obstetrics and Gynecology, Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan.

Since the publication of this paper, the authors noticed that Yosuke Fujii was assigned to the incorrect affiliation. The affiliation information is provided correctly, above.
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http://dx.doi.org/10.1038/s10038-018-0500-5DOI Listing
November 2018

11-oxygenated C19 steroids as circulating androgens in women with polycystic ovary syndrome.

Endocr J 2018 Oct 14;65(10):979-990. Epub 2018 Jul 14.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.

11-oxygenated C19 steroids (11oxC19s) are newly specified human androgens. Although median serum levels of 11oxC19 were reported to be higher in patients with polycystic ovary syndrome (PCOS) than in unaffected women, inter-individual variations in androgen levels among PCOS patients have poorly been investigated. Here, we quantified four 11oxC19s, i.e., 11-ketotestosterone (11KT), 11β-hydroxytestosterone (11OHT), 11β-hydroxyandrostenedione (11OHΔ4A), and 11-ketoandrostenedione (11KΔ4A), in blood samples of 28 PCOS patients and 31 eumenorrheic women using liquid chromatography-tandem mass spectrometry. We referred to our previous data of classic androgens in these individuals. We found that 11OHT levels were higher in the PCOS group than in the eumenorrheic group. Moreover, although the median values of 11KT, 11KΔ4A, and 11OHΔ4A were comparable between the two groups, these steroids were markedly increased in some patients. Of the 28 patients, 8 had high levels of both 11oxC19s and classic androgens, whereas 4 had an increase only in 11oxC19 levels, and 12 had an increase only in classic androgen levels. Intragroup variations in androgen levels were relatively large in the PCOS group. Levels of 11OHT and 11KT were significantly higher in overweight/obese patients than in normal weight patients and correlated with body mass indexes. These results highlight the clinical significance of 11oxC19s as circulating androgens in PCOS patients and indicate that the accumulation of 11oxC19s and/or classic androgens is an essential feature of PCOS. The profiles of circulating androgens appear to vary among patients. In particular, overweight/obesity likely enhances the 11oxC19s accumulation in PCOS, although this notion awaits further validation.
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http://dx.doi.org/10.1507/endocrj.EJ18-0212DOI Listing
October 2018

Genitopatellar syndrome: the first reported case in Japan.

Hum Genome Var 2018 28;5. Epub 2018 May 28.

4Education Center, Asahikawa Medical University, Asahikawa, Japan.

Genitopatellar syndrome (GPS) is mainly characterized by an absence of patellae, congenital flexion contractures of the lower limbs, psychomotor retardation, and anomalies of the external genitalia and kidneys. We report an 18-year-old female with a novel heterozygous truncating mutation in exon 17 of the gene [MC_000010.11:c.3603_3606 del, p.Arg1201fs]. This is the first report of typical GPS in a Japanese individual. The details of our findings may contribute to elucidating the mechanism underlying GPS-specific clinical features.
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http://dx.doi.org/10.1038/s41439-018-0010-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972145PMC
May 2018

Maternal Uniparental Disomy for Chromosome 20: Physical and Endocrinological Characteristics of Five Patients.

J Clin Endocrinol Metab 2018 06;103(6):2083-2088

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

Context: Maternal uniparental disomy for chromosome 20 [UPD(20)mat], resulting in aberrant expression of imprinted transcripts at the GNAS locus, is a poorly characterized condition. These patients manifested a phenotype similar to that of Silver-Russell syndrome (SRS) and small for gestational age-short stature (SGA-SS); however, the etiological relationship between UPD(20)mat and SRS/SGA-SS remains unclear. Moreover, no report has described endocrinological assessment of UPD(20)mat patients, although paternal UPD(20), the mirror image entity of UPD(20)mat, is known to cause multiple hormone resistance reflecting reduced α-subunit of the stimulatory G protein expression.

Participants: Patients 1 to 5 showed nonmosaic heterodisomy and/or isodisomy for the entire chromosome 20. Patients 1 to 3 and 4 were identified through UPD(20)mat screening for 55 patients with etiology-unknown SRS and 96 patients with SGA-SS, respectively. Patient 5 was identified through molecular analysis for patients with developmental defects. Patients 1 to 5 manifested postnatal growth failure and feeding problems, with or without developmental delay, and other clinical features. Patients 1 to 4 were born SGA. Patients 4 and 5 exhibited hypercalcemia and low or low-normal parathyroid hormone levels. Patient 1 showed constantly decreased thyroid-stimulating hormone (TSH) levels after 12 years of age, although she had a normal TSH level at 5.2 years of age.

Conclusion: The results suggest that UPD(20)mat underlies growth failure and feeding problems with additional features and could account for >5% of etiology-unknown SRS and small percentages of SGA-SS. Most important, this study provides an indication that UPD(20)mat can be associated with hypersensitivity of hormone receptors, which may gradually develop with age.
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http://dx.doi.org/10.1210/jc.2017-02780DOI Listing
June 2018

An unclassified variant of activates a cryptic splice site in a patient with CHARGE syndrome.

Hum Genome Var 2018 8;5:18006. Epub 2018 Mar 8.

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

CHARGE syndrome is a rare autosomal dominant disease that is typically caused by heterozygous mutations. A variant in a splicing acceptor site (NM_017780.3:c.7165-4A>G) was identified in a Japanese boy with CHARGE syndrome. This variant has been considered to be an "unclassified variant" due to its position outside the consensus splicing sites. In this study, abnormal splicing derived from this known variant was confirmed by cDNA sequencing.
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http://dx.doi.org/10.1038/hgv.2018.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842149PMC
March 2018

Loss-of-function and gain-of-function mutations in PPP3CA cause two distinct disorders.

Hum Mol Genet 2018 04;27(8):1421-1433

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.

Calcineurin is a calcium (Ca2+)/calmodulin-regulated protein phosphatase that mediates Ca2+-dependent signal transduction. Here, we report six heterozygous mutations in a gene encoding the alpha isoform of the calcineurin catalytic subunit (PPP3CA). Notably, mutations were observed in different functional domains: in addition to three catalytic domain mutations, two missense mutations were found in the auto-inhibitory (AI) domain. One additional frameshift insertion that caused premature termination was also identified. Detailed clinical evaluation of the six individuals revealed clinically unexpected consequences of the PPP3CA mutations. First, the catalytic domain mutations and frameshift mutation were consistently found in patients with nonsyndromic early onset epileptic encephalopathy. In contrast, the AI domain mutations were associated with multiple congenital abnormalities including craniofacial dysmorphism, arthrogryposis and short stature. In addition, one individual showed severe skeletal developmental defects, namely, severe craniosynostosis and gracile bones (severe bone slenderness and perinatal fractures). Using a yeast model system, we showed that the catalytic and AI domain mutations visibly result in decreased and increased calcineurin signaling, respectively. These findings indicate that different functional effects of PPP3CA mutations are associated with two distinct disorders and suggest that functional approaches using a simple cellular system provide a tool for resolving complex genotype-phenotype correlations.
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http://dx.doi.org/10.1093/hmg/ddy052DOI Listing
April 2018

Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis.

EBioMedicine 2018 Jan 6;27:138-150. Epub 2017 Dec 6.

Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan. Electronic address:

Costello syndrome is a "RASopathy" that is characterized by growth retardation, dysmorphic facial appearance, hypertrophic cardiomyopathy and tumor predisposition. >80% of patients with Costello syndrome harbor a heterozygous germline G12S mutation in HRAS. Altered metabolic regulation has been suspected because patients with Costello syndrome exhibit hypoketotic hypoglycemia and increased resting energy expenditure, and their growth is severely retarded. To examine the mechanisms of energy reprogramming by HRAS activation in vivo, we generated knock-in mice expressing a heterozygous Hras G12S mutation (Hras mice) as a mouse model of Costello syndrome. On a high-fat diet, Hras mice developed a lean phenotype with microvesicular hepatic steatosis, resulting in early death compared with wild-type mice. Under starvation conditions, hypoketosis and elevated blood levels of long-chain fatty acylcarnitines were observed, suggesting impaired mitochondrial fatty acid oxidation. Our findings suggest that the oncogenic Hras mutation modulates energy homeostasis in vivo.
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http://dx.doi.org/10.1016/j.ebiom.2017.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828294PMC
January 2018

Efficacy and safety of two doses of Norditropin (somatropin) in short stature due to Noonan syndrome: a 2-year randomized, double-blind, multicenter trial in Japanese patients.

Endocr J 2018 Feb 7;65(2):159-174. Epub 2017 Nov 7.

Thyroid and Endocrine Center, Fukushima Global Medical Science Center, Fukushima Medical University, Fukushima 960-1295, Japan.

This randomized double-blind multicenter trial (NCT01927861) evaluated the growth-promoting effect and safety of Norditropin (NN220; somatropin) in Japanese children with short stature due to Noonan syndrome. Prepubertal children aged 3-<11 years (boys) or 3-<10 years (girls) with Noonan syndrome were randomized to receive GH 0.033 mg/kg/day (n = 25, mean age 6.57 years, 11 females) or 0.066 mg/kg/day (n = 26, mean age 6.06 years, eight females) for 104 weeks. Change in height standard deviation score (HSDS) from baseline was analyzed based on an ANCOVA model. Baseline HSDS was -3.24. Estimated change in HSDS [95% CI] after 104 weeks' treatment was 0.84 [0.66, 1.02] and 1.47 [1.29, 1.64] for the lower and higher doses, respectively; estimated mean difference 0.63 [0.38, 0.88], p < 0.0001. Rates and patterns of adverse events (AEs) were similar between groups. Most were mild and reported as unlikely to be related to Norditropin. There were no withdrawals due to AEs. Insulin-like growth factor-I SDS increased from -1.71 to -0.64 (0.033 mg/kg/day) and to 0.63 (0.066 mg/kg/day). HbA increased slightly (0.033 mg/kg/day: +0.14%; 0.066 mg/kg/day: +0.13%); glucose profiles were almost unchanged; insulin profiles increased in both groups in the oral glucose tolerance test. There were no clinically significant abnormal electrocardiogram or echocardiography findings. We conclude that Norditropin at doses of 0.033 mg/kg/day or 0.066 mg/kg/day for 104 weeks increases height in Japanese children with short stature due to Noonan syndrome, with a favorable safety profile. The effect was greater with 0.066 mg/kg/day compared with 0.033 mg/kg/day.
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http://dx.doi.org/10.1507/endocrj.EJ17-0313DOI Listing
February 2018