Publications by authors named "Tetsuya Niihori"

77 Publications

Duplications in the G3 domain or switch II region in HRAS identified in patients with Costello syndrome.

Hum Mutat 2021 Oct 7. Epub 2021 Oct 7.

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

Costello syndrome (CS) is an autosomal-dominant disorder characterized by distinctive facial features, hypertrophic cardiomyopathy, skeletal abnormalities, intellectual disability, and predisposition to cancers. Germline variants in HRAS have been identified in patients with CS. Intragenic HRAS duplications have been reported in three patients with a milder phenotype of CS. In this study, we identified two known HRAS variants, p.(Glu63_Asp69dup), p.(Glu62_Arg68dup), and one novel HRAS variant, p.(Ile55_Asp57dup), in patients with CS, including a patient with craniosynostosis. These intragenic duplications are located in the G3 domain and the switch II region. Cells expressing cDNA with these three intragenic duplications showed an increase in ELK-1 transactivation. Injection of wild-type or mutant HRAS mRNAs with intragenic duplications in zebrafish embryos showed significant elongation of the yolk at 11 h postfertilization, which was improved by MEK inhibitor treatment, and a variety of developmental abnormalities at 3 days post fertilization was observed. These results indicate that small in-frame duplications affecting the G3 domain and switch II region of HRAS increase the activation of the ERK pathway, resulting in developmental abnormalities in zebrafish or patients with CS.
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http://dx.doi.org/10.1002/humu.24287DOI Listing
October 2021

Detection of intracellular histological abnormalities using cardiac magnetic resonance T1 mapping in patients with Danon disease: a case series.

Eur Heart J Case Rep 2021 May 4;5(5):ytab145. Epub 2021 May 4.

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

Background: Danon disease is an X-linked dominant disorder with defects in the lysosome-associated membrane protein 2 (LAMP2) gene and is characterized histologically by intracellular autophagic vacuoles in skeletal and cardiac muscles. Cardiac magnetic resonance (CMR) T1 mapping potentially allows to differentiate intracellular and extracellular cardiac abnormalities with a combination of native T1 value and extracellular volume (ECV) fraction.

Case Summary: We assessed CMR T1 mapping in two Danon disease patients (a 22-year-old man and his 48-year-old mother), who had a LAMP2 c.864G>A p. Val288Val mutation, and two blood relatives without Danon disease (his 47-year-old maternal aunt and 49-year-old father). The male patient underwent a left ventricular (LV) assist device implantation at 15 months after the image acquisition because he was inotrope dependent (INTERMACS profile 3) and had no noticeable psychological or musculoskeletal symptoms. His mother was in New York Heart Association Class II with mildly reduced LV ejection fraction (46%). The Danon group showed late gadolinium enhancement (LGE) in the anterior and posterolateral LV walls. In the interventricular wall, where evident LGE was not noted, the Danon group had high native T1 value, compared with the T1 value in the non-Danon group, and normal ECV fraction. Cardiac biopsy from the interventricular wall showed intracytoplasmic autophagic vacuoles, which are characteristics of Danon disease.

Discussion: This characteristic pattern of high native T1 and normal ECV fraction in the areas without LGE, which may reflect the existence of intracytoplasmic autophagic vacuoles, may support the differential diagnosis of Danon disease from other cardiomyopathies.
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http://dx.doi.org/10.1093/ehjcr/ytab145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276603PMC
May 2021

A novel deletion in the C-terminal region of HSPB8 in a family with rimmed vacuolar myopathy.

J Hum Genet 2021 Oct 20;66(10):965-972. Epub 2021 Mar 20.

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

Heat shock protein family B member 8, encoded by HSPB8, is an essential component of the chaperone-assisted selective autophagy complex, which maintains muscle function by degrading damaged proteins in the cells. Mutations in HSPB8 have been reported to cause Charcot-Marie-Tooth type 2L, distal hereditary motor neuropathy IIa, and rimmed vacuolar myopathies (RVM). In this study, we identified a novel heterozygous frameshift variant c.525_529del in HSPB8 in a large Japanese family with RVM, using whole exome sequencing. Three affected individuals had severe respiratory failure, which has not been addressed by previous studies. Muscle atrophy in the paraspinal muscles was also a clinical feature of the individuals affected with RVM in this study. The frameshift mutation was located in the last coding exon, and the mutated protein was predicted to harbor an isoleucine-leucine-valine (ILV) sequence, which corresponds to the IXI/V (isoleucine, X amino acids, and isoleucine or valine) motif. The IXI/V motif is essential for assembly into larger oligomers in other small heat shock proteins and all frameshift mutants of HSPB8 were predicted to share the ILV sequence in the C-terminal extension. The in silico prediction tools showed low protein solubility and increased aggregation propensity for the region around the ILV sequence. The IXI/V motif might be associated with the pathogenesis of HSPB8-related RVM.
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http://dx.doi.org/10.1038/s10038-021-00916-yDOI Listing
October 2021

A de novo CHD3 variant in a child with intellectual disability, autism, joint laxity, and dysmorphisms.

Brain Dev 2021 Apr 24;43(4):563-565. Epub 2020 Dec 24.

Department of Medical Genetics and Genomics, Sapporo Medical University School of Medicine, Sapporo, Japan.

Background: Chromodomain helicase DNA-binding (CHD) proteins play important roles in developmental processes. CHD3, a member of the CHD family of proteins, was reported to be a cause of a neurodevelopmental syndrome by Snijders Blok et al., but only a small number of probands have been reported.

Case Report: The patient was a 9-year-old female with severe intellectual disability, speech impairment, autism, joint laxity and dysmorphisms. Whole exome sequencing revealed a de novo missense variant in CHD3 (NM_001005273:exon18: c.2896C > T:p.R966W).

Conclusion: We report a case with a pathogenic variant in the CHD3 gene. Our report indicates that CHD3 analysis is helpful for diagnosis of the cases with neurodevelopmental disorders, joint laxity, and coarse facial phenotype.
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http://dx.doi.org/10.1016/j.braindev.2020.12.004DOI Listing
April 2021

Costello syndrome model mice with a Hras mutation are susceptible to develop house dust mite-induced atopic dermatitis.

Cell Death Dis 2020 08 13;11(8):617. Epub 2020 Aug 13.

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

Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations. Patients with Costello syndrome present craniofacial abnormalities, cardiac defects, and cancer predisposition, as well as skin abnormalities, including papillomas, keratosis pilaris, and eczematous dermatitis. However, the mechanisms underlying the dermatological abnormalities remain unclear. Here, we demonstrated that knock-in mice expressing an Hras G12S mutation (Hras mice) are susceptible to develop atopic dermatitis (AD)-like skin lesions, including eczema, pruritus, elevated serum IgE levels, acanthosis, and the infiltration of mast cells, basophils, and type-2 innate lymphoid cells in the dermis, after stimulation with house dust mite allergens (Dermatophagoides farinae, Dfb). Reduced skin barrier function, increased proliferation of phosphorylated ERK (p-ERK)-positive epidermal cells, and increased Th2-type cytokines as well as epithelial cell-derived cytokines, including IL-33, were observed in the skin tissue of Hras mice compared with Hras mice. Cultured Hras keratinocytes exhibited increased IL-33 expression after Dfb stimulation. PD0325901, an MEK inhibitor, ameliorated AD-like symptoms in Hras mice, showing decreased proliferation of p-ERK-positive epidermal cells and decreased expression of IL-33. Our findings indicate that the epidermis of Hras mice stimulated by Dfb strongly induced IL-33 expression and type-2 innate lymphoid cells, resulting in AD-like skin lesions. These results suggest that the epidermis of Hras mice are prone to development of eczematous dermatitis stimulated with house dust mite allergens.
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http://dx.doi.org/10.1038/s41419-020-02845-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426869PMC
August 2020

A somatic activating KRAS variant identified in an affected lesion of a patient with Gorham-Stout disease.

J Hum Genet 2020 Nov 26;65(11):995-1001. Epub 2020 Jun 26.

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

Gorham-Stout disease (GSD), a rare disorder of unknown etiology, is characterized by massive osteolysis that is associated with proliferation and dilation of lymphatic vessels. Variants in cancer-associated genes have been described in complex lymphatic anomalies. To explore the pathogenesis of GSD, we performed the amplicon-based deep sequencing on 50 cancer-related genes to assay affected tissues from the six patients with GSD. In one patient, a somatic activating KRAS c.182A > G variant (p.Q61R) was detected in 1% of the tissue sample. Conversely, the mutant allele was not detected in uninvolved normal skin and blood samples. Histopathology of the patient's tissue sample showed proliferation of abnormal lymphatic and blood vascular endothelial cells, osteoclasts, and activated macrophages. The activating KRAS variant is a known 'hotspot' variant, frequently identified in several types of human cancer. This is the first report of identifying a pathogenic variant in a patient with GSD. This finding may set the stage for elucidation of pathophysiology and the development of novel therapies for GSD.
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http://dx.doi.org/10.1038/s10038-020-0794-yDOI Listing
November 2020

The genetic profile of dysferlinopathy in a cohort of 209 cases: Genotype-phenotype relationship and a hotspot on the inner DysF domain.

Hum Mutat 2020 09 5;41(9):1540-1554. Epub 2020 Jul 5.

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

Dysferlinopathy is a group of autosomal recessive muscular dystrophies caused by variants in the dysferlin gene (DYSF), with variable proximal and distal muscle involvement. We performed DYSF gene analyses of 200 cases suspected of having dysferlinopathy (Cohort 1), and identified diagnostic variants in 129/200 cases, including 19 novel variants. To achieve a comprehensive genetic profile of dysferlinopathy, we analyzed the variant data from 209 affected cases from unrelated 209 families, including 80 previously diagnosed and 129 newly diagnosed cases (Cohort 2). Among the 90 types of variants identified in 209 cases, the NM_003494.3: c.2997G>T; p.Trp999Cys, was the most frequent (96/420; 22.9%), followed by c.1566C>G; p.Tyr522* (45/420; 10.7%) on an allele base. p.Trp999Cys was found in 70/209 cases (33.5%), including 20/104 cases (19.2%) with the Miyoshi muscular phenotype and 43/82 cases (52.4%) with the limb-girdle phenotype. In the analysis of missense variants, p.Trp992Arg, p.Trp999Arg, p.Trp999Cys, p.Ser1000Phe, p.Arg1040Trp, and p.Arg1046His were located in the inner DysF domain, representing in 113/160 missense variants (70.6%). This large cohort highlighted the frequent missense variants located in the inner DysF domain as a hotspot for missense variants among our cohort of 209 cases (>95%, Japanese) and hinted at their potential as targets for future therapeutic strategies.
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http://dx.doi.org/10.1002/humu.24036DOI Listing
September 2020

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

PLoS Genet 2020 02 26;16(2):e1008628. Epub 2020 Feb 26.

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

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.
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http://dx.doi.org/10.1371/journal.pgen.1008628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062289PMC
February 2020

Biallelic variants/mutations of IL1RAP in patients with steroid-sensitive nephrotic syndrome.

Int Immunol 2020 04;32(4):283-292

Department of Microbiology and Immunology, Endocrinology and Vascular Medicine, Sendai, Japan.

Nephrotic syndrome (NS) is a renal disease characterized by severe proteinuria and hypoproteinemia. Although several single-gene mutations have been associated with steroid-resistant NS, causative genes for steroid-sensitive NS (SSNS) have not been clarified. While seeking to identify causative genes associated with SSNS by whole-exome sequencing, we found compound heterozygous variants/mutations (c.524T>C; p.I175T and c.662G>A; p.R221H) of the interleukin-1 receptor accessory protein (IL1RAP) gene in two siblings with SSNS. The siblings' parents are healthy, and each parent carries a different heterozygous IL1RAP variant/mutation. Since IL1RAP is a critical subunit of the functional interleukin-1 receptor (IL-1R), we investigated the effect of these variants on IL-1R subunit function. When stimulated with IL-1β, peripheral blood mononuclear cells from the siblings with SSNS produced markedly lower levels of cytokines compared with cells from healthy family members. Moreover, IL-1R with a variant IL1RAP subunit, reconstituted on a hematopoietic cell line, had impaired binding ability and low reactivity to IL-1β. Thus, the amino acid substitutions in IL1RAP found in these NS patients are dysfunctional variants/mutations. Furthermore, in the kidney of Il1rap-/- mice, the number of myeloid-derived suppressor cells, which require IL-1β for their differentiation, was markedly reduced although these mice did not show significantly increased proteinuria in acute nephrotic injury with lipopolysaccharide treatment. Together, these results identify two IL1RAP variants/mutations in humans for the first time and suggest that IL1RAP might be a causative gene for familial NS.
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http://dx.doi.org/10.1093/intimm/dxz081DOI Listing
April 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

Detection of NRAS mutation in cell-free DNA biological fluids from patients with kaposiform lymphangiomatosis.

Orphanet J Rare Dis 2019 09 11;14(1):215. Epub 2019 Sep 11.

Department of Pediatrics, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu, 501-1194, Japan.

Background: Kaposiform lymphangiomatosis (KLA) has recently been distinguished as a novel subtype of generalized lymphatic anomaly (GLA) with foci of spindle endothelial cells. All cases of KLA involve multiple organs and have an unfavorable prognosis. However, the molecular pathogenesis is unknown, and there are no useful biomarkers. In the present study, we performed genetic analysis to elucidate the cause of this disease and detect biomarkers for it.

Methods: We performed whole-exome sequencing of DNA samples from leukocytes and a biopsy specimen and analyzed cell-free DNA (cfDNA) from plasma and pleural effusion of patients to identify the NRAS c.182A > G (p.Q61R) mutation using the droplet digital polymerase chain reaction (ddPCR).

Results: All KLA patients (patients 1-5) had invasive and aggressive features (hemorrhagic pleural effusions, coagulation disorder, and thrombocytopenia) and characteristic findings of KLA in their pathological examinations. In whole exome sequencing for patient 1, c.182A > G missense variant (p.Q61R) in NRAS was identified in fresh frozen samples of a mass on the left chest wall at a frequency of 5% of total alleles but not in his blood leukocytes. Furthermore, the same mutation was detected in cfDNA isolated from plasma and pleural effusion by using ddPCR. ddPCR analysis of plasma/pleural effusion samples from an additional four KLA patients showed that the same mutation was detected in isolated cfDNA in three of the four, as well as in a tissue sample from one of the three plasma/effusion-positive patients that had been obtained to confirm the mutation.

Conclusion: These results provide the first evidence that NRAS oncogenic variant was identified in DNA samples from KLA patients from not only two affected lesions but also plasma and pleural effusion.
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http://dx.doi.org/10.1186/s13023-019-1191-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737666PMC
September 2019

LZTR1 facilitates polyubiquitination and degradation of RAS-GTPases.

Cell Death Differ 2020 03 23;27(3):1023-1035. Epub 2019 Jul 23.

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

Leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a member of the BTB-Kelch superfamily, which interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Mutations in LZTR1 have been identified in glioblastoma, schwannomatosis, and Noonan syndrome. However, the functional role of LZTR1 in carcinogenesis or human development is not fully understood. Here, we demonstrate that LZTR1 facilitates the polyubiquitination and degradation of RAS via the ubiquitin-proteasome pathway, leading to the inhibition of the RAS/MAPK signaling. The polyubiquitination and degradation of RAS was also observed in cells expressing MRAS, HRAS, NRAS, and KRAS as well as oncogenic RAS mutants and inhibited the activation of ERK1/2 and cell growth. In vivo ubiquitination assays showed that MRAS-K127 and HRAS-K170 were ubiquitinated by LZTR1 and that the polyubiquitinated-chains contained mainly Ub-K48, K63, and K33-linked chains, suggesting its possible involvement in autophagy. Immunoprecipitation analyses showed the interaction of LZTR1 and RAS-GTPases with autophagy-related proteins, including LC3B and SQSTM1/p62. Co-expression of LZTR1 and RAS increased the expression of lipidated form of LC3B. However, long-term treatment with chloroquine had little effect on RAS protein levels, suggesting that the contribution of autophagy to LZTR1-mediated RAS degradation is minimal. Taken together, these results show that LZTR1 functions as a "RAS killer protein" mainly via the ubiquitin-proteasome pathway regardless of the type of RAS GTPase, controlling downstream signal transduction. Our results also suggest a possible association of LZTR1 and RAS-GTPases with the autophagy. These findings provide clues for the elucidation of the mechanisms of RAS degradation and regulation of the RAS/MAPK signaling cascade.
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http://dx.doi.org/10.1038/s41418-019-0395-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206011PMC
March 2020

Genotype-phenotype correlation analysis in Japanese patients with Noonan syndrome.

Endocr J 2019 Nov 10;66(11):983-994. Epub 2019 Jul 10.

Department of Gastroenterology and Endocrinology, Osaka Women's and Children's Hospital, Izumi, Osaka 594-1101, Japan.

Noonan syndrome (NS) is a heterogeneous disorder with multiple congenital malformations. Recent advances in molecular and genetic approaches have identified a number of responsible genes for NS, most of which are components of the RAS/MAPK signaling pathway, and genotype-phenotype correlation analyses have been extensively performed; however, analysis of Japanese NS patients is limited. Here, we evaluated clinical characteristics in genetically diagnosed NS patients and their relationships to genotypes. A total of 48 clinically diagnosed NS were included, and responsible mutations were identified in 39 patients (81.3%) with PTPN11 mutations being the most prevalent followed by SOS1 mutations. Cardiac anomalies including pulmonary stenosis and hypertrophic cardiomyopathy were most prevalent (87.2%), and the prevalence of hypertrophic cardiomyopathy was greater in patients without PTPN11 mutations than in those with PTPN11 mutations. Short stature was the second-most prevalent (69.2%) characteristic, and present height SD score was significantly associated with height SD score at 1 year old. Patients with SOS1 mutations had greater present height SD score and better growth during infancy. These findings suggest the presence of a genotype-phenotype correlation in Japanese patients with NS, which enables us to use genetic information to predict the clinical course and may allow for genotype-based medical interventions.
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http://dx.doi.org/10.1507/endocrj.EJ18-0564DOI Listing
November 2019

Germline-Activating RRAS2 Mutations Cause Noonan Syndrome.

Am J Hum Genet 2019 06 23;104(6):1233-1240. Epub 2019 May 23.

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

Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.
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http://dx.doi.org/10.1016/j.ajhg.2019.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562005PMC
June 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

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

Leucine-485 deletion variant of BRAF may exhibit the severe end of the clinical spectrum of CFC syndrome.

J Hum Genet 2019 May 6;64(5):499-504. Epub 2019 Mar 6.

Department of Pediatric Neurology, Miyagi Children's Hospital, Sendai, 989-3126, Japan.

The genotype-phenotype correlation in BRAF variant in cardio-facio-cutaneous (CFC) syndrome is not clearly defined. Here we report a case with a severe clinical phenotype and a novel BRAF variant, p.Leu485del. The present case showed severe intellectual disability, impaired awareness, hyperekplexia, involuntary movements, early onset refractory seizures, and delayed myelination on brain magnetic resonance imaging as well as a polycystic and dysplastic kidney, which are previously unreported anomalies in CFC or RAS/mitogen-activated protein kinase syndromes related to BRAF variant. CFC syndrome, especially caused by BRAF variant, should be included in the differential diagnosis of patients with developmental and epileptic encephalopathies and hyperekplexia. Furthermore, we need to keep in mind that missense variants or the deletion of Leucine-485 may be associated with severe symptoms.
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http://dx.doi.org/10.1038/s10038-019-0579-3DOI Listing
May 2019

Biallelic GALM pathogenic variants cause a novel type of galactosemia.

Genet Med 2019 06 19;21(6):1286-1294. Epub 2018 Oct 19.

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

Purpose: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia.

Methods: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants.

Results: The highest blood galactose levels observed in each patient were 17.3-41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between β- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients' peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins.

Conclusion: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.
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http://dx.doi.org/10.1038/s41436-018-0340-xDOI Listing
June 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

Novel IARS2 mutations in Japanese siblings with CAGSSS, Leigh, and West syndrome.

Brain Dev 2018 Nov 2;40(10):934-938. Epub 2018 Jul 2.

Department of Pediatrics, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.

Background: IARS2 encodes isoleucine-tRNA synthetase, which is aclass-1 amino acyl-tRNA synthetase. IARS2 mutations are reported to cause Leigh syndrome or cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysphasia syndrome (CAGSSS). To our knowledge, IARS2 mutations and diseases related to it have only been reported in three families. Here we report a case of two Japanese siblings with Leigh syndrome, some features of CAGSSS, and West syndrome that are found to have compound heterozygous novel IARS2 mutations.

Case Report: A 7-month-old Japanese girl presented with infantile spasms. Brain magnetic resonance imaging (MRI) revealed diffuse brain atrophy and hyperintensity in the bilateral basal ganglia. Three years later, her younger sister also presented with infantile spasms. MRI revealed diffuse brain atrophy and hyperintensity of the bilateral ganglia, suggesting Leigh syndrome. The siblings were identified with compound heterozygous missense mutations in IARS2, p.[(Phe227Ser)];[(Arg817His)].

Conclusion: This is the first case study reporting Leigh syndrome concomitant with some features of CAGSSS in siblings with novel IARS2 mutations, thereby broadening the phenotypic spectrum of IARS2-related disorders. Further studies are warranted to elucidate the nature of these disorders.
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http://dx.doi.org/10.1016/j.braindev.2018.06.010DOI Listing
November 2018

Co-occurrence of hypertrophic cardiomyopathy and juvenile myelomonocytic leukemia in a neonate with Noonan syndrome, leading to premature death.

Clin Case Rep 2018 Jul 8;6(7):1202-1207. Epub 2018 May 8.

Department of Hematology and Oncology Kobe Children's Hospital Kobe Japan.

We report a case of a neonate with Noonan syndrome presenting with concurrent hypertrophic cardiomyopathy and juvenile myelomonocytic leukemia, which resulted in premature death. Cases with Noonan syndrome diagnosed during the neonatal period might not necessarily show mild clinical course, and premature death is a possible outcome to be considered.
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http://dx.doi.org/10.1002/ccr3.1568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028379PMC
July 2018

Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment.

Nat Commun 2018 05 17;9(1):1960. Epub 2018 May 17.

Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan.

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.
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http://dx.doi.org/10.1038/s41467-018-04193-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958119PMC
May 2018

Genomic analysis identifies masqueraders of full-term cerebral palsy.

Ann Clin Transl Neurol 2018 May 26;5(5):538-551. Epub 2018 Mar 26.

Department of Pediatrics Tohoku University School of Medicine 2-1 Seiryo-machi, Aoba-ku Sendai Miyagi 980-8575 Japan.

Objective: Cerebral palsy is a common, heterogeneous neurodevelopmental disorder that causes movement and postural disabilities. Recent studies have suggested genetic diseases can be misdiagnosed as cerebral palsy. We hypothesized that two simple criteria, that is, full-term births and nonspecific brain MRI findings, are keys to extracting masqueraders among cerebral palsy cases due to the following: (1) preterm infants are susceptible to multiple environmental factors and therefore demonstrate an increased risk of cerebral palsy and (2) brain MRI assessment is essential for excluding environmental causes and other particular disorders.

Methods: A total of 107 patients-all full-term births-without specific findings on brain MRI were identified among 897 patients diagnosed with cerebral palsy who were followed at our center. DNA samples were available for 17 of the 107 cases for trio whole-exome sequencing and array comparative genomic hybridization. We prioritized variants in genes known to be relevant in neurodevelopmental diseases and evaluated their pathogenicity according to the American College of Medical Genetics guidelines.

Results: Pathogenic/likely pathogenic candidate variants were identified in 9 of 17 cases (52.9%) within eight genes: ,,,,,,, and . Five identified variants had previously been reported. No pathogenic copy number variations were identified. The missense variant and the splice-site variants in and were validated by in vitro functional experiments.

Interpretation: The high rate of detecting causative genetic variants (52.9%) suggests that patients diagnosed with cerebral palsy in full-term births without specific MRI findings may include genetic diseases masquerading as cerebral palsy.
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http://dx.doi.org/10.1002/acn3.551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945967PMC
May 2018

Rett-like features and cortical visual impairment in a Japanese patient with HECW2 mutation.

Brain Dev 2018 May 1;40(5):410-414. Epub 2018 Feb 1.

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

Numerous genetic syndromes that include intellectual disability (ID) have been reported. Recently, HECW2 mutations were detected in patients with ID and growth development disorders. Four de novo missense mutations have been reported. Here, we report a Japanese girl with Rett-like symptoms of severe ID, hypotonia, refractory epilepsy, and stereotypical hand movement (hand tapping, flapping, and wringing) after the age of 1 year. Characteristically, she had cortical visual impairment. She had difficulty swallowing since the age of 4 years, and diminished activity was noticeable since the age of 12 years, suggesting neurodevelopmental regression. She has no acquired microcephaly, and brain magnetic resonance imaging showed non-specific mild cerebral and cerebellar atrophy without progression over time. Genetic analyses of MECP2, CDKL5, and FOXG1 were negative. Whole-exome sequencing analysis revealed a known de novo mutation (c.3988C > T) in HECW2. The characteristics of her clinical symptoms are severe cortical visual impairment and Rett-like phenotype such as involuntary movements and regression. This is the first report that patients with HECW2 mutation could show Rett-like feature.
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http://dx.doi.org/10.1016/j.braindev.2017.12.015DOI Listing
May 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

Activated Braf induces esophageal dilation and gastric epithelial hyperplasia in mice.

Hum Mol Genet 2017 12;26(23):4715-4727

Department of Medical Genetics.

Germline mutations in BRAF are a major cause of cardio-facio-cutaneous (CFC) syndrome, which is characterized by heart defects, characteristic craniofacial dysmorphology and dermatologic abnormalities. Patients with CFC syndrome also commonly show gastrointestinal dysfunction, including feeding and swallowing difficulties and gastroesophageal reflux. We have previously found that knock-in mice expressing a Braf Q241R mutation exhibit CFC syndrome-related phenotypes, such as growth retardation, craniofacial dysmorphisms, congenital heart defects and learning deficits. However, it remains unclear whether BrafQ241R/+ mice exhibit gastrointestinal dysfunction. Here, we report that BrafQ241R/+ mice have neonatal feeding difficulties and esophageal dilation. The esophagus tissues from BrafQ241R/+ mice displayed incomplete replacement of smooth muscle with skeletal muscle and decreased contraction. Furthermore, the BrafQ241R/+ mice showed hyperkeratosis and a thickened muscle layer in the forestomach. Treatment with MEK inhibitors ameliorated the growth retardation, esophageal dilation, hyperkeratosis and thickened muscle layer in the forestomach in BrafQ241R/+ mice. The esophageal dilation with aberrant skeletal-smooth muscle boundary in BrafQ241R/+ mice were recovered after treatment with the histone H3K27 demethylase inhibitor GSK-J4. Our results provide clues to elucidate the pathogenesis and possible treatment of gastrointestinal dysfunction and failure to thrive in patients with CFC syndrome.
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http://dx.doi.org/10.1093/hmg/ddx354DOI Listing
December 2017

Long-term outcome of a 26-year-old woman with West syndrome and an nuclear receptor subfamily 2 group F member 1 gene (NR2F1) mutation.

Seizure 2017 Aug 20;50:144-146. Epub 2017 Jun 20.

Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Electronic address:

Long-term outcome of West syndrome with a NR2F1 mutation.
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http://dx.doi.org/10.1016/j.seizure.2017.06.018DOI Listing
August 2017

Craniosynostosis in patients with RASopathies: Accumulating clinical evidence for expanding the phenotype.

Am J Med Genet A 2017 Sep 26;173(9):2346-2352. Epub 2017 Jun 26.

Department of Medical Genetics, Osaka Women's and Children's Hospital, Osaka Prefectural Hospital Organization, Osaka, Japan.

RASopathies are phenotypically overlapping genetic disorders caused by dysregulation of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. RASopathies include Noonan syndrome, cardio-facio-cutaneous (CFC) syndrome, Costello syndrome, Neurofibromatosis type 1, Legius syndrome, Noonan syndrome with multiple lentigines, Noonan-like syndrome, hereditary gingival fibromatosis, and capillary malformation/arteriovenous malformation syndrome. Recently, six patients with craniosynostosis and Noonan syndrome involving KRAS mutations were described in a review, and a patient with craniosynostosis and Noonan syndrome involving a SHOC2 mutation has also been reported. Here, we describe patients with craniosynostosis and Noonan syndrome due to de novo mutations in PTPN11 and patients with craniosynostosis and CFC syndrome due to de novo mutations in BRAF or KRAS. All of these patients had cranial deformities in addition to the typical phenotypes of CFC syndrome and Noonan syndrome. In RASopathy, patients with cranial deformities, further assessments may be necessary to look for craniosynostosis. Future studies should attempt to elucidate the pathogenic mechanism responsible for craniosynostosis mediated by the RAS/MAPK signaling pathway.
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http://dx.doi.org/10.1002/ajmg.a.38337DOI Listing
September 2017
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