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Congenital hypothyroidism due to thyroid dysgenesis (TD), presented as thyroid aplasia, hypoplasia or ectopia, is one of the most prevalent rare diseases with an isolated organ malformation. The pathogenesis of TD is largely unknown, although a genetic predisposition has been suggested. We performed a genome-wide association study (GWAS) with 142 Japanese TD cases and 8380 controls and found a significant locus at 2q33.3 (top single nucleotide polymorphism, rs9789446: P = 4.4 × 10-12), which was replicated in a German patient cohort (P = 0.0056). A subgroup analysis showed that rs9789446 confers a risk for thyroid aplasia (per allele odds ratio = 3.17) and ectopia (3.12) but not for hypoplasia. Comprehensive epigenomic characterization of the 72-kb disease-associated region revealed that it was enriched for active enhancer signatures in human thyroid. Analysis of chromosome conformation capture data showed long-range chromatin interactions of this region with promoters of two genes, FZD5 and CCNYL1, mediating Wnt signaling. Moreover, rs9789446 was found to be a thyroid-specific quantitative trait locus, adding further evidence for a cis-regulatory function of this region in thyroid tissue. Specifically, because the risk rs9789446 allele is associated with increased thyroidal expression of FDZ5 and CCNYL1 and given the recent demonstration of perturbed early thyroid development following overactivation of Wnt signaling in zebrafish embryos, an enhanced Wnt signaling in risk allele carriers provides a biologically plausible TD mechanism. In conclusion, our work found the first risk locus for TD, exemplifying that in rare diseases with relatively low biological complexity, GWAS may provide mechanistic insights even with a small sample size.
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Estudo de Associação Genômica Ampla , Disgenesia da Tireoide , Animais , Humanos , Peixe-Zebra/genética , Via de Sinalização Wnt/genética , Doenças Raras , Disgenesia da Tireoide/genética , Predisposição Genética para DoençaRESUMO
Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.
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Divisão Celular Assimétrica/genética , Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Oryza , Zigoto/citologia , Divisão Celular/genética , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteína Quinase 6 Ativada por Mitógeno/genética , Oryza/embriologia , Oryza/enzimologia , Oryza/genética , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismoRESUMO
To overcome a limitation to the breeding of autogamous crops, recurrent selection using transgenic male sterility (RSUTMS) has been proposed. In this system, negatively or positively selectable marker traits are required along with dominant transgenic male sterility. Anthocyanin pigmentation is an excellent marker trait. Two regulatory genes for MYB and bHLH and a structural gene for DFR are required for anthocyanin pigmentation in rice. Therefore, to apply anthocyanin pigmentation as a marker trait in various rice genotypes, coordinated expression of the three genes is required. In this study, we developed a leaf sheath-specific promoter and introduced three genes-DFR and C1/Myb, driven by the 35S promoter, and OsB2/bHLH, driven by the leaf sheath-specific promoter-into the rice genome. Leaf sheath-specific pigmentation was confirmed in all seven genotypes tested, which included japonica and indica cultivars. Analysis of genome sequence data from 25 cultivars showed that the strategy of conferring leaf sheath-specific anthocyanin pigmentation by introduction of these three genes would be effective for a wide range of genotypes and will be applicable to RSUTMS.
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Because genomic selection is designed for the population breeding of allogamous species, a successive outcrossing system is required for efficient use of genomic selection in autogamous crops, such as Oryza sativa L. (rice). Transgenic and dominant male-sterility is a suitable tool for efficient outcrossing of autogamous crops. Though there have been some reports of dominant male-sterile rice developed using transgenic technology, the flowering habit was substandard. Here, to isolate promoters that, when linked to a lethal gene, induce dominant male-sterility while retaining a good flowering habit, we identified 38 candidate genes with anther-specific expression by using the 'RiceXPro' database. We then evaluated the abilities of the near-upstream regions of these genes to induce male-sterility when linked to the lethal gene barnase and introduced into the rice cultivar 'Nipponbare'. Seven of the 38 promoters induced clear dominant male-sterility; promoters expressed in the later stage of anther development induced male-sterility while retaining better flowering habits when compared to ones expressed in the early stage. These seven promoters could potentially be used to facilitate development of an efficient outcross-based breeding system in rice.
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Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice (Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9. Barnase-induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice.
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Mutations in DUOXA2, encoding dual oxidase maturation factor 2, is a rare genetic cause of congenital hypothyroidism. Only four biallelic DUOXA2 mutation carriers have been described to date. This study was conducted to report the clinical and genetic findings of a DUOXA2 mutation-carrying family, and to review the previously reported cases. The proband was a 4-year-old girl, who was diagnosed as having congenital hypothyroidism in the frame of newborn screening. She had a high serum TSH level (138 mU/L) and a low free T4 level (0.4 ng/dL). Ultrasonography revealed goiter. She was immediately treated with levothyroxine. At age 3 years, reevaluation of her thyroid function showed a slightly elevated serum TSH level (11.0 mU/L) with normal free T4 level. Screening of the eleven congenital hypothyroidism-related genes demonstrated a previously reported nonsense DUOXA2 mutation (p.Tyr138*) in the homozygous state. Unexpectedly, we also found that the elder brother of the proband, who had no significant past medical history, had the identical homozygous mutation. Using expression experiments with HEK293 cells, we confirmed that p.Tyr138* was a loss-of-function mutation. In the literature, clinical courses of three patients were described, showing characteristic age-dependent improvement of the thyroid function. In conclusion, The proband showed comparable clinical phenotype to previously reported cases, while her brother was unaffected. The phenotypic spectrum of DUOXA2 mutations could be broader than currently accepted.
Assuntos
Hipotireoidismo Congênito/genética , Proteínas de Membrana/genética , Glândula Tireoide/diagnóstico por imagem , Tireotropina/sangue , Tiroxina/uso terapêutico , Pré-Escolar , Hipotireoidismo Congênito/sangue , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/tratamento farmacológico , Feminino , Células HEK293 , Homozigoto , Humanos , Recém-Nascido , Masculino , Mutação , Triagem Neonatal , Irmãos , Tiroxina/sangue , Resultado do Tratamento , UltrassonografiaRESUMO
Thyroid peroxidase (TPO) deficiency, caused by biallelic TPO mutations, is a well-established genetic form of congenital hypothyroidism (CH). More than 100 patients have been published, and the patients have been diagnosed mostly in the frame of newborn screening (NBS) programs. Correlation between clinical phenotypes and TPO activity remains unclear. Here, we report clinical and molecular findings of two unrelated TPO mutation-carrying mildly hypothyroid patients. The two patients were born at term after an uneventful pregnancy and delivery, and were NBS negative. They sought medical attention due to goiter at age 8 years. Evaluation of the thyroid showed mild elevation of serum TSH levels, normal or slightly low serum T4 levels, high serum T3 to T4 molar ratio, high serum thyroglobulin levels, and high thyroidal 123I uptake. We performed next-generation sequencing-based genetic screening, and found that one patient was compound heterozygous for two novel TPO mutations (p.Asp224del; c.820-2A>G), and the other was homozygous for a previously known mutation (p.Trp527Cys). In vitro functional analyses using HEK293 cells showed that the two amino acid-altering mutations (p.Asp224del and p.Trp527Cys) caused partial loss of the enzymatic activity. In conclusion, we report that TPO mutations with residual activity are associated with mild TPO deficiency, which is clinically characterized by marked goiter, mild TSH elevation, high serum T3 to T4 molar ratio, and high serum thyroglobulin levels. Our findings illuminate the hitherto under-recognized correlation between clinical phenotypes and residual enzymatic activity among patients with TPO deficiency.
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Autoantígenos/genética , Autoantígenos/metabolismo , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Iodeto Peroxidase/deficiência , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Mutação , Criança , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/patologia , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Testes Genéticos , Células HEK293 , Humanos , Recém-Nascido , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Masculino , Triagem Neonatal , Linhagem , Fenótipo , Índice de Gravidade de DoençaRESUMO
Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ciclopentanos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oxilipinas/farmacologia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Perfilação da Expressão Gênica , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salinidade , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVES: Mutations in the dual oxidase 2 gene (DUOX2) is the most common genetic cause of congenital hypothyroidism (CH) in Japan. All previously described DUOX2 mutation-carrying families have followed autosomal recessive inheritance. We report a nonconsanguineous Japanese family harbouring biallelic DUOX2 mutations, which presented an apparently dominant inheritance of nonautoimmune hypothyroidism. DESIGN AND METHODS: The proband and her two sisters had been diagnosed as having CH on newborn screening and were treated with levothyroxine. Their mother had subclinical hypothyroidism. We sequenced DUOX2 in the proband and her family members. Pathogenicity of the identified novel mutation (p.Y1347C) was verified in vitro. RESULTS: We found that the proband and her sisters were compound heterozygous for a novel DUOX2 mutation p.Y1347C and a previously reported functional variant p.H678R. Unexpectedly, we found that the mother was homozygous for p.H678R. Expression experiments showed that the p.Y1347C mutant had reduced H2 O2 -producing activity, although there was no significant difference in the level of protein expression or localization, between wild type and p.Y1347C. CONCLUSIONS: We report a DUOX2 mutation-carrying pedigree presenting pseudodominant inheritance of nonautoimmune hypothyroidism. We speculate that the relatively high frequency of DUOX2 mutations could lead to pseudodominant inheritance in Japan.
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Hipotireoidismo Congênito/genética , Genes Dominantes , Padrões de Herança/genética , Mutação de Sentido Incorreto , NADPH Oxidases/genética , Alelos , Hipotireoidismo Congênito/metabolismo , Análise Mutacional de DNA , Oxidases Duais , Saúde da Família , Feminino , Genótipo , Humanos , Immunoblotting , Masculino , Microscopia de Fluorescência , NADPH Oxidases/metabolismo , LinhagemRESUMO
During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.
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Proteínas de Arabidopsis/genética , Arabidopsis , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico/genética , Rad51 Recombinase/genética , Recombinases Rec A/genética , Recombinação Genética , Arabidopsis/genética , Centrômero/genética , Segregação de Cromossomos , Cromossomos/genética , Fragmentação do DNA , Eucromatina/genética , Heterocromatina , Meiose/genética , Proteínas Mutantes/genética , Infertilidade das Plantas , Recombinação Genética/genéticaRESUMO
Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.
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Hipotireoidismo Congênito , Oxidases Duais , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Hipotireoidismo Congênito/genética , Oxidases Duais/genética , Mutação , NADPH Oxidases/genética , Tiroxina/uso terapêutico , Adulto JovemRESUMO
Insufficient thyroid hormone production in newborns is referred to as congenital hypothyroidism. Multinodular goiter (MNG), characterized by an enlarged thyroid gland with multiple nodules, is usually seen in adults and is recognized as a separate disorder from congenital hypothyroidism. Here we performed a linkage analysis of a family with both nongoitrous congenital hypothyroidism and MNG and identified a signal at 15q26.1. Follow-up analyses with whole-genome sequencing and genetic screening in congenital hypothyroidism and MNG cohorts showed that changes in a noncoding TTTG microsatellite on 15q26.1 were frequently observed in congenital hypothyroidism (137 in 989) and MNG (3 in 33) compared with controls (3 in 38,722). Characterization of the noncoding variants with epigenomic data and in vitro experiments suggested that the microsatellite is located in a thyroid-specific transcriptional repressor, and its activity is disrupted by the variants. Collectively, we presented genetic evidence linking nongoitrous congenital hypothyroidism and MNG, providing unique insights into thyroid abnormalities.
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Cromossomos Humanos Par 15 , Hipotireoidismo Congênito , Repetições de Microssatélites , Linhagem , Humanos , Hipotireoidismo Congênito/genética , Repetições de Microssatélites/genética , Feminino , Masculino , Cromossomos Humanos Par 15/genética , Bócio Nodular/genética , Adulto , Glândula Tireoide/patologia , Glândula Tireoide/metabolismo , Ligação GenéticaRESUMO
CONTEXT: Thyroglobulin (Tg), encoded by TG, is essential for thyroid hormone synthesis. TG defects result in congenital hypothyroidism (CH). Most reported patients were born before the introduction of newborn screening (NBS). OBJECTIVE: We aimed to clarify the phenotypic features of patients with TG defects diagnosed and treated since the neonatal period. METHODS: We screened 1061 patients with CH for 13 CH-related genes and identified 30 patients with TG defects. One patient was diagnosed due to hypothyroidism-related symptoms and the rest were diagnosed via NBS. Patients were divided into 2 groups according to their genotypes, and clinical characteristics were compared. We evaluated the functionality of the 7 missense variants using HEK293 cells. RESULTS: Twenty-seven rare TG variants were detected, including 15 nonsense, 3 frameshift, 2 splice-site, and 7 missense variants. Patients were divided into 2 groups: 13 patients with biallelic truncating variants and 17 patients with monoallelic/biallelic missense variants. Patients with missense variants were more likely to develop thyroid enlargement with thyrotropin stimulation than patients with biallelic truncating variants. Patients with biallelic truncating variants invariably required full hormone replacement, whereas patients with missense variants required variable doses of levothyroxine. Loss of function of the 7 missense variants was confirmed in vitro. CONCLUSION: To our knowledge, this is the largest investigation on the clinical presentation of TG defects diagnosed in the neonatal period. Patients with missense variants showed relatively mild hypothyroidism with compensative goiter. Patients with only truncating variants showed minimal or no compensative goiter and required full hormone replacement.
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Hipotireoidismo Congênito , Estudos de Associação Genética , Triagem Neonatal , Tireoglobulina , Humanos , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/diagnóstico , Feminino , Masculino , Tireoglobulina/genética , Recém-Nascido , Japão/epidemiologia , Pré-Escolar , Mutação de Sentido Incorreto , Lactente , Criança , Fenótipo , Genótipo , Células HEK293 , População do Leste AsiáticoRESUMO
BACKGROUND: Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. RESULTS: We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin--an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases--on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. CONCLUSIONS: These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.
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Recombinação Homóloga , Meristema/enzimologia , Oryza/enzimologia , Proteínas de Plantas/genética , RecQ Helicases/genética , Pontos de Checagem da Fase S do Ciclo Celular , Morte Celular , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Meristema/citologia , Meristema/genética , Mutação , Oryza/citologia , Oryza/genética , Proteínas de Plantas/metabolismo , RecQ Helicases/metabolismoRESUMO
In plants, mitogen activated protein kinases (MPKs) are involved in various signaling pathways that lead to biotic and abiotic responses as well as that regulate developmental processes. Among them, MPK6 and its closely related homologue, MPK3, act redundantly and are known to be involved in asymmetric cell divisions of meristemoid mother cells in stomata development and of zygotes in Arabidopsis. Loss-of-function mutants of GLE4/OsMPK6, which is an orthologue of MPK6 in rice, showed a defect in polarity establishment in early stage of embryogenesis. However, because of the embryo lethality of the mutations, the function of GLE4/OsMPK6 in post-embryonic development is not clarified. Here, we report the analysis of post embryonic function of GLE4/OsMPK6 in vegetative stage of rice using regenerated gle4/osmpk6 homozygous plants from tissue culture. The regenerated plants are dwarf and produce multiple shoots with small leaves. These shoots never develop into reproductive stage, instead, proliferate vegetative shoots repeatedly. Leaves of gle4/osmpk6 have small leaf blade at the tip and blade-sheath boundary become obscure. Stomata arrangement is also disturbed in gle4/osmpk6 leaf blade. The shape of shoot apical meristem of gle4/osmpk6 become disorganized. Thus, GLE4/OsMPK6 functions in shoot organization and stomata patterning in the post embryonic development in rice.
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During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Exodesoxirribonucleases/genética , Recombinação Homóloga/genética , Oryza/genética , RecQ Helicases/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Genes Reporter , Marcadores Genéticos , Glucuronidase , Modelos Moleculares , Oryza/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RecQ Helicases/metabolismo , Plântula/enzimologia , Plântula/genéticaRESUMO
Context: Thyroid hormone has been shown to have a protective role in neuronal injury, although the mechanisms have not been established. The cellular response to stress that promotes adaptation and survival has been shown to involve epigenetic modifications. Objective: We hypothesized that the neuroprotective role of thyroid hormone was associated with epigenetic modifications of histone proteins. We used hypoxic neurons as a model system for hypoxia-induced brain injury. Methods: Mouse primary cortical neurons were exposed to 0.2% oxygen for 7â hours, with or without, treatment with triiodothyronine (T3). We analyzed the expression of histone-modifying enzymes by RNA-seq and the post-translationally modified histone 3 proteins by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results: We found that methylation of H3K27, associated with inactive promoters, was highly induced in hypoxic neurons, and this histone methylation was reduced by T3 treatment. H3K4 methylation is the hallmark of active promoters. The expression of 3 (Set1db, Kmta2c, and Kmt2e) out of 6 H3K4 methyltransferases was downregulated by hypoxia and expression was restored by T3 treatment. H3K4me3 protein, measured by ELISA, was increased 76% in T3-treated hypoxic neurons compared with the levels without T3 treatment. H3K56ac plays a critical role in transcription initiation and was markedly increased in T3-treated hypoxic neurons compared with those without T3 treatment, indicating stimulation of gene transcription. Additionally, T3 treatment restored hypoxia-induced downregulation of histone acetyltransferase, Kat6a, Kat6b, and Crebbp, which function as transcription factors. Conclusion: These findings indicate that T3 treatment mitigates hypoxia-induced histone modifications and protects neurons from hypoxia-induced injury.
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PAX8 is a transcription factor that is expressed in the thyroid gland and kidneys. Monoallelic loss-of-function PAX8 variants cause congenital hypothyroidism (CH), and urogenital malformations are infrequent complications seen in less than 10% of PAX8 variant carriers. Herein, we report the case of a 3-yr-old female patient with CH who was diagnosed during newborn screening. She was treated with levothyroxine, and she showed normal growth and development at a minimal dose (0.7 µg/kg/d of levothyroxine at 3 yr of age). At 5 mo of age, she visited an emergency department for fever and was incidentally found to have differently sized kidneys by ultrasonography, which was subsequently diagnosed as unilateral multicystic dysplastic kidney. Her serum creatinine and cystatin C levels were normal. Next-generation sequencing-based genetic analysis revealed that the patient was heterozygous for a PAX8 frameshift variant (p.Thr320ProfsTer106) and a DUOX2 missense variant (p.Arg885Gln). Our patient is the first truncating PAX8 variant carrier to have a urogenital malformation with CH. Genetic analysis for PAX8 should be considered in patients with CH and urogenital malformations.
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Glycogen storage disease type I is an autosomal recessive disorder caused by the defect in the glucose-6-phosphate enzyme system. Frequent intake of glucose-containing glycogen storage disease formula, uncooked cornstarch, or both, are usually needed to maintain normal blood glucose level. We report a glycogen storage disease type 1b girl with biotin deficiency caused by an exclusive glucose-containing glycogen storage disease formula for years, presenting with the appearance of severe skin lesions, and diagnosed by urinary organic acid analysis by gas chromato-spectrometry, and blood acylcarnitine analysis by tandem mass-spectrometry.