RESUMO
We used N-ethyl-N-nitrosurea-induced germline mutagenesis combined with automated meiotic mapping to identify specific systolic blood pressure (SBP) and heart rate (HR) determinant loci. We analyzed 43,627 third-generation (G3) mice from 841 pedigrees to assess the effects of 45,378 variant alleles within 15,760 genes, in both heterozygous and homozygous states. We comprehensively tested 23% of all protein-encoding autosomal genes and found 87 SBP and 144 HR (with 7 affecting both) candidates exhibiting detectable hypomorphic characteristics. Unexpectedly, only 18 of the 87 SBP genes were previously known, while 26 of the 144 genes linked to HR were previously identified. Furthermore, we confirmed the influence of two genes on SBP regulation and three genes on HR control through reverse genetics. This underscores the importance of our research in uncovering genes associated with these critical cardiovascular risk factors and illustrate the effectiveness of germline mutagenesis for defining key determinants of polygenic phenotypes that must be studied in an intact organism.
Assuntos
Etilnitrosoureia , Camundongos , Animais , Pressão Sanguínea/genética , Frequência Cardíaca/genética , Mutagênese , Etilnitrosoureia/toxicidade , AlelosRESUMO
O camundongo mutante recessivo bate-palmas (bapa) originou-se de mutagênese química induzida por ENU (N-ethyl-N-nitrosourea) e apresenta alterações posturais com movimentos anormais dos membros posteriores quando levantado pela cauda. No sequenciamento do exoma identificou-se uma mutação no gene Kmt2d, localizado no cromossomo 15, que foi confirmada pelo sequenciamento do DNA pelo método de Sanger. A perda da função do gene KMT2D localizada no cromossomo 12 em humanos foi descrita como responsável pela síndrome de Kabuki, que é uma anomalia congênita rara, autossômica dominante. O fenótipo clínico da doença é variável, mas algumas características mais comuns são face dismórfica, anormalidades esqueléticas, alterações nas impressões digitais, leve a moderado retardo mental e retardo do crescimento pós-natal. O presente trabalho teve como objetivo a análise do comportamento e da morfologia craniofacial dos camundongos bapa comparando com modelos de mutação do gene Kmt2d descritos na literatura.(AU)
The recessive mutant mouse named bate-palmas (bapa) claps in Portuguese, originates from an ENU (N-ethyl-N-nitrosourea) mutagenesis program, presenting balance impairment and motor incoordination. Exome sequencing identified a mutation in the KMT2D gene, located on chromosome 15, which was confirmed by DNA sequence by the Sanger method. The loss of function of the gene KMT2D, located on chromosome 12 in humans, was described as being responsible for Kabuki syndrome, also known as Niikawa-Koruki syndrome, which is a rare congenital anomaly, autosomal dominant. The clinical phenotype of the disease is variable, but some common characteristics are dysmorphic facial features, skeletal abnormalities, fingerprint alterations, mild to moderate cognitive problems and postnatal growth retardation. The objective os this study was to analyze the behavior and craniofacial morphology of bapa mice comparing to KMT2D gene mutation models described on literature.(AU)
Assuntos
Animais , Masculino , Camundongos , Camundongos Mutantes/anatomia & histologia , Camundongos Mutantes/psicologia , Comportamento Animal , Mutagênicos , Mutação Puntual , EtilnitrosoureiaRESUMO
O camundongo mutante recessivo bate-palmas (bapa) originou-se de mutagênese química induzida por ENU (N-ethyl-N-nitrosourea) e apresenta alterações posturais com movimentos anormais dos membros posteriores quando levantado pela cauda. No sequenciamento do exoma identificou-se uma mutação no gene Kmt2d, localizado no cromossomo 15, que foi confirmada pelo sequenciamento do DNA pelo método de Sanger. A perda da função do gene KMT2D localizada no cromossomo 12 em humanos foi descrita como responsável pela síndrome de Kabuki, que é uma anomalia congênita rara, autossômica dominante. O fenótipo clínico da doença é variável, mas algumas características mais comuns são face dismórfica, anormalidades esqueléticas, alterações nas impressões digitais, leve a moderado retardo mental e retardo do crescimento pós-natal. O presente trabalho teve como objetivo a análise do comportamento e da morfologia craniofacial dos camundongos bapa comparando com modelos de mutação do gene Kmt2d descritos na literatura.
The recessive mutant mouse named bate-palmas (bapa) claps in Portuguese, originates from an ENU (N-ethyl-N-nitrosourea) mutagenesis program, presenting balance impairment and motor incoordination. Exome sequencing identified a mutation in the KMT2D gene, located on chromosome 15, which was confirmed by DNA sequence by the Sanger method. The loss of function of the gene KMT2D, located on chromosome 12 in humans, was described as being responsible for Kabuki syndrome, also known as Niikawa-Koruki syndrome, which is a rare congenital anomaly, autosomal dominant. The clinical phenotype of the disease is variable, but some common characteristics are dysmorphic facial features, skeletal abnormalities, fingerprint alterations, mild to moderate cognitive problems and postnatal growth retardation. The objective os this study was to analyze the behavior and craniofacial morphology of bapa mice comparing to KMT2D gene mutation models described on literature.
Assuntos
Masculino , Animais , Camundongos , Camundongos Mutantes/anatomia & histologia , Camundongos Mutantes/psicologia , Comportamento Animal , Etilnitrosoureia , Mutagênicos , Mutação PuntualRESUMO
Development of the eyelid requires coordination of the cellular processes involved in proliferation, cell size alteration, migration, and cell death. C57BL/6J-corneal opacity (B6-Co) mice are mutant mice generated by the administration of N-ethyl-N-nitrosourea (100 mg/kg). They exhibit the eyelids open at birth phenotype, abnormal round cell shape from tightened F-actin bundles in leading edge keratinocytes at E16.5, and gradual corneal opacity with neovessels. The tip of the leading edge in B6-Co mice did not move forward, and demonstrated a sharp peak shape without obvious directionality. Analysis of the biological characteristics of B6-Co mice demonstrated that abnormal migration of keratinocytes could affect eyelid development, but proliferation and apoptosis in B6-Co mice had no effect. Mutant gene mapping and sequence analysis demonstrated that in B6-Co mice, adenosine was inserted into the untranslated regions, between 3030 and 3031, in the mRNA 3'-terminal of Fgf10. In addition, guanine 7112 was substituted by adenine in the Mtap1B mRNA, and an A2333T mutation was identified in Mtap1B. Quantitative real-time polymerase chain reaction analysis showed that expression of the Hbegf gene was significantly down-regulated in the eyelids of B6- Co mice at E16.5, compared to B6 mice. However, the expression of Rock1, Map3k1, and Jnk1 genes did not show any significant changes. Abnormal keratinocyte migration and down-regulated expression of the Hbegf gene might be associated with impaired eyelid development in B6-Co mice.
Assuntos
Córnea/metabolismo , Neovascularização da Córnea/genética , Opacidade da Córnea/genética , Pálpebras/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Queratinócitos/metabolismo , Regiões 3' não Traduzidas , Actinas/genética , Actinas/metabolismo , Animais , Movimento Celular , Polaridade Celular , Proliferação de Células , Forma Celular , Córnea/anormalidades , Córnea/crescimento & desenvolvimento , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Embrião de Mamíferos , Etilnitrosoureia , Pálpebras/anormalidades , Pálpebras/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutagênicos , Fenótipo , Cultura Primária de CélulasRESUMO
Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks but also display potentially deleterious mutagenic phenotypes that contribute to antibiotic resistance in bacteria and lead to human disease. Effective activity of these enzymes requires association with ring-shaped processivity factors, which dictate their access to sites of DNA synthesis. Here, we show for the first time that the mismatch repair protein MutS plays a role in regulating access of the conserved Y-family Pol IV to replication sites. Our biochemical data reveals that MutS inhibits the interaction of Pol IV with the ß clamp processivity factor by competing for binding to the ring. Moreover, the MutS-ß clamp association is critical for controlling Pol IV mutagenic replication under normal growth conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the regulation of a replication activity.
Assuntos
DNA Polimerase beta/metabolismo , Replicação do DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pseudomonas aeruginosa/metabolismo , Biocatálise , DNA/biossíntese , DNA/química , DNA Polimerase III/metabolismo , Etilnitrosoureia , Mutagênese/genética , Peptídeos/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/crescimento & desenvolvimento , Resposta SOS em Genética/genética , Especificidade por SubstratoRESUMO
PURPOSE: To evaluate the effects of letrozole (Ltz) in carcinogen+estrogen-induced endometrial hyperplasia. METHODS: BALB/c female mice were divided into four groups of 12 animals each receiving an intrauterine dose of N-ethyl-N-nitrosourea (ENU) and weekly subcutaneous injections of estradiol hexaidrobenzoate (EHB), except for group I(control). The groups were divided in I (control), II (ENU+EHB), III (ENU+EHB+MPA) and IV (ENU+EHB+Ltz). Group III also received intramuscular injections of MPA (medroxy progesterone acetate) every four weeks, while group IV received oral doses of Ltz daily. At the end of 16 weeks, the animals were sacrificed, and blood samples were collected for the measurement of serum estradiol and progesterone levels. Uterine histological sections were made to evaluate the presence of endometrial proliferative lesions. Differences between groups were evaluated with student's t test, ANOVA and chi-square test. RESULTS: Groups ENU+EHB, ENU+EHB+MPA and ENU+EHB+Ltz showed varying degrees of endometrial hyperplasia. The incidence of hyperplasia in groups ENU+EHB and ENU+EHB+Ltz was higher and more severe than in group ENU+EHB+MPA. Control group showed lower levels of serum estradiol than the other groups. CONCLUSION: There was no evidence that letrozole could act as an antiestrogenic drug in the development of endometrial proliferative lesions.
Assuntos
Inibidores da Aromatase/farmacologia , Carcinogênese/efeitos dos fármacos , Hiperplasia Endometrial/tratamento farmacológico , Nitrilas/farmacologia , Triazóis/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/etiologia , Animais , Antineoplásicos Hormonais/farmacologia , Inibidores da Aromatase/uso terapêutico , Carcinogênese/patologia , Hiperplasia Endometrial/induzido quimicamente , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/etiologia , Endométrio/efeitos dos fármacos , Endométrio/patologia , Estradiol/sangue , Etilnitrosoureia , Feminino , Letrozol , Acetato de Medroxiprogesterona/farmacologia , Camundongos Endogâmicos BALB C , Nitrilas/uso terapêutico , Progesterona/sangue , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do Tratamento , Triazóis/uso terapêuticoRESUMO
PURPOSE: To evaluate the effects of letrozole (Ltz) in carcinogen+estrogen-induced endometrial hyperplasia. METHODS: BALB/c female mice were divided into four groups of 12 animals each receiving an intrauterine dose of N-ethyl-N-nitrosourea (ENU) and weekly subcutaneous injections of estradiol hexaidrobenzoate (EHB), except for group I(control). The groups were divided in I (control), II (ENU+EHB), III (ENU+EHB+MPA) and IV (ENU+EHB+Ltz). Group III also received intramuscular injections of MPA (medroxy progesterone acetate) every four weeks, while group IV received oral doses of Ltz daily. At the end of 16 weeks, the animals were sacrificed, and blood samples were collected for the measurement of serum estradiol and progesterone levels. Uterine histological sections were made to evaluate the presence of endometrial proliferative lesions. Differences between groups were evaluated with student's t test, ANOVA and chi-square test. RESULTS: Groups ENU+EHB, ENU+EHB+MPA and ENU+EHB+Ltz showed varying degrees of endometrial hyperplasia. The incidence of hyperplasia in groups ENU+EHB and ENU+EHB+Ltz was higher and more severe than in group ENU+EHB+MPA. Control group showed lower levels of serum estradiol than the other groups. CONCLUSION: There was no evidence that letrozole could act as an antiestrogenic drug in the development of endometrial proliferative lesions.
Assuntos
Animais , Feminino , Triazóis/farmacologia , Inibidores da Aromatase/farmacologia , Hiperplasia Endometrial/tratamento farmacológico , Carcinogênese/efeitos dos fármacos , Nitrilas/farmacologia , Progesterona/sangue , Fatores de Tempo , Triazóis/uso terapêutico , Adenocarcinoma/etiologia , Adenocarcinoma/tratamento farmacológico , Reprodutibilidade dos Testes , Resultado do Tratamento , Neoplasias do Endométrio/etiologia , Neoplasias do Endométrio/tratamento farmacológico , Acetato de Medroxiprogesterona/farmacologia , Antineoplásicos Hormonais/farmacologia , Inibidores da Aromatase/uso terapêutico , Hiperplasia Endometrial/induzido quimicamente , Hiperplasia Endometrial/patologia , Endométrio/efeitos dos fármacos , Endométrio/patologia , Estradiol/sangue , Etilnitrosoureia , Carcinogênese/patologia , Camundongos Endogâmicos BALB C , Nitrilas/uso terapêuticoRESUMO
In the present study, we described the phenotype, histologic morphology, and molecular etiology of a mouse model of unstable hemoglobin Santa Ana. Hematologic evaluation of anemic mice (Anem/+) discovered after N-ethyl-N-nitrosourea mutagenesis revealed moderate anemia with intense reticulocytosis and polychromasia, followed by anisocytosis, macrocytosis, hypochromia, and intraerythrocytic inclusion and Heinz bodies. The mice also demonstrated hemoglobinuria, bilirubinemia, and erythrocytic populations with differing resistance to osmotic lysis. Splenomegaly (particularly in older mutant mice) and jaundice were apparent at necropsy. Histopathologic examination revealed dramatically increased hematopoiesis and hemosiderosis in hematopoietic organs and intracellular iron deposition in tubular renal cells. These data are characteristic of a congenital hemolytic regenerative anemia, similar to human anemias due to unstable hemoglobin. Genetic mapping assigned the affected gene to mouse chromosome 7, approximately 50 cM from the Hbb locus. The sequence of the mutant Hbb gene exhibited a TâC transversion at nucleotide 179 in Hbb-b1, leading to the substitution of proline for leucine at amino acid residue 88 and thus homologous to the genetic defect underlying Santa Ana anemia in humans.
Assuntos
Anemia Hemolítica Congênita/sangue , Hemoglobinas Anormais/análise , Animais , Mapeamento Cromossômico , Modelos Animais de Doenças , Etilnitrosoureia , Feminino , Genótipo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MutaçãoRESUMO
BACKGROUND: Congenital heart disease (CHD) has a multifactorial pathogenesis, but a genetic contribution is indicated by heritability studies. To investigate the spectrum of CHD with a genetic pathogenesis, we conducted a forward genetic screen in inbred mice using fetal echocardiography to recover mutants with CHD. Mice are ideally suited for these studies given that they have the same four-chamber cardiac anatomy that is the substrate for CHD. METHODS AND RESULTS: Ethylnitrosourea mutagenized mice were ultrasound-interrogated by fetal echocardiography using a clinical ultrasound system, and fetuses suspected to have cardiac abnormalities were further interrogated with an ultrahigh-frequency ultrasound biomicroscopy. Scanning of 46 270 fetuses revealed 1722 with cardiac anomalies, with 27.9% dying prenatally. Most of the structural heart defects can be diagnosed using ultrasound biomicroscopy but not with the clinical ultrasound system. Confirmation with analysis by necropsy and histopathology showed excellent diagnostic capability of ultrasound biomicroscopy for most CHDs. Ventricular septal defect was the most common CHD observed, whereas outflow tract and atrioventricular septal defects were the most prevalent complex CHD. Cardiac/visceral organ situs defects were observed at surprisingly high incidence. The rarest CHD found was hypoplastic left heart syndrome, a phenotype never seen in mice previously. CONCLUSIONS: We developed a high-throughput, 2-tier ultrasound phenotyping strategy for efficient recovery of even rare CHD phenotypes, including the first mouse models of hypoplastic left heart syndrome. Our findings support a genetic pathogenesis for a wide spectrum of CHDs and suggest that the disruption of left-right patterning may play an important role in CHD.
Assuntos
Ecocardiografia Doppler , Coração Fetal/diagnóstico por imagem , Testes Genéticos , Cardiopatias Congênitas/diagnóstico por imagem , Cardiopatias Congênitas/genética , Microscopia Acústica , Mutação , Ultrassonografia Pré-Natal/métodos , Animais , Modelos Animais de Doenças , Ecocardiografia Doppler em Cores , Etilnitrosoureia/toxicidade , Feminino , Coração Fetal/anormalidades , Predisposição Genética para Doença , Cardiopatias Congênitas/embriologia , Hereditariedade , Ensaios de Triagem em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linhagem , FenótipoRESUMO
Patterning of the limb anterior-posterior axes depends on several signals that derive from the three signaling centers of the limb bud. These signals interact to constitute a complex and ordered network that critically contributes to the development of limb buds. Preaxial polydactyly in mouse is predominantly caused by ectopic expression of the zone of polarizing activity or Sonic hedgehog in the anterior region of the limb bud. In this study, we describe an N-ethyl-N-nitrosourea-induced polydactylous mouse (Alx4m1Yzcm) with an extra digit on the anterior aspect of one or two hinddigits. The mutation was mapped to chromosome 2, between markers D2Mit45 and D2Mit184. The Alx4 gene was identified as a potential candidate gene in this location. Sequence analysis of the Alx4 gene for polydactylous heterozygotes revealed an A/T transversion mutation that resulted in substitution of a lysine codon with a stop (nonsense) codon at position 145. Alx4m1Yzcm homozygous mice exhibited multiple abnormalities, including extensive preaxial polydactyly of all four limbs (up to seven digits) and the formation of omphalocele.