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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 866-869, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515777

RESUMO

OBJECTIVE: To determine the CGG repeat number and methylation status of FMR1 gene for fetuses whose mothers have carried a FMR1 mutation. METHODS: For 30 pregnant women, the fetal CGG repeat number was determined with a GC-rich PCR system by using chorionic villus, amniotic fluid or umbilical blood samples. The methylation status of the FMR1 gene was confirmed with Southern blotting. RESULTS: In total 30 prenatal diagnoses were performed for 29 carriers of FMR1 gene mutations and 1 with FMR1 gene deletion mosaicism. Three fetuses were found to carry premutations, 9 were with full mutations and 1 with mosaicism of premutation and full mutations. Eighteen fetuses were normal. CONCLUSION: Considering the genetic complexity of Fragile X syndrome (FXS), single method may not suffice accurate determination of their genetic status. The pitfalls and technical limitations of protocols requires adoption of personalized strategy for its prenatal diagnosis.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Diagnóstico Pré-Natal , Feminino , Heterozigoto , Humanos , Mutação , Gravidez
2.
An Acad Bras Cienc ; 91(3): e20180882, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553368

RESUMO

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by dynamic mutations of a CGG repetition segment in an X chromosome's single gene. It is considered the leading hereditary cause of both Autism Spectrum Disorders and Intellectual Disability. Some authors suggest that all individuals diagnosed with some of these latter conditions to be clinically and molecularly trialled for FXS due to the high levels of comorbidity between both conditions and also due to the variable expressiveness of this syndrome. This study has focused on verifying the presence of FMR1 expanded alleles since there is a lack of information about this kind of mutation in autism patients from the northern region of Brazil. The presence of large alleles for this gene could offer new therapeutic or pharmacological methods for the treatment of these patients. Both the presence and the frequency of CGG expansions were verified in 90 autism males by molecular analysis. Four of them had intermediate alleles and four others presented premutated alleles. Premutation carriers are on the propensity of developing the late onset Fragile X-associated tremor/ataxia syndrome. No full mutation alleles were found. Further studies are necessary to obtain more accurate statistical data about this kind of dynamic mutation.


Assuntos
Transtorno do Espectro Autista/genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Mutação/genética , Adolescente , Alelos , Criança , Pré-Escolar , Predisposição Genética para Doença , Testes Genéticos , Humanos , Masculino , Adulto Jovem
3.
Adv Exp Med Biol ; 1155: 155-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468394

RESUMO

Fragile X syndrome is an X-linked dominant disorder and the most common cause of inherited mental retardation. It is caused by trinucleotide repeat expansion in the fragile X mental retardation 1 gene (FMR1) at the Xq27.3. The expansion blocks expression of the gene product, Fragile X Mental Retardation Protein (FMRP). The syndrome includes mild to moderate mental retardation and behavioral manifestations such as tactile defensiveness, gaze avoidance, repetitive motor mannerisms, perseverative (repetitive) speech, hyperarousal and it frequently includes seizures. This behavioral phenotype overlaps significantly with autism spectrum disorder. The knockout mice lack normal Fmr1 protein and show macro-orchidism, learning deficits, and hyperactivity. Consequently, this knockout mouse may serve as a valuable tool in the elucidation of the physiological role of FMR1 and the mechanisms involved in macroorchidism, abnormal behavior, abnormalities comparable to those of human fragile X patients. In this study we evaluated the effects of taurine on the testicular physiology to better understand the cellular mechanisms underlying macro-orchidism. We found that there was a significant decrease in the number of Leydig cells in the testis of fragile X mouse. Furthermore, the expression of somatostatin was drastically decreased and differential expression pattern of CDK5 in fragile X mouse testis. In the control testis, CDK is expressed in primary and secondary spermatids whereas in the Fmr1 ko mice CDK 5 is expressed mainly in spermatogonia. Taurine supplementation led to an increase in CDK5 expression in both controls and Ko mice. CDKs (Cyclin-dependent kinases) are a group of serine/threonine protein kinases activated by binding to a regulatory subunit cyclin. Over 20 functionally diverse proteins involved in cytoskeleton dynamics, cell adhesion, transport, and membrane trafficking act as CDK5 substrates elucidating the molecular mechanisms of CDK5 function. CDK5 phosphorylates a diverse list of substrates, implicating it in the regulation of a range of cellular processes. CDK5 is expressed in Leydig cells, Sertoli cells, spermatogonia and peritubular cells indicating a role in spermatogenesis. In this study we examined the expression levels of CDK5 and how it is affected by taurine supplementation in the testes and found that taurine plays an important role in testicular physiology and corrected some of the pathophysiology observed in the fragile x mouse testis.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Taurina/farmacologia , Testículo/fisiopatologia , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Síndrome do Cromossomo X Frágil/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Expansão das Repetições de Trinucleotídeos
4.
Artigo em Russo | MEDLINE | ID: mdl-31464297

RESUMO

The objective of this study is to provide a detailed description of clinical characteristics of disorders associated with FMR1 gene, which is located on the long arm of chromosome X. The most frequent FMR1 mutations are related to CGG-repeat expansion in the promoter region: premutation (from 55 to 199), full mutation (more than 200 repeats). The first section of the article is devoted to the fragile X mental retardation syndrome (FX syndrome) caused by FMR1 full mutation. The clinical characteristics of FX syndrome are presented. The second section provides information about specific phenotypes associated with FMR1 premutation that can be observed in maternal relatives (grandmother, mother's siblings, grandfather) of the child. The most frequent symptoms that observed in permutation carriers are mild cognitive impairment, ASD, ADHD in children, fragile X-associated tremor/ataxia syndrome (FXTAS) in older carries, fragile X-associated primary ovarian insufficiency (FXPOI) in women. The last section provides information about screening diagnostic instruments that help to identify the risk of fragile X syndrome. It also presents the key questions to be asked to family members in order to identify the risk of the permutation.


Assuntos
Proteína do X Frágil de Retardo Mental , Síndrome do Cromossomo X Frágil , Mutação , Insuficiência Ovariana Primária , Idoso , Ataxia , Criança , Feminino , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Humanos , Insuficiência Ovariana Primária/diagnóstico , Insuficiência Ovariana Primária/genética , Fatores de Risco , Tremor
5.
J Clin Neurosci ; 66: 269-270, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178302

RESUMO

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late onset neurodegenerative disorder associated with dysfunction of movement, memory, and the peripheral nervous system. We report an 82 years old male who presented with tremors and difficulty with balance that started at 65 years of age. His motor examination revealed decreased strength in left lower extremity. Tremors were seen in both the upper limbs at rest that worsened with movement. Bilateral lower extremities showed absent vibration and proprioception sensations, absent reflexes and upgoing toes. Electrodiagnostic studies revealed sensory predominant axonal sensory-motor peripheral polyneuropathy. Brain MRI revealed microvascular ischemic changes. The cervical and lumbar MRI showed diffuse degenerative changes. Genetic test for heritable causes of ataxia revealed a premutation in Fragile X gene (84 CGG repeats), confirming the diagnosis of FXTAS. On further genetic testing three out of his four daughters also tested positive for the FMR1 premutation. In appropriate clinical setting, Fragile X-associated tremor/ataxia syndrome (FXTAS) should be considered in every middle aged/elderly patient who presented with slowly progressive ataxia, tremor and peripheral polyneuropathy without any history of cognitive or neurological disabilities in childhood.


Assuntos
Ataxia/diagnóstico , Ataxia/genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Tremor/diagnóstico , Tremor/genética , Idoso de 80 Anos ou mais , Testes Genéticos/métodos , Humanos , Masculino
6.
BMC Med Genet ; 20(1): 81, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096929

RESUMO

BACKGROUND: The prevalence of CGG repeat expansion mutation in FMR1 gene varies among different populations. In this study, we investigated the prevalence of this mutation in women of reproductive age from northern China. METHODS: A total of 11,891 pre-conceptional or pregnant women, including 5037 pregnant women and 7357 women with the history of spontaneous abortion or induced abortion due to delayed growth of the embryos, were recruited. The number of CGG repeats in FMR1 was measured by the TRP-PCR method. We also offered genetic counseling and prenatal diagnosis to the women carrying pre-mutation or full mutation alleles. RESULTS: The prevalence of pre-mutation in reproductive women in northern China was 1/410, higher than that in southern China and Korea but lower than that in western countries. We also found that the prevalence of pre-mutation was relatively high (1/320) in women with abortion history. CONCLUSION: Screening for CGG repeat expansion mutation in FMR1 should be recommended to the women with the history of spontaneous abortion or induced abortion due to delayed growth of the embryos.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Mutação , Reprodução , Repetições de Trinucleotídeos , Adolescente , China , Estudos de Coortes , Feminino , Humanos , Gravidez , Resultado da Gravidez , Adulto Jovem
7.
Mol Autism ; 10: 19, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31011411

RESUMO

Background: Autism spectrum disorders (ASD) affect around 1.5% of people worldwide. Symptoms start around age 2, when children fail to maintain eye contact and to develop speech and other forms of communication. Disturbances in glutamatergic and GABAergic signaling that lead to synaptic changes and alter the balance between excitation and inhibition in the developing brain are consistently found in ASD. One of the hallmarks of these disorders is hypersensitivity to sensory stimuli; however, little is known about its underlying causes. Since the retina is the part of the CNS that converts light into a neuronal signal, we set out to study how it is affected in adolescent mice prenatally exposed to valproic acid (VPA), a useful tool to study ASD endophenotypes. Methods: Pregnant female mice received VPA (600 mg/kg, ip) or saline at gestational day 11. Their male adolescent pups (P29-35) were behaviorally tested for anxiety and social interaction. Proteins known to be related with ASD were quantified and visualized in their retinas by immunoassays, and retinal function was assessed by full-field scotopic electroretinograms (ERGs). Results: Early adolescent mice prenatally exposed to VPA displayed impaired social interest and increased anxiety-like behaviors consistent with an ASD phenotype. The expression of GABA, GAD, synapsin-1, and FMRP proteins were reduced in their retinas, while mGluR5 was increased. The a-wave amplitudes of VPA-exposed were smaller than those of CTR animals, whereas the b-wave and oscillatory potentials were normal. Conclusions: This study establishes that adolescent male mice of the VPA-induced ASD model have alterations in retinal function and protein expression compatible with those found in several brain areas of other autism models. These results support the view that synaptic disturbances with excitatory/inhibitory imbalance early in life are associated with ASD and point to the retina as a window to understand their subjacent mechanisms.


Assuntos
Transtorno do Espectro Autista/fisiopatologia , Retina/metabolismo , Potenciais de Ação , Animais , Transtorno do Espectro Autista/etiologia , Transtorno do Espectro Autista/metabolismo , Endofenótipos , Feminino , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Retina/fisiopatologia , Comportamento Social , Sinapsinas/genética , Sinapsinas/metabolismo , Ácido Valproico/toxicidade
8.
Methods Mol Biol ; 1942: 3-10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900171

RESUMO

Fragile X syndrome (FXS) is one of the most common reasons for intellectual disability (ID). First described in the 1940s, it took many years to understand the disease. The awe-inspiring breakthroughs in both science and technology facilitated the recognition of the unique inheritance pattern and the genetic mechanism of fragile X. In this chapter we describe the history and evolution of our understanding of FXS as mirrored by advances in genetics.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/história , Testes Genéticos , Mutação , Síndrome do Cromossomo X Frágil/genética , História do Século XX , Humanos
9.
Methods Mol Biol ; 1942: 11-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900172

RESUMO

Fragile X syndrome (FXS) is characterized by mental retardation and in the vast majority of cases it is caused by expansion of CGG trinucleotide repeats in the 5' untranslated region (or UTR) in the FMR1 gene on the X chromosome. The size and methylation status of CGG repeats are correlated with the clinical phenotype of FMR1-related disorders. The methods used for clinical genetic testing of FXS include polymerase chain reaction (PCR) amplification and Southern blot analyses, which effectively detect alleles with repeats in the normal, intermediate, premutation, and full mutation size ranges, as well as the methylation status of FMR1 promoter region.


Assuntos
Southern Blotting/métodos , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Testes Genéticos , Mutação , Reação em Cadeia da Polimerase/métodos , Metilação de DNA , Feminino , Síndrome do Cromossomo X Frágil/genética , Humanos , Masculino , Repetições de Trinucleotídeos
10.
Methods Mol Biol ; 1942: 29-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900173

RESUMO

The vast majority of fragile X affected patients do not transcribe FMR1 due to a CGG repeat expansion in the 5'-untranslated region of the FMR1 gene. When the CGGs considerably expand, it elicits abnormal DNA methylation and histone modifications, which are responsible for FMR1 transcriptional silencing. In this chapter, we describe in detail two commonly used protocols for monitoring the epigenetic state of the FMR1 gene that bypass the difficulty in directly analyzing the CGGs. One protocol is for accurately measuring DNA methylation levels and the other is for profiling histone modifications.


Assuntos
Metilação de DNA , Epigênese Genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Repetições de Trinucleotídeos , Regiões 5' não Traduzidas , Inativação Gênica , Humanos
11.
Methods Mol Biol ; 1942: 49-59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900174

RESUMO

Knowledge of the CGG•CCG-repeat number, AGG interruption status, and the extent of DNA methylation of the FMR1 gene are vital for both diagnosis of the fragile X-related disorders and for basic research into disease mechanisms. We describe here assays that we use in our laboratory to assess these parameters. Our assays are PCR-based and include one for repeat size that can also be used to assess the extent of methylation and a related assay that allows the AGG interruption pattern to be reliably determined even in women. A second more quantitative methylation assay is also described. We also describe our method for cloning of repeats to generate the reference standards necessary for the accurate determination of repeat number and AGG interruption status.


Assuntos
Metilação de DNA , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Expansão das Repetições de Trinucleotídeos , Humanos
12.
Methods Mol Biol ; 1942: 71-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900176

RESUMO

The neurosphere assay is a widely used method to culture neural precursor cells (NPCs), which include mixed populations of neural stem and progenitor cells, from the mammalian central nervous system. Fmr1-knockout (KO) mice generated to model fragile X syndrome (FXS) recapitulate the major phenotype of FXS. Neurosphere differentiation of cortical progenitors derived from brains of Fmr1-KO mice has been shown to reflect disordered mechanisms during cortical development in FXS in vivo. The cellular composition of neurospheres is heterogeneous, but robust FXS-specific alterations can be identified when culturing conditions are kept constant.


Assuntos
Encéfalo/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/genética , Células-Tronco Neurais/metabolismo , Neurogênese , Animais , Encéfalo/citologia , Células Cultivadas , Proteína do X Frágil de Retardo Mental/genética , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia
13.
Methods Mol Biol ; 1942: 141-153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900182

RESUMO

Drosophila melanogaster is an ideal model to study Fragile X syndrome (FXS) as it presents us with a toolbox to identify genetic modifiers and to investigate the molecular mechanisms of FXS. Here we describe some of the methods that have been used to study FXS, ranging from reverse genetic screening using the GAL4-UAS system, to mushroom body staining and courtship behavioral assays to examine the learning and memory deficits associated with FXS.


Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Transtornos de Aprendizagem/fisiopatologia , Transtornos da Memória/fisiopatologia , Animais , Comportamento Animal , Corte , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Olho/fisiopatologia , Feminino , Proteína do X Frágil de Retardo Mental/genética , Masculino , Corpos Pedunculados/fisiopatologia
14.
Methods Mol Biol ; 1942: 155-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900183

RESUMO

Despite almost 30 years of biomedical research, a treatment or cure for fragile X syndrome (FXS) is not yet available. The reasons behind this are varied, and among them are discrepancies in both research methodologies and research models. For many years, the fmr1 knockout mouse model dominated the field, and was used to draw important conclusions. The establishment of FXS-human cellular models called these conclusions into question, showing conflicting evidence. Discrepancies in FXS research, between mouse and human, might arise from differences inherent to each species, and from the use of different methodologies. This chapter summarizes these discrepancies and evaluates their impact on the current status of clinical trials.


Assuntos
Pesquisa Biomédica , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Células-Tronco Pluripotentes/citologia , Animais , Células-Tronco Embrionárias/metabolismo , Feminino , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismo
15.
Methods Mol Biol ; 1942: 165-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900184

RESUMO

Women who carry the FMR1 premutation may suffer from ongoing deterioration of ovarian function. The lucidity of the molecular mechanism of FXTAS is emerging and findings from research in the field of FXTAS could elucidate the pathogenesis of FXPOI. To date there are three possible mechanisms for ovarian dysfunction in FMR1 permutation carriers. The first is the RNA toxic gain-of-function mechanism initiating loss of function of over 30 specific RNA-binding proteins. The second is associated to the formation of an abnormal polyglycine-containing protein (FMRpolyG), and the third is related to novel lncRNAs, named FMR4 and FMR6. Herein we describe our laboratory methodology, focusing on the culturing and manipulation of granulosa cells from human female premutation carriers, trying to reveal the actual possible mechanisms liable to FXPOI. Detecting the precise pathways in premutation carrier might facilitate in offering these women the opportunity to make an informed decision regarding their reproductive and family planning.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Células da Granulosa/patologia , Mutação , Insuficiência Ovariana Primária/fisiopatologia , Animais , Portador Sadio , Feminino , Síndrome do Cromossomo X Frágil/genética , Células da Granulosa/metabolismo , Humanos , Camundongos , Insuficiência Ovariana Primária/genética , Repetições de Trinucleotídeos
16.
Methods Mol Biol ; 1942: 173-189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900185

RESUMO

Individuals carrying an FMR1 expansion between 55 and 200 CGG repeats, are at risk of developing the Fragile X-associated tremor/ataxia syndrome (FXTAS), a late onset neurodegenerative disorder characterized by cerebellar gait ataxia, intentional tremor, neuropathy, parkinsonism, cognitive decline, and psychological disorders, such as anxiety and depression. In addition, brain atrophy, white matter disease, and hyperintensities of the middle cerebellar peduncles can also be present. The neuropathological distinct feature of FXTAS is represented by the presence of eosinophilic intranuclear inclusions in neurons and astrocytes throughout the brain and in other tissues. In this chapter, protocols for available diagnostic tools, in both humans and mice, the clinical features and the basic molecular mechanisms leading to FXTAS and the animal models proposed to study this disorder are discussed.


Assuntos
Ataxia/diagnóstico , Encéfalo/patologia , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Mutação , Tremor/diagnóstico , Animais , Ataxia/genética , Encéfalo/metabolismo , Feminino , Síndrome do Cromossomo X Frágil/genética , Humanos , Corpos de Inclusão Intranuclear , Masculino , Camundongos , Tremor/genética , Repetições de Trinucleotídeos
17.
Methods Mol Biol ; 1942: 61-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900175

RESUMO

Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.


Assuntos
Sistemas CRISPR-Cas , Proteína do X Frágil de Retardo Mental/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Luciferases/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Síndrome do Cromossomo X Frágil/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
18.
Methods Mol Biol ; 1972: 199-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30847793

RESUMO

Fragile X mental retardation 1 (FMR1) CGG repeat expansions cause fragile X syndrome-the leading monogenic form of intellectual disability-and increase the risk for fragile X-associated tremor ataxia syndrome and fragile X-associated primary ovarian insufficiency. Southern blot (SB) analysis is the current gold standard test for FMR1 molecular diagnosis. Several polymerase chain reaction (PCR)-based methods are now available for sizing FMR1 CGG repeat expansions. These methods offer higher diagnostic sensitivity and specificity compared to SB analysis, significantly reduce the turnaround time and increase throughput. In this chapter, we describe a triplet-repeat primed PCR protocol that employs capillary electrophoresis to resolve the derived amplicon products, enabling precise determination of the FMR1 genotypes in both males and females and characterization of the CGG repeat structure.


Assuntos
Primers do DNA/metabolismo , Eletroforese Capilar/métodos , Proteína do X Frágil de Retardo Mental/genética , Reação em Cadeia da Polimerase/métodos , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genética , Humanos , Mutação/genética
19.
Nat Neurosci ; 22(3): 386-400, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742117

RESUMO

Fragile X syndrome results from a loss of the RNA-binding protein fragile X mental retardation protein (FMRP). How FMRP regulates neuronal development and function remains unclear. Here we show that FMRP-deficient immature neurons exhibit impaired dendritic maturation, altered expression of mitochondrial genes, fragmented mitochondria, impaired mitochondrial function, and increased oxidative stress. Enhancing mitochondrial fusion partially rescued dendritic abnormalities in FMRP-deficient immature neurons. We show that FMRP deficiency leads to reduced Htt mRNA and protein levels and that HTT mediates FMRP regulation of mitochondrial fusion and dendritic maturation. Mice with hippocampal Htt knockdown and Fmr1-knockout mice showed similar behavioral deficits that could be rescued by treatment with a mitochondrial fusion compound. Our data unveil mitochondrial dysfunction as a contributor to the impaired dendritic maturation of FMRP-deficient neurons and suggest a role for interactions between FMRP and HTT in the pathogenesis of fragile X syndrome.


Assuntos
Dendritos/metabolismo , Giro Denteado/metabolismo , Proteína do X Frágil de Retardo Mental/metabolismo , Proteína Huntingtina/metabolismo , Dinâmica Mitocondrial , Animais , Giro Denteado/crescimento & desenvolvimento , Feminino , Proteína do X Frágil de Retardo Mental/genética , Técnicas de Silenciamento de Genes , Genes Mitocondriais , Proteína Huntingtina/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo
20.
Neuron ; 101(4): 648-661.e4, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30679017

RESUMO

Distinct genetic forms of autism are hypothesized to share a common increase in excitation-inhibition (E-I) ratio in cerebral cortex, causing hyperexcitability and excess spiking. We provide a systematic test of this hypothesis across 4 mouse models (Fmr1-/y, Cntnap2-/-, 16p11.2del/+, Tsc2+/-), focusing on somatosensory cortex. All autism mutants showed reduced feedforward inhibition in layer 2/3 coupled with more modest, variable reduction in feedforward excitation, driving a common increase in E-I conductance ratio. Despite this, feedforward spiking, synaptic depolarization, and spontaneous spiking were largely normal. Modeling revealed that E and I conductance changes in each mutant were quantitatively matched to yield stable, not increased, synaptic depolarization for cells near spike threshold. Correspondingly, whisker-evoked spiking was not increased in vivo despite detectably reduced inhibition. Thus, elevated E-I ratio is a common circuit phenotype but appears to reflect homeostatic stabilization of synaptic drive rather than driving network hyperexcitability in autism.


Assuntos
Transtorno Autístico/fisiopatologia , Potenciais Somatossensoriais Evocados , Potenciais Pós-Sinápticos Excitadores , Potenciais Pós-Sinápticos Inibidores , Córtex Somatossensorial/fisiopatologia , Animais , Transtorno Autístico/genética , Cromossomos Humanos Par 16/genética , Proteína do X Frágil de Retardo Mental/genética , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Córtex Somatossensorial/fisiologia , Proteína 2 do Complexo Esclerose Tuberosa/genética
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