Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

In the present study,Fmr1 knockout mice （KO mice） were used as the model for fragile X syndrome.The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts,indicating a learning and memory disorder.After treatment with 30,60,90,120,or 200 mg/kg lithium chloride,the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated,in particular,the 200 mg/kg lithium chloride treatment had the most significant effect.Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta,an inactive form of glycogen synthase kinase 3 beta,in the cerebral cortex and hippocampus of the Fmr1 KO mice.These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice,possibly by inhibiting glycogen synthase kinase 3 beta activity.

Mixed Connective Tissue Disease Complicated by Retinal Microvasculopathy and Its Relationship with Fragile-X Syndrome

Purpose: To report an unusual case of retinal microvasculopathy secondary to mixed connective tissue disease (MCTD) on a background history of fragile-X syndrome (FXS). Methods: Case report and literature review. Results: A cotton wool spot was discovered in a 29-year-old female who presented with an ischaemic digit secondary to Raynaud’s phenomenon. She also has a background history of MCTD and FXS. Fundus examination and automated perimetry findings were normal. Magnet resonance imaging and computed tomography aortogram did not demonstrate any evidence of vasculitis in the head and neck. She was tested positively for U1-ribonuclear peptide. Interestingly, the re-distribution of Fragile-X related gene 1 has been suggested to trigger autoimmune responses in experiments. This finding makes the case peculiar as it suggests an alternate explanation for this patient’s clinical findings. Conclusion: Retinal vasculopathy is a rare complication of MCTD. The background history of FXS potentially highlights an alternate autoimmune pathogenetic mechanism.

A Rapid Screening and Diagnosis on Fragile X Syndrome by PCR

Polymerase chain reaction (PCR) technique combined with direct detection by silver staining on denaturing DNA sequencing gel was used to analyze the (CGG)n repeats within the FMR1 gene on 169 suspected patients with mental retardation and 33 kindreds of 6 fragile X families. The results showed that : (1) No PCR products were detected in 3 males in the suspected group. (2) In the fragile X family studies, the 5 male probands failed to show any PCR products. (3) Diplex PCR with the primers flanking the FRAXE locus was used to serve as an internal control for the 8 above mentioned males and only normal products of the FRAXE locus were detected, indicating that the possibility of false negative results of the FRAXA locus could be eliminated. These findings suggested that analysis of (CGG)n repeat within the FMR1 gene by PCR technique could efficiently detect premutation carriers and that negative PCR products in mentally retarded males might highly imply the diagnosis of fragile X syndrome after the false negative results have been excluded by diplex PCR. This PCR assay is suitable for the screening and diagnosis of fragile X syndrome in a large number of populations due to its rapidity, simplicity, stability and reliability.

Reducing histone acetylation rescues cognitive deficits in mouse model of fragile X syndrome

Fragile X syndrome(FXS)is the most prevalent inherited intellectual disability,resulting from a loss of fragile X mental retardation protein(FMRP).Patients with FXS suffer lifelong cognitive disabilities,but the function of FMRP in the adult brain and the mechanism underlying age-related cognitive decline in FXS is not fully understood.Here,we report that a loss of FMRP results in increased protein synthesis of histone acetyltransferase EP300 and ubiquitinationmediated degradation of histone deacetylase HDAC1 in adult hippocampal neural stem cells(NSCs).Consequently,FMRPdeficient NSCs exhibit elevated histone acetylation and age-related NSC depletion,leading to cognitive impairment in mature adult mice.Reducing histone acetylation rescues both neurogenesis and cognitive deficits in mature adult FMRPdeficient mice.Our work reveals a role for FMRP and histone acetylation in cognition and presents a potential novel ther⁃apeutic strategy for treating adult FXS patients.

Dental treatment of twin monozygotic brothers with Fragile X syndrome

Fragile X syndrome (FXS) is the main cause of inherited mental retardation and is the result of transcriptional silencing of the fragile X mental retardation gene FMR1. An absence of the associated protein FMRP leads to the deregulation of many genes, which results in phenotypes of Attention-Deficit Hyperactivity Disorder (ADHD), anxiety, epilepsy and autism. The aim of this article is to report the clinical case of twin siblings affected by FXS and to describe the procedures for dental treatment with intravenous sedation. Information regarding the characteristic manifestations of FXS not only aided in the handling of the patients but also enabled us to develop clinical programs to promote and maintain oral health using individualized and specific dental procedures.

Prevalence of fragile X syndrome in males and females in Indonesia

AIM: To investigate the prevalence of fragile X syndrome(FXS) in intellectually disabled male and female Indonesians.METHODS: This research is an extension of a previously reported study on the identification of chromosomal aberrations in a large cohort of 527 Indonesians with intellectual disability(ID). In this previous study,87 patients had a chromosomal abnormality, five of whom expressed fragile sites on Xq27.3. Since FXS cannot always be identified by cytogenetic analysis, molecular testing of the fragile X mental retardation 1 CGG repeat was performed in 440 samples. The testing was also conducted in the five previously identified samples to confirm the abnormality. In total, a molecular study was conducted in 445 samples(162 females and 283 males).RESULTS: In the cohort of Indonesian ID population, the prevalence of FXS is 9/527(1.7%). The prevalence in males and females is 1.5%(5/329) and 2%(4/198), respectively. Segregation analysis in the families and X-inactivation studies were performed. We performed the first comprehensive genetic survey of a representative sample of male and female ID individuals from institutions and special schools in Indonesia. Our findings show that a comprehensive study of FXS can be performed in a developing country like Indonesia where diagnostic facilities are limited.CONCLUSION: The prevalence of FXS is equal in females and males in our study, which suggests that the prevalence of FXS in females could be underestimated.

GABAB receptor upregulates fragile X mental retardation protein expression in neurons

Aim Fragile X mental retardation protein （FMRP） is an RNA-binding protein important for the control of translation and synaptic function. The mutation or silencing of FMRP causes Fragile X syndrome （FXS） , which leads to intellectual disability and social impairment. γ-aminobutyric acid （GABA） is the major inhibitory neuro- transmitter of the mammalian central nervous system, and its metabotropic GABAB receptor has been implicated in various mental disorders. The GABAB receptor agonist baclofen has been shown to improve FXS symptoms in a mouse model and in human patients, suggesting the role of GABAB receptor on FMRP regulation. Here we investi- gated the signaling events linking the GABAB receptor and FMRP. Methods Western blot was used in this study to detect protein expression and kinase phosphorylation in cerebellar granule neurons. For key molecules in signal- ling pathway, RNAi was used in MEFs to confirm the results in neurons. Results GABAB receptor activation up- regulated cAMP response element binding protein-dependent Fmrp expression in cultured mouse cerebellar granule neurons via two distinct mechanisms： the transactivation of insulin-like growth factor-1 receptor and activation of protein kinase C. In addition, a positive allosteric modulator of the GABAB receptor, CGP7930, stimulated Fmrp expression in neurons. Conclusion These results suggest a role for GABAB receptor in Fmrp regulation and a po- tential interest of GABAB receptor signaling in FXS improvement.

Erectile dysfunction in Fragile X patients

Aim:To study a possible defect in spermatogenesis of Fragile X syndrome(FXS)patients.Methods:Two different polymerase chain reaction(PCR)based methods were used for the molecular diagnosis of FXS.Sperm collection was done mostly according to the laboratory manual of the World Health Organization.Results:We failed to collect sperm samples from five Fragile X subjects aged 18-60 years as a result of an unexpected erectile dysfunction(ED). Multiple examinations of the same subject at different times,and of different subjects from different provinces by different physicians,showed the same result consistently in all five subjects examined.Conclusion:Erectile reflex is an instinctive response in all healthy males.The absence of erection can be caused by hormonal,physical or neuronal malfunction.As hormonal profiles were reported to be generally normal in Fragile X men,we propose that an unknown physical factor or the neuronal circuit,or both,underlying the erection is compromised.The finding of ED in Fragile X patients may help better understand the clinical spectrum and pathogenesis of the disease.(Asian J Androl 2006 Jul;8:483-487)

利用酵母双杂交系统筛选与FXR1P相互作用的蛋白

目的筛选FXR1P相互作用蛋白,探讨FXR1P生物学功能。方法应用酵母双杂交技术,构建诱饵表达载体pGBKT7/FXR1,转化酵母菌AH109,与转化了人胎脑cDNA文库的酵母菌Y187交配筛选阳性克隆并测序。结果重组表达载体pGBKT7/FXR1构建成功;从人胎脑cDNA文库中筛选出5个与FXR1P结合的蛋白基因,分别与5种已知的编码蛋白基因序列同源:胞苷一磷酸-N-乙酰神经氨酸合成酶(CMAS)、铁蛋白重链多肽1(FTH1)、高尔基体自身抗原golgina亚类4(GOLGA4)、17-β羟类固醇脱氢酶1(HSD17B1)、绒毛膜生长催乳激素1(CSH1)。结论为进一步研究FXR1P的功能及脆性X综合征发病机制提供新的线索。

FXR1P相互作用RNA的研究

目的:本研究利用酵母三杂交系统从人脑海马回cDNA文库中筛选FXR1P的靶RNA,以进一步阐明FXR1基因的功能。方法:将表达FXR1P全长的质粒pYESTrp3/FXR1转化酵母菌株L40ura3/pHybLex/Zeo-MS2,检测毒性和自激活性;应用酵母三杂交技术从人脑海马回pRH3′-cDNA文库中筛选FXR1P的相互作用RNA;分离初步筛选的结果,再次转化含有诱饵质粒的融合菌株L40ura3/pHybLex/Zeo-MS2/pYESTrp3/FXR1,重新验证阳性结果;最后对阳性结果的外源插入片段进行测序和生物信息学分析。结果:酵母三杂交的筛选得到了3个阳性结果,经过测序和同源性分析,其中一个阳性结果中的插入片段为K-ALPHA-1 mRNA的部分序列。结论:K-ALPHA-1 mRNA可能是一种新的FXR1P的靶RNA。

FXR基因家族

FXR基因家族是与脆性X综合征发病相关的一个基因家族。有3个家族成员。在进化上高度保守，氨基酸序列高度相似，有共同的功能结构域：两个KH结构域、RGG盒、NES和NLS。能够与RNA和多聚核糖体结合。通过NES和NLS携带其mRNA在细胞核和细胞质之间穿梭。不同的蛋白亚型其组织分布各异。该家族成员在大脑和神经发育过程中发挥重要作用，影响认知过程和智力发育。

Developmental Impairments of Synaptic Refinement in the Thalamus of a Mouse Model of Fragile X Syndrome

While somatosensory over-reactivity is a common feature of autism spectrum disorders such as fragile X syndrome(FXS),the thalamic mechanisms underlying this remain unclear.Here,we found that the developmental elimination of synapses formed between the principal nucleus of V(PrV)and the ventral posterior medial nucleus(VPm)of the somatosensory system was delayed in fragile X mental retardation 1 gene knockout(Fmr1 KO)mice,while the developmental strengthening of these synapses was disrupted.Immunohistochemistry showed excessive VGluT2 puncta in mutants at P12–13,but not at P7–8 or P15–16,confirming a delay in somatic pruning of PrV-VPm synapses.Impaired synaptic function was associated with a reduction in the frequency of quantal AMPA events,as well as developmental deficits in presynaptic vesicle size and density.Our results uncovered the developmental impairment of thalamic relay synapses in Fmr1 KO mice and suggest that a thalamic contribution to the somatosensory over-reactivity in FXS should be considered.

FXR2酵母双杂交饵载体的构建及酵母菌转化

目的构建FXR2酵母双杂交系统的“饵”质粒,并转化酵母菌,为筛选FXR2P互作蛋白作基础。方法提取pMD18-T-FXR2,酶切后将FXR2基因连接到MATCHMAKERLexATwo-HybridSystem中的plexA质粒上,得到“诱饵”(Bait)-plexA-FXR2,导入大肠杆菌扩增,筛选阳性克隆并提取重组Bait质粒,再转入酵母菌EGY48(p8op-lacZ)。结果通过遗传学方法筛选得到构建成功的plexA-FXR2;通过营养缺陷筛选,证明Bait重组质粒转入酵母菌中。结论成功构建Bait质粒并使酵母菌转化。

IQCE mRNA与FXR1P在酵母三杂交体系中的相互作用

脆性X相关基因1编码蛋白FXR1P是一种RNA结合蛋白,其所结合的靶RNA目前所知甚少。本研究应用酵母三杂交技术对本课题组从pRH3-cDNA人脑海马RNA表达文库中筛选到的一种可能与FXR1P存在相互作用的RNA IQCE进行研究,以验证该RNA与FXR1P的相互作用。方法为:提取利用酵母三杂交技术初步筛选得到的酵母阳性克隆的质粒,转化大肠杆菌Top10,利用其质粒不相容性分离插入了目的片段的pRH3′-cDNA质粒,将该质粒转化入含目的基因FXR1的酵母菌株L40-ura3/pHyb lex/Zeo-MS2/pYESTrp3/FXR1,进行一对一的酵母三杂交验证,最后将该片段进行测序。测序结果为IQCE的一段编码序列,而目前尚无研究报导FXR1与IQCE的相互关系。结论:提示FXR1P与IQCE mRNA存在相互作用,IQCE可能是FXR1P发育调控网络组成成员之一。

脆性X综合征携带者筛查遗传咨询专家共识

脆性X综合征是遗传性智力障碍和孤独症谱系障碍最常见的单基因病,以X连锁不完全显性方式遗传。大约99%的脆性X综合征为FMR1基因CGG扩增动态突变导致,CGG扩增达到前突变或全突变时,可能出现相应的表型。近年来,脆性X综合征携带者筛查在我国逐渐开展起来,遗传咨询能力相对不足。为了更加规范地进行遗传咨询并提供后续生育指导,单基因病携带者筛查共识专家组、中华医学会医学遗传学分会遗传咨询学组通过多次讨论,对脆性X综合征携带者筛查的适用人群、检测前和检测后的遗传咨询内容形成了共识,并通过德尔菲法形成了推荐意见。

伴有癫痫的脆性X综合征家系1例

脆性X综合征(fragile X syndrome,FXS)是FMR1基因CGG异常重复扩增导致的疾病。本文报告1对经基因检测诊断为FXS的兄弟,2例患者分别为15岁和14岁,均存在语言障碍、智力障碍、注意力缺陷障碍、孤独症谱系障碍和FXS特征性面容等临床表现,其中先证者伴有罕见的晚发性癫痫发作,经左乙拉西坦治疗效果良好,而其弟弟经反复随访未见脑电图异常。该对病例提示FXS临床表型具有多样性和异质性。

FXR1真核表达载体的构建及鉴定

目的构建携带人脆性X相关基因1(FXR1)的真核表达载体pCMV-HA,为研究FXR1P互作蛋白奠定基础。方法以pYESTrp3-FXR1为模板进行聚合酶链反应(PCR)特异扩增FXR1基因片断,将扩增片段经EcoR I和XhoI双酶切后克隆到pCMV-HA载体,酶切鉴定阳性克隆并测序鉴定。结果成功构建了pC-MV-HA/FXR1真核表达载体。结论pCMV-HA/FXR1真核表达载体的构建为进一步在细胞内鉴定FXR1互作蛋白奠定了基础,从而对研究FXR1在脆性X综合征中的作用机理具有深远意义。

脆性X综合征遗传学诊断方法研究进展

脆性X综合征(FXS)是导致智力障碍和发育障碍的主要单基因病之一,呈X连锁不完全显性遗传。FXS的病因是FMR1基因内(CGG)n重复序列的不稳定扩展及其上游CpG岛的异常甲基化,进而导致脆性X智力低下蛋白(FMRP)减少或缺乏,FMRP的水平直接关系到临床表型的严重程度。临床表现和基因检测是诊断FXS的主要依据。然而,FMR1基因分子结构和遗传模式的特殊性使得FXS的分子诊断和遗传咨询面临挑战。因此,如何简便而准确地进行FMR1基因检测一直是临床关注的焦点。文章针对FXS遗传学诊断方法的研究进展进行综述,旨在促进FXS的规范诊断,为临床提供帮助。

脆性X相关震颤/共济失调综合征

脆性X相关震颤/共济失调综合征(FXTAS)是一种由于X染色体智能低下1型(FMR1)基因前突变(PM)引起的神经系统变性疾病,由于其发病率低,临床表现复杂多样,往往导致误诊漏诊的情况出现,因此,广大临床医生需提高对该病的认知。本文旨在综述FXTAS的发病机制、临床表现、病理特征、诊断要点等方面的研究进展。

Wnt信号通路在FXTAS中的研究进展

Wnt信号转导通路是一种在真核生物进化过程中所具有高度保守的信号通路,包括经典β-catenin依赖性通路和非经典β-catenin非依赖性通路。Wnt信号转导通路不仅参与生长、发育以及代谢等各类重要的生命过程,也和阿兹海默病、帕金森病、糖尿病以及肿瘤等常见疾病有密切的联系。脆性X相关震颤/共济失调综合征(fragile X-associated tremor/ataxia syndrome,FXTAS)是脆性X智力低下基因FMR1(fragile X mental retardation 1)基因的前突变扩增(CGG重复55～200次)所导致,该疾病的主要临床特征是进行性意向震颤、步态共济失调、痴呆等,表现出和帕金森病的某些运动症状类似,FXTAS的5'端非编码区(CGG) n重复次数在人群中存在多态性。近些年来,研究发现其与Wnt信号通路之间存在相关联系,本研究就Wnt信号通路对FXTAS的相关研究进展进行综述。