FMRP检测在儿童脆性X综合征中诊断价值的实验评价

目的对脆性X智力低下蛋白（fragile X mental retardation protein,FMRP）免疫细胞化学诊断方法并进行临床流行病学评价;利用ROC曲线确定适合本地筛查脆性X综合征的外周血淋巴细胞FMRP免疫组化法的诊断临界点。方法对临床拟诊为不明原因智力低下的的41例不明原因的智力低下患儿,同时进行免疫细胞化学方法进行外周血淋巴细胞FMRP表达检测和7-deza—dGTP PCR法两种方法检查,以目前较为公认的7-deza—dGTP PCR法为诊断金标准,对外周血淋巴细胞FMRP细胞免疫组化法（SP法）进行实验评价,计算其灵敏度、特异度、阳性似然比、阴性似然比等统计学指标。采用ROC曲线确定适合本地筛查脆性X综合征的外周血淋巴细胞FMRP表达率的诊断临界点。结果41例不明原因智力低下患儿中染色体核型分析均正常,其中染色体脆性位点检查异常的1例,约为2．4％。7-deza—dGTP PCR法诊断FXS患者11例,约占26．2％,其中男9例,女2例;ROC计算得出最佳的外周血淋巴细胞FMRP表达率的诊断临界点为46％,灵敏度为90．9％,特异度为93．5％。结论外周血淋巴细胞FMRP免疫细胞化学检测是一种可靠的FXS诊断和筛查方法,具有快速、简便、廉价、相对无创伤性等优点,可进行大样本筛查及标本邮寄快递检测,适宜在我国基层开展早期FXS患者诊断和筛查。     

Mapping nonverbal IQ in young boys with fragile X syndrome

This study examines the developmental changes in nonverbal intellectual functioning evident in males with fragile X syndrome (FXS) measured by the Leiter International Performance Scales-Revised (Leiter-R). The Leiter-R provides both IQ scores and associated growth scores which permit the examination of both age-based IQ scores and overall intellectual growth. Participants were 45 males with full mutation FXS and ranged in age from 4.0 to 13.8 years. Each child was assessed annually using the Leiter-R as part of a larger longitudinal battery for an average of 3.5 assessments per child and a range of 2-6 assessments, representing a total of 156 assessment occasions. Longitudinal analyzes of Leiter scores consisted primarily of hierarchical linear modeling, with the impact of chronological age, maternal education, fragile X mental retardation 1 protein (FMRP), autistic behaviors also being assessed. Findings revealed a significant linear decline in nonverbal IQ scores, with no effects of maternal education, autistic behaviors, or FMRP on mean level or rate of change in IQ scores over time. The decline slowed significantly around 8 years of age, but scores continued to decline into the 12th year of age. In contrast, a significant linear increase was observed in Leiter-R growth scores, which was negatively influenced by autistic behaviors. The rate of increase did not change over time, and neither mean level nor rate of increase was influenced by maternal education or FMRP levels. These findings suggest that declines in IQ are the result of steady, but suboptimal intellectual growth, rather than a true deterioration in overall intellectual functioning.     

FMR1 gene and fragile X syndrome

Taxonomic features of fragile X syndrome (FXS) associated with the fragile X mutation have evolved over several decades. Males are more severely impacted cognitively than females, but both show declines in IQ scores as they age. Although many males with FXS exhibit autistic-like features, autism does not occur more frequently in males with FXS than among males with mental retardation (MR). FXS is caused by inactivation of the FMR1 gene located on Xq27.3. FMRP, the protein produced by FMR1, has been detected in most organs and in brain. In cells, it is located primarily in cytoplasm and contains motifs found in RNA-binding proteins. The FMRP N-terminal contains a functional nuclear localization signal which permits the protein to shuttle between cytoplasm and nucleus. FMR1 knockout mice show subtle behavioral and visual-spatial difficulties. Analysis of their brain tissue suggests absence of FMRP impairs synaptic maturation. Individuals with the fragile premutation produce FMRP, and the phenotype associated with the premutation has been controversial. However, there seems to be a higher incidence of premature ovarian failure in women with the premutation than is found in the general female population. This may be related to unusual increases in mRNA levels in premutation carriers.     

Problem behavior in boys with fragile X syndrome

This study examines problem behavior over time in 59 boys with fragile X syndrome (FXS), aged 4-12 years, using the Child Behavior Checklist (CBCL). Approximately 49% of the boys scored within the borderline or clinical range on total problem behavior, while 56-57% scored in the borderline or clinical range on the attention and thought problems subscales, and 26% scored in this range on the social problems subscale. With a mean of 2.5 assessments per child, behavior problems were stable during the 3-year period of study. Total problem behavior was higher for children who displayed autistic behavior, were rated as low in adaptability, had mothers with higher maternal education levels, and were on medication. Mothers with more education also rated their children as having more attention, thought, and total problems. Children taking medication differed from boys who were not taking medication on social problems, but not on attention and thought problems. Low adaptability and more autistic characteristics predicted thought problems.     

Fragile X syndrome in Korea: a case series and a review of the literature

The purposes of this study were to present DNA analysis findings of our case series of fragile X syndrome (FXS) based on methylation-specific polymerase chain reaction (MS-PCR), PCR, and Southern blotting alongside developmental characteristics including psychological profiles and to review the literature on FXS in Korea. The reports of 65 children (male:female, 52:13; age, 6.12+/-4.00 yrs) referred for the diagnosis of FXS over a 26-months period were retrospectively reviewed for the identification of full mutation or premutation of fragile X mental retardation 1 (FMR1). Among the 65 children, there were 4 boys with full mutation, and one boy showed premutation of FMR1, yielding a 6.15% positive rate of FXS. All 4 children with full mutation showed significant developmental delay, cognitive dysfunction, and varying degrees of autistic behaviors. The boys with premutation showed also moderate mental retardation, severe drooling, and behavioral problems as severe as the boys with full mutation. Thirteen articles on FXS in Korea have been published since 1993, and they were reviewed. The positive rate of FXS was in the range of 0.77-8.51%, depending on the study groups and the method of diagnosis. Finally, the population-based prevalence study on FXS in Korea is required in the near future.     

Chronological age, but not FMRP levels, predicts neuropsychological performance in girls with fragile X syndrome.

The effect of FMRP levels and chronological age on executive functioning, visual-spatial abilities ,and verbal fluency tasks were examined in 46 school-age girls with fragile X syndrome (FXS). Results indicated that FMRP levels were not predictive of outcome on the neuropsychological tests but that performance on the executive functioning task tended to worsen with chronological age. This age effect was not observed on the tests of visual-spatial abilities or verbal fluency. These data indicate that relative deficits in executive functioning in girls with FXS become more pronounced with age. In contrast, the relative deficits in spatial and verbal abilities of these girls did not appear to increase with age, suggesting that these abilities may be spared.     

Identification of Fragile X Syndrome Specific Molecular Markers in Human Fibroblasts: A Useful Model to Test the Efficacy of Therapeutic Drugs

Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, fragile X mental retardation protein (FMRP), an RNA‐binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p‐mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p‐S6K1). The treatment with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3‐kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.     

ADHD symptoms in children with FXS

Parent- and teacher-report of attention-deficit/hyperactivity disorder (ADHD) symptoms were examined using problem behavior and DSM-IV symptom inventory questionnaires for 63 children with full mutation fragile X syndrome (FXS) and 56 children without disabilities matched on mental age (MA). Prevalence rates of ADHD symptoms varied depending on type of measure (problem behavior or DSM-IV criteria), subscale (ADHD-inattentive or ADHD-hyperactive), scoring method (continuous T-scores or categorical scores based on DSM-IV algorithm), and rater (parent or teacher). Overall, 54-59% of boys with FXS met diagnostic behavioral criteria for either ADHD-inattentive type only, ADHD-hyperactive type only, or ADHD-combined type based on parent or teacher report. Boys with FXS were rated as having clinically high scores or met diagnostic criteria at higher rates than expected for the general population and had higher raw scores than their MA-matched peers. Parent ratings of boys with FXS resulted in higher ADHD-inattentive type and ADHD-hyperactive type T-scores than teachers. Boys who were rated as meeting DSM-IV criteria were more likely to be taking psychotropic medication and to have younger mental ages. Parents were substantially more likely than teachers to rate boys as meeting DSM-IV criteria for ADHD-inattentive type, while teachers were only slightly more likely than parents to rate boys as meeting DSM-IV criteria for ADHD-hyperactive type. Teachers were more likely than parents to rate boys as meeting DSM-IV criteria for ADHD when boys had lower levels of FMRP.     

Altered maturation of the primary somatosensory cortex in a mouse model of fragile X syndrome

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and results from the loss of the fragile X mental retardation protein (FMRP). Many fragile X-related cognitive and behavioral features emerge during childhood and are associated with abnormal synaptic and cellular organization of the cerebral cortex. Identifying the roles of FMRP in cortical development will provide a basis for understanding the pathogenesis of the syndrome. However, how the loss of FMRP influences the developmental trajectory of cortical maturation remains unclear. We took advantage of the stereotyped and well-characterized development of the murine primary somatosensory cortex to examine cortical maturation during a time-window that corresponds to late embryonic and early postnatal development in the human. In the Fmr1 knockout mouse, we find a delay in somatosensory map formation, alterations in the morphology profile of dendrites and spines of layer 4 neurons and a decrease in the synaptic levels of proteins involved in glutamate receptor signaling at times corresponding to the highest levels of FMRP expression. In contrast, cortical arealization, synaptic density in layer 4 and early postnatal regulation of mRNAs encoding synaptic proteins are not altered in Fmr1 knockout mice. The specificity of the developmental delay in Fmr1 knockout mice indicates that the loss of FMRP does not result in a general stalling of cerebral cortex maturation. Instead, our results suggest that inaccurate timing of developmental processes caused by the loss of FMRP may lead to alterations in neural circuitry that underlie behavioral and cognitive dysfunctions associated with FXS.     

A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability

Fragile X syndrome (FXS) results from the loss of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates a variety of cytoplasmic mRNAs. FMRP regulates mRNA translation and may be important in mRNA localization to dendrites. We report a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, we found that FMRP binds, in vivo, the mRNA encoding PSD-95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3' untranslated region of the PSD-95 (also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. Although we also found that the PSD-95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through our functional analysis of this FMRP target we provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS.     

Quantitative measurement of FMRP in blood platelets as a new screening test for fragile X syndrome

Lessard M, Chouiali A, Drouin R, Sébire G, Corbin F. Quantitative measurement of FMRP in blood platelets as a new screening test for fragile X syndrome. The fragile X syndrome usually results from CGG repeats expansion and methylation of the FMR1 gene leading to the absence of expression of its encoded protein, fragile X mental retardation protein (FMRP). Therefore, its diagnosis is traditionally based on the detection of these molecular alterations. As an alternative, FMRP-based screening methods have been proposed over the years. Most of them are based on immunohistochemistry analyses applied to a restricted number of lymphocytes (100) or hair roots (10-20) with limited diagnosis potential. In this study, we describe a truly quantitative approach using a new model, the blood platelet, which can be recovered easily with very high purity (99.9%). FMRP levels in platelets were first measured in a control population (n = 124) and reference values were established. FMRP measurements were also performed in confirmed fragile X subjects. Receiver operating characteristic curve analysis has shown that our test can easily discriminate fragile X males and females from controls (area under curve, AUC = 0.948). Cognitive functions were also assessed in these individuals using age-specific Wechsler Intelligence Scales for Children and the Vineland Adaptive Behavior Scales. A proportional relationship between FMRP levels, intelligence quotient and adaptive behavior was observed among fragile X individuals, suggesting that our test would be able to detect fragile X cases and may predict cognitive functions.     

CGG repeat in the FMR1 gene: size matters

The FMR1 gene contains a CGG repeat present in the 5'-untranslated region which can be unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In patients with fragile X syndrome (FXS), a repeat length exceeding 200 CGGs (full mutation: FM) generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The absence of FMR1 protein, FMRP, seen in FM is the cause of the mental retardation in patients with FXS. The premutation (PM) is defined as 55-200 CGGs. Female PM carriers are at risk of developing primary ovarian insufficiency. Elderly PM carriers might develop a progressive neurodegenerative disorder called fragile X-associated tremor/ataxia syndrome (FXTAS). Although arising from the mutations in the same gene, distinct mechanisms lead to FXS (absence of FMRP), FXTAS (toxic RNA gain-of-function) and FXPOI. The pathogenic mechanisms thought to underlie these disorders are discussed. This review gives insight on the implications of all possible repeat length categories seen in fragile X families.     

Inhibition of GSK3β improves hippocampus-dependent learning and rescues neurogenesis in a mouse model of fragile X syndrome

Fragile X syndrome (FXS), a common inherited form of intellectual disability with learning deficits, results from a loss of fragile X mental retardation protein (FMRP). Despite extensive research, treatment options for FXS remain limited. Since FMRP is known to play an important role in adult hippocampal neurogenesis and hippocampus-dependent learning and FMRP regulates the adult neural stem cell fate through the translational regulation of glycogen synthase kinase 3β (GSK3β), we investigated the effects of a GSK3β inhibitor, SB216763, on Fmr1 knockout mice (Fmr1 KO). We found that the inhibition of GSK3β could reverse the hippocampus-dependent learning deficits and rescue adult hippocampal neurogenesis at multiple stages in Fmr1 KO mice. Our results point to GSK3β inhibition as a potential treatment for the learning deficits seen in FXS.     

Metabotropic receptor-dependent long-term depression persists in the absence of protein synthesis in the mouse model of fragile X syndrome

Fragile X syndrome (FXS), a form of human mental retardation, is caused by loss of function mutations in the fragile X mental retardation gene (FMR1). The protein product of FMR1, fragile X mental retardation protein (FMRP) is an RNA-binding protein and may function as a translational suppressor. Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) in hippocampal area CA1 is a form of synaptic plasticity that relies on dendritic protein synthesis. mGluR-LTD is enhanced in the mouse model of FXS, Fmr1 knockout (KO) mice, suggesting that FMRP negatively regulates translation of proteins required for LTD. Here we examine the synaptic and cellular mechanisms of mGluR-LTD in KO mice and find that mGluR-LTD no longer requires new protein synthesis, in contrast to wild-type (WT) mice. We further show that mGluR-LTD in KO and WT mice is associated with decreases in AMPA receptor (AMPAR) surface expression, indicating a similar postsynaptic expression mechanism. However, like LTD, mGluR-induced decreases in AMPAR surface expression in KO mice persist in protein synthesis inhibitors. These results are consistent with recent findings of elevated protein synthesis rates and synaptic protein levels in Fmr1 KO mice and suggest that these elevated levels of synaptic proteins are available to increase the persistence of LTD without de novo protein synthesis.     

Biological and environmental contributions to adaptive behavior in fragile X syndrome

Fragile X syndrome is the most common cause of inherited developmental disability. The purpose of the present study is to understand how both biological and environmental influences affect the development of adaptive behavior in children with fragile X. In-home assessments were conducted on 120 children (80 boys and 40 girls) with the fragile X full mutation and their unaffected siblings (58 boys and 62 girls). Hierarchical multiple regression analyses were utilized. Independent variables included biological or demographic variables specific to the child (age, gender, full-scale IQ, and FMRP percentage), as well as factors specific to his/her environment (household income, home environment, maternal psychopathology, and effectiveness of educational/therapeutic services). Results of these analyses showed that for both boys with fragile X and the control sibling group, adaptive behavior was predicted by IQ, age, gender, and home environment. For girls with fragile X, adaptive behavior was most strongly associated with IQ. Adaptive behavior was not significantly associated with FMRP in boys or girls with fragile X. By examining the relative influences of biological and environmental factors on adaptive behavior in children with fragile X, we begin to lay the foundation for the development of more specific treatment studies in children with this disorder.     

Social behavior in Fmr1 knockout mice carrying a human FMR1 transgene

Fragile X syndrome (FXS) results from the loss of expression of the fragile X mental retardation (FMR1) gene. Individuals affected by FXS experience many behavioral problems, including cognitive impairment, hyperactivity, social anxiety, and autistic-like behaviors. A mouse model of Fmr1 deficiency (Fmr1KO) exhibits several similar behavioral phenotypes, including alterations in social behavior. In an earlier study, Fmr1 knockout mice carrying a yeast-artificial chromosome (YAC) transgene that over-expresses normal human FMRP (KOYAC) showed a correction or overcorrection of some behavioral responses, such as hyperactivity and anxiety-related responses. This report presents results from a study examining transgenic rescue of abnormal social responses in Fmr1KO mice. Relative to their wild-type (WT) littermates, Fmr1KO mice made more active social approaches to standard C57BL/6 partner mice in a direct social interaction test, a result consistent with a previous study. KOYAC mice showed fewer active approaches to partners than the WT or Fmr1KO littermates, indicating correction of this phenotype. This finding expands the number of murine behavioral responses caused by Fmr1 deficiency and corrected by overexpression of human FMRP.     

A double-blind, parallel, multicenter comparison of L-acetylcarnitine with placebo on the attention deficit hyperactivity disorder in fragile X syndrome boys

Attention deficit hyperactivity disorder (ADHD) is a frequent behavioral problem in young boys with fragile X syndrome (FXS), and its treatment is critical for improving social ability. The short-term efficacy of stimulant medications like methylphenidate (MPH) is well established in children with ADHD. FXS boys treated with MPH have improved attention span and socialization skills; however their mood becomes unstable at higher doses. Therefore, alternative pharmacological treatment of ADHD symptoms is desirable. A recent study showed that carnitine has a beneficial effect on the hyperactive-impulsive behavior in boys with ADHD without side effects. Our previous placebo-controlled trial indicated that L-acetylcarnitine (LAC) reduces hyperactivity in FXS boys. The objective of this study was to determine the efficacy of LAC in a larger sample of FXS boys with ADHD. The study design was randomized, double blind placebo controlled, parallel, and multicenter (with eight centers involved in Italy, France, and Spain). Sixty-three FXS males with ADHD (aged 6-13 years) were enrolled; 7 patients dropped out, 56 completed the one-year treatment, and 51 were included in the statistical analysis. Both groups improved their behavior, showing that psychosocial intervention has a significant therapeutic effect. However, we observed a stronger reduction of hyperactivity and improvement of social behavior in patients treated with LAC, compared with the placebo group, as determined by the Conners' Global Index Parents and the Vineland Adaptive Behavior Scale. Our results show that LAC (20-50 mg/kg/day) represents a safe alternative to the use of stimulant drugs for the treatment of ADHD in FXS children.     

FMRP expression as a potential prognostic indicator in fragile X syndrome.

Absence or deficit of FMR1 protein (FMRP) resulting from methylation of full mutation genes is the fundamental defect in fragile X syndrome. We used FMRP immunocytochemistry and detailed phenotypic assessment to investigate the relationship between degree of FMRP expression and the broad clinical spectrum of impairment in 80 individuals affected with fragile X syndrome. FMRP expression correlated with IQ in mosaic males (P=0.043), males with a partially methylated full mutation (P=0.0005), and females with a full mutation (P=0.046). In the females, FMRP expression also correlated with the number of fragile X physical features (P=0.0003). Even modest deficits in FMRP result in some manifestations of fragile X syndrome. In this initial study of 53 males, FMRP expression testing had a very high positive predictive value (100%, confidence interval of 29-100%) for a nonretarded IQ among males with expression of FMRP in > or = 50% of lymphocytes (3 males), suggesting that FMRP expression may have potential as a prognostic indicator in males with fragile X syndrome.