Animal Model for Fragile X Syndrome

The fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by an expansion of a polymorphic CGG repeat upstream of the coding region in the FMR1 gene. The expansion blocks expression of the FMR1 gene due to methyla-tion of the FMR1 promoter. Functional studies on the FMR1 protein have shown that the protein can bind RNA and might be involved in transport of RNAs from the nucleus to the cytoplasm. A role of FMR1 protein on translation of certain mRNAs has been suggested. An animal model for fragile X syndrome exists and these mice show some behavioural difficulties mimicking the human fragile X syndrome phenotype. This review presents what is known about the protein and what is learned from the animal model for fragile X syndrome.     

Disentangling Attentional Biases and Attentional Deficits in Depression: An Event-Related Potential P300 Analysis

Electroencephalographic event-related potentials (ERPs) were used to investigate maladaptive attentional processes in depression. Specifically, we measured the ERP P300 component—a waveform that reflects the real-time allocation of attention to stimuli of high informational salience—as it was elicited by neutral and negatively valent words among currently depressed, previously depressed, and never-depressed participants. The study design allowed us to clarify the degree to which the oft-reported attenuation of P300 response in depression should be regarded as evidence of: (a) a general, pervasive impairment in depressive attentional function; or (b) the operation of depressotypic attentional biases, which may give rise to attentional deficits only regarding stimuli of non-negative emotional valence. Consistent with the latter possibility, depressed individuals were observed in this study to experience, on average, a robust P300 response to negatively valent target words—a response of larger magnitude than that observed among previously depressed and never-depressed controls. This enhanced P300 response to negative stimuli in depression appears to be a statelike, rather than traitlike, phenomenon.

Enhanced perfusion in eyes and cerebral perfusion defects in a patient with Fragile X Syndrome

Fragile X syndrome (FXS) is known as the most common form of inherited mental retardation. In our study, brain perfusion single photon emission computed tomography (SPECT) was performed in a 6 year-old boy diagnosed with FXS. Diffuse bilateral uptake of Technetium-99m hexamethyl propylene amine oxime (99mTc-HMPAO) was noted in his orbits, as well as cortical perfusion defects (hypoperfusion in the right parietal and the left temporal lobe). Ophthalmologic examination showed no pathological findings. Magnetic resonance imaging (MRI) revealed no abnormality in the orbital structures, although hypoplasia of cerebellum and vermis was visualized. Since the patient was crying during the injection, the increased blood flow or the increased metabolism of the eyes and/or ocular muscles may be responsible for this orbital finding. Alternatively, the enhanced uptake of HMPAO in the orbits may reflect the pathology associated with FXS, because patients with FXS might have visual-motor abnormalities. To the best of our knowledge, there has been no report documenting such an orbital uptake of HMPAO. Moreover, the visualization of decreased cerebral perfusion, with the normal findings of MRI, indicates that brain SPECT imaging with HMPAO is helpful for detecting brain abnormalities in children with FXS.     

Genetic Testing for Fragile X

Ask the Expert provides research-based answers to practice questions submitted by JSPN readers. Where possible, evidence from research is used to support the experts' recommendations.     

Syndrome X

Insulin resistance is an early and major feature in the development of non-insulin-dependent diabetes mellitus(NIDDM). It is also associated with hyperlipidemia, hypertension, obesity and cardiovascular disease. It is the clustor of the risk factors for atherosclerosis and recognized as 'insulin-resistance syndrome' (Syndrome X). Central (abdominal) obesity is much more strongly associated with insulin resistance than overall obesity. The increase of both the influx of free fatty acid to liver and the production of TNF-alpha in adipose tissue may play an important role in mechanism of insulin resistance associated with central obesity. Calorie restriction and weight loss improve insulin sensitivity in overweight humans. Exercise training also improves insulin sensitivity via increased oxidative enzymes, glucose transporters (GLUT4) and capillarity in muscle as well as by reducing abdominal fat. The new 'glitazones' (thiazolidinediones) is used clinically to improve insulin sensitivity.     

Finding FMR1 mosaicism in Fragile X syndrome

OBJECTIVE: Almost all patients with Fragile X Syndrome (FXS) exhibit a CGG repeat expansion (full mutation) in the Fragile Mental Retardation 1 gene (FMR1). Here, the authors report five unrelated males with FXS harboring a somatic full mutation/deletion mosaicism.

METHODS: Mutational profiles were only elucidated by using a combination of molecular approaches (CGG-based PCR, Sanger sequencing, MS-MLPA, Southern blot and mPCR).

RESULTS: Four patients exhibited small deletions encompassing the CGG repeats tract and flanking regions, whereas the remaining had a larger deletion comprising at least exon 1 and part of intron 1 of FMR1 gene. The presence of a 2-3 base pairs microhomology in proximal and distal non-recurrent breakpoints without scars supports the involvement of microhomology mediated induced repair (MMBIR) mechanism in three small deletions.

CONCLUSION: The authors data highlights the importance of using different research methods to elucidate atypical FXS mutational profiles, which are clinically undistinguishable and may have been underestimated.     

Size and methylation mosaicism in males with Fragile X syndrome

Background: Size and methylation mosaicism are a common phenomenon in Fragile X syndrome (FXS). Here, the authors report a study on twelve fragile X males with atypical mosaicism, seven of whom presented with autism spectrum disorder.

Methods: A combination of Southern Blot and PCR analysis was used for CGG allele sizing and methylation. FMR1 mRNA and FMRP expression were measured by qRT-PCR and by Homogeneous Time Resolved Fluorescence methodology, respectively.

Results: DNA analysis showed atypical size- or methylation-mosaicism with both, full mutation and smaller (normal to premutation) alleles, as well as a combination of methylated and unmethylated alleles. Four individuals carried a deletion of the CGG repeat and portions of the flanking regions. The extent of methylation among the participants was reflected in the lower FMR1 mRNA and FMRP expression levels detected in these subjects.

Conclusion: Decreased gene expression is likely the main contributor to the cognitive impairment observed in these subjects; although the presence of a normal allele did not appear to compensate for the presence of the full mutation, it correlated with better cognitive function in some but not all of the reported cases emphasizing the complexity of the molecular and clinical profile in FXS.     

Validity of analysis of FMRP expression in blood smears as a screening test for Fragile X Syndrome in the Indian population

Molecular diagnosis of Fragile X Syndrome (FXS) is carried out by PCR or Southern blot analysis on DNA isolated from leukocytes. These DNA analyses are time consuming and expensive, making it impractical for mass screening programs. We have recently standardized and tested the diagnostic potential of a rapid antibody test on blood smears, based on the presence of FMRP, the protein product of the FMR1 gene, in lymphocytes from normal individuals and the absence of FMRP in lymphocytes in patients with FXS. This test is essentially similar to the one developed at Erasmus University in the Netherlands, with suitable modifications. The diagnostic power of the antibody test is perfect for males, whereas the results are less specific for females. The cutoff value for affected male individuals, expressed as the percentage of FMRP-positive cells, was 20%. In normal individuals, the cutoff value was 85%. The results of the antibody test correlated well with that of Southern blots. Sensitivity of the test was 100% and specificity was 97.5%. This noninvasive test requires one or two drops of blood and is rapid, simple, and cheap, making it an ideal choice for large screening large groups of male mental retardates and neonates for FXS in developing countries such as India.     

Behavioural Excesses and Deficits Associated with Dementia in Adults who have Down Syndrome

Background Informant‐based assessment of behavioural change and difference in dementia in Down syndrome can aid diagnosis and inform service delivery. To date few studies have examined the impact of different types of behavioural change. Methods The Assessment for Adults with Developmental Disabilities (AADS), developed for this study, assesses behavioural excesses (11 items) and deficits (17 items) associated with dementia. Inter‐informant reliability, internal consistency and concurrent validity were evaluated and found to be robust. Results A comparison of the AADS subscale scores for three groups (n = 12) of adults with Down syndrome demonstrated more frequent deficits and excesses and greater management difficulty and effects on the individual in a dementia group than age comparable and younger groups. Conclusion The AADS is a promising dementia specific measure for people with intellectual disability. Further research should evaluate change as dementia progresses and the nature of management difficulty and effects on the individual.     

A Quantitative ELISA Assay for the Fragile X Mental Retardation 1 Protein

Non-coding (CGG-repeat) expansions in the fragile X mental retardation 1 (FMR1) gene result in a spectrum of disorders involving altered neurodevelopment (fragile X syndrome), neurodegeneration (late-onset fragile X-associated tremor/ataxia syndrome), or primary ovarian insufficiency. While reliable and quantitative assays for the number of CGG repeats and FMR1 mRNA levels are now available, there has been no scalable, quantitative assay for the FMR1 protein (FMRP) in non-transformed cells. Using a combination of avian and murine antibodies to FMRP, we developed a sensitive and highly specific sandwich enzyme-linked immunosorbent assay (ELISA) for FMRP in peripheral blood lymphocytes. This ELISA method is capable of quantifying FMRP levels throughout the biologically relevant range of protein concentrations and is specific for the intact FMRP protein. Moreover, the ELISA is well-suited for replicate protein determinations across serial dilutions in non-transformed cells and is readily scalable for large sample numbers. The FMRP ELISA is potentially a powerful tool in expanding our understanding of the relationship between FMRP levels and the various FMR1-associated clinical phenotypes.Fragile X syndrome (OMIM #300624), the most common heritable form of intellectual impairment and the leading known single-gene form of autism,^1,2 is nearly always caused by lowered (or absence of) expression of the fragile X mental retardation 1 (FMR1) protein (FMRP) in individuals who harbor FMR1 alleles in the full mutation range (>200 CGG repeats) or high premutation range (premutation, 55 to 200 CGG repeats).^3,4,5,6,7 Most individuals with a premutation allele have IQs that fall within the normal range, although some children experience attention deficit hyperactivity disorder and autism spectrum disorders.^8,9 Moreover, in adults, there is an increased risk of primary ovarian insufficiency,^10,11 emotional problems including depression and anxiety,¹² and the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome.^13,14 Although reduction or loss of FMRP is generally believed to be the basis for fragile X syndrome, as well as many of the neurodevelopmental problems in the upper premutation range, quantitative comparisons of molecular (FMRP) and clinical phenotypes are generally lacking due to the absence of a quantitative measure of the protein.Thus far, the main approaches for measuring protein levels have been indirect, involving immunohistochemical staining of peripheral blood lymphocytes or hair roots. A rapid immunohistochemical test of blood smears, using a mouse monoclonal antibody, was developed by Willemsen et al^15,16 In that approach, measurement of FMRP was assessed by counting positively stained lymphocytes, with the fraction of positively staining lymphocytes representing a measure of protein level. Microscopic evaluation of smears is necessary to distinguish positively stained lymphocytes from non-specifically stained monocytes. Moreover, as there is no weighting for the degree or intensity of staining, a weakly stained cell is counted the same as a cell that is intensely stained. Thus, the method is best suited for establishing the absence of FMRP in full mutation males, or the fraction of FMRP positive lymphocytes (a reflection of X activation ratio) in full mutation females. Expression of FMRP has also been studied by immunohistochemical analysis of hair roots.^17,18 One potential advantage of this method over the use of blood smears is that skin and neural cells both arise from the ectodermal germ layer, reducing the potential for discordance between cell types, particularly in the case of size- or methylation-mosaicism. However, this method also is not capable of quantifying protein levels.FMRP levels have been quantified by immunoblot analysis, either in Epstein Barr Virus-transformed lymphoblastoid cells⁵ or in non-transformed cells.¹⁹ The major caveat with the use of transformed cells, other than the effort required for the transformation process per se, is the uncertainty in comparing FMRP expression (or even allele size and methylation status) with the corresponding molecular measures in non-transformed lymphocytes. By contrast, Kaufmann et al¹⁹ did quantify FMRP levels in untransformed peripheral blood leukocytes using a Western blot analysis. The protein measures were well-controlled and it is therefore somewhat surprising that additional studies did not follow this methodologic line. One concern may have related to the potential cross-reactivity of the single anti-FMRP antibody with the paralogous proteins, FXR1P and FXR2P.Given the central importance of FMRP to the presence and severity of the clinical phenotype in fragile X syndrome, a method for accurately and rapidly quantifying FMRP levels is necessary. To this end, we have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for FMRP that precisely determines levels of the protein in circulating lymphocytes in humans. The assay is sensitive to small changes in protein levels, targets intact FMRP specifically, and is a reliable method for the measurement of FMRP in blood. Of course, the caveat of measuring a peripheral protein level for a central nervous system disorder remains incompletely resolved. Notwithstanding this concern, a truly quantitative measure of FMRP will allow a better assessment of its importance in various clinical settings.     

3D pattern of brain abnormalities in Fragile X syndrome visualized using tensor-based morphometry

Fragile X syndrome (FraX), a genetic neurodevelopmental disorder, results in impaired cognition with particular deficits in executive function and visuo-spatial skills. Here we report the first detailed 3D maps of the effects of the Fragile X mutation on brain structure, using tensor-based morphometry. TBM visualizes structural brain deficits automatically, without time-consuming specification of regions-of-interest. We compared 36 subjects with FraX (age: 14.66+/-1.58 S.D., 18 females/18 males), and 33 age-matched healthy controls (age: 14.67+/-2.2 S.D., 17 females/16 males), using high-dimensional elastic image registration. All 69 subjects' 3D T1-weighted brain MRIs were spatially deformed to match a high-resolution single-subject average MRI scan in ICBM space, whose geometry was optimized to produce a minimal deformation target. Maps of the local Jacobian determinant (expansion factor) were computed from the deformation fields. Statistical maps showed increased caudate (10% higher; p = 0.001) and lateral ventricle volumes (19% higher; p = 0.003), and trend-level parietal and temporal white matter excesses (10% higher locally; p = 0.04). In affected females, volume abnormalities correlated with reduction in systemically measured levels of the Fragile X mental retardation protein (FMRP; Spearman's r < -0.5 locally). Decreased FMRP correlated with ventricular expansion (p = 0.042; permutation test), and anterior cingulate tissue reductions (p = 0.0026; permutation test) supporting theories that FMRP is required for normal dendritic pruning in fronto-striatal-limbic pathways. No sex differences were found; findings were confirmed using traditional volumetric measures in regions of interest. Deficit patterns were replicated by performing statistics after logarithmic transformation, which may be more appropriate for tensor-valued data. Investigation of how these anomalies emerge over time will accelerate our understanding of FraX and its treatment.     

The Neuropsychological Assessment of Age Related Cognitive Deficits in Adults with Down's Syndrome

There is substantial evidence that older adults who have Down's syndrome are at risk for developing Alzheimer's disease. However, whilst the neuropathological signs of Alzheimer's disease are almost always apparent in those over the age of 30, the clinical signs may not be evident. This may be because the neuropathological changes are not sufficiently advanced to impair performance or because detecting neuropsychological deficits is more difficult in the presence of substantial, global cognitive impairment. In this study, 70 adults with Down's syndrome were assessed using a battery of neuropsychological tests, some of which had been used to assess dementia arising from Alzheimer's disease in the general population. Participants were screened to rule out advanced dementia or a pre-existing degree of cognitive impairment which would preclude participation in the tests. Analysis of test results for those under the age of 40, showed significant negative correlations between the Vineland Adaptive Behavior Scales and performance on all neuropsychological tests, thus demonstrating the effect of pre-existing global cognitive impairment on these particular tests. The results of a cross-sectional age group comparison, showed no difference between age groups on neuropsychological deficits which may indicate advanced dementia. This was almost certainly due to the screening procedure. However, the older age groups showed significantly impaired performance on memory tests in comparison to the younger age group. It is concluded that the more subtle cognitive impairments which associate to Alzheimer's disease can be identified in the presence of a global cognitive impairment with sufficiently sensitive tests.