Common DNA methylation alterations in multiple brain regions in autism.

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders defined clinically by a triad of features including impairment in social interaction, impairment in communication in social situations and restricted and repetitive patterns of behavior and interests, with considerable phenotypic heterogeneity among individuals. Although heritability estimates for ASD are high, conventional genetic-based efforts to identify genes involved in ASD have yielded only few reproducible candidate genes that account for only a small proportion of ASDs. There is mounting evidence to suggest environmental and epigenetic factors play a stronger role in the etiology of ASD than previously thought. To begin to understand the contribution of epigenetics to ASD, we have examined DNA methylation (DNAm) in a pilot study of postmortem brain tissue from 19 autism cases and 21 unrelated controls, among three brain regions including dorsolateral prefrontal cortex, temporal cortex and cerebellum. We measured over 485,000 CpG loci across a diverse set of functionally relevant genomic regions using the Infinium HumanMethylation450 BeadChip and identified four genome-wide significant differentially methylated regions (DMRs) using a bump hunting approach and a permutation-based multiple testing correction method. We replicated 3/4 DMRs identified in our genome-wide screen in a different set of samples and across different brain regions. The DMRs identified in this study represent suggestive evidence for commonly altered methylation sites in ASD and provide several promising new candidate genes.

Social impairments in Rett syndrome: characteristics and relationship with clinical severity.

BACKGROUND: While behavioural abnormalities are fundamental features of Rett syndrome (RTT), few studies have examined the RTT behavioural phenotype. Most of these reports have focused on autistic features, linked to the early regressive phase of the disorder, and few studies have applied standardised behavioural measures. We used a battery of standardised measures of behaviour and functioning to test the following hypotheses: (1) autistic behaviour is prominent throughout childhood in RTT; (2) autistic features are more salient in individuals with milder presentation; (3) severity of autistic behaviour is associated with a wider range of behavioural problems; and (4) specific MECP2 mutations are linked to more severe autistic behaviour. METHODS: Eighty MECP2 mutation-positive girls with RTT (aged 1.6-14.9 years) were administered: (1) the Screen for Social Interaction (SSI), a measure of autistic behaviour suited for individuals with severe communication and motor impairment; (2) the Rett Syndrome Behaviour Questionnaire (RSBQ), covering a wide range of abnormal behaviours in RTT; (3) the Vineland Adaptive Behavior Scales (VABS); and (4) a modified version of the Rett Syndrome Severity Scale (RSSS). Regression analyses examined the predictive value of age and RSSS on autistic behaviour and other behavioural abnormalities. T-tests further characterised the behavioural phenotype of individual MECP2 mutations. RESULTS: While age had no significant effect on SSI or RSBQ total scores in RTT, VABS Socialization and Composite scores decreased over time. Clinical severity (i.e. RSSS) also increased with age. Surprisingly, SSI performance was not related to either RSSS or VABS Composite scores. Autistic behaviour was weakly linked with the RSBQ Hand behaviour factor scores, but not with the RSBQ Fear/Anxiety factor. Clinical (neurological) severity did not predict RSBQ scores, as evidenced by the analysis of individual MECP2 mutations (e.g. p.R106W, p.R270X and p.R294X). CONCLUSIONS: Our data suggest that in RTT, autistic behaviour persists after the period of regression. It also demonstrated that neurological and behavioural impairments, including autistic features, are relatively independent of one another. Consistent with previous reports of the RTT phenotype, individual MECP2 mutations demonstrate complex associations with autistic features. Evidence of persistent autistic behaviour throughout childhood, and of a link between hand function and social skills, has important implications not only for research on the RTT behavioural phenotype, but also for the clinical management of the disorder.

Autism spectrum disorder in Down syndrome: cluster analysis of Aberrant Behaviour Checklist data supports diagnosis.

BACKGROUND: The diagnostic validity of autism spectrum disorder (ASD) based on Diagnostic and Statistical Manual of Mental Disorders (DSM) has been challenged in Down syndrome (DS), because of the high prevalence of cognitive impairments in this population. Therefore, we attempted to validate DSM-based diagnoses via an unbiased categorisation of participants with a DSM-independent behavioural instrument. METHODS: Based on scores on the Aberrant Behaviour Checklist - Community, we performed sequential factor (four DS-relevant factors: Autism-Like Behaviour, Disruptive Behaviour, Hyperactivity, Self-Injury) and cluster analyses on a 293-participant paediatric DS clinic cohort. The four resulting clusters were compared with DSM-delineated groups: DS + ASD, DS + None (no DSM diagnosis), DS + DBD (disruptive behaviour disorder) and DS + SMD (stereotypic movement disorder), the latter two as comparison groups. RESULTS: Two clusters were identified with DS + ASD: Cluster 1 (35.1%) with higher disruptive behaviour and Cluster 4 (48.2%) with more severe autistic behaviour and higher percentage of late onset ASD. The majority of participants in DS + None (71.9%) and DS + DBD (87.5%) were classified into Cluster 2 and 3, respectively, while participants in DS + SMD were relatively evenly distributed throughout the four clusters. CONCLUSIONS: Our unbiased, DSM-independent analyses, using a rating scale specifically designed for individuals with severe intellectual disability, demonstrated that DSM-based criteria of ASD are applicable to DS individuals despite their cognitive impairments. Two DS + ASD clusters were identified and supported the existence of at least two subtypes of ASD in DS, which deserve further characterisation. Despite the prominence of stereotypic behaviour in DS, the SMD diagnosis was not identified by cluster analysis, suggesting that high-level stereotypy is distributed throughout DS. Further supporting DSM diagnoses, typically behaving DS participants were easily distinguished as a group from those with maladaptive behaviours.

Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids.

Prostaglandins play important and diverse roles in the CNS. The first step in prostaglandin synthesis involves enzymatic oxidation of arachidonic acid, which is catalyzed by prostaglandin H(PGH) synthase, also referred to as cyclooxygenase. We have cloned an inducible form of this enzyme from rat brain that is nearly identical to a murine, mitogen-inducible cyclooxygenase identified from fibroblasts. Our studies indicate that this gene, here termed COX-2, is expressed throughout the forebrain in discrete populations of neurons and is enriched in the cortex and hippocampus. Neuronal expression is rapidly and transiently induced by seizures or NMDA-dependent synaptic activity. No expression is detected in glia or vascular endothelial cells. Basal expression of COX-2 appears to be regulated by natural synaptic activity in the developing and adult brain. Both basal and induced expression of COX-2 are inhibited by glucocorticoids, consistent with COX-2 regulation in peripheral tissues. Our studies indicate that COX-2 expression may be important in regulating prostaglandin signaling in brain. The marked inducibility in neurons by synaptic stimuli suggests a role in activity-dependent plasticity.

Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites.

Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.

Development of a video-based evaluation tool in Rett syndrome.

This paper describes the development of a video-based evaluation tool for use in Rett syndrome (RTT). Components include a parent-report checklist, and video filming and coding protocols that contain items on eating, drinking, communication, hand function and movements, personal care and mobility. Ninety-seven of the 169 families who initially agreed to participate returned a videotape within 8 months of the first request. Subjects whose videos were returned had a similar age profile to those who did not provide a video but were more likely to have classical than atypical RTT. Evidence of the content and social validity and inter-rater reliability on 11 videos is provided. Video may provide detailed, objective assessment of function and behaviour in RTT.

Cycloxygenase-2 activity promotes cognitive deficits but not increased amyloid burden in a model of Alzheimer's disease in a sex-dimorphic pattern.

Administration of non-steroidal anti-inflammatory agents reduces the risk of developing Alzheimer's disease in normal aging populations, an effect that may occur from inhibition of the cyclooxygenases, the rate-limiting enzymes in the formation of prostaglandins. In this study, we investigated whether increased activity of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, potentiates disease progression in a transgenic mouse model of Alzheimer's disease. To study the functional effects of COX-2 activity, male and female bigenic mice (amyloid precursor protein with Swedish mutation [APPswe]-presenilin-1 protein with deletion of exon 9 [PS1dE9] and trigenic COX-2/APPswe-PS1dE9) were behaviorally tested +/-administration of the selective COX-2 inhibitor celecoxib. Behavioral testing included a three-trial Y maze that measures spatial working and recognition memories and an open field task that tested levels of hyperactivity. Overexpression of COX-2 in APPswe-PS1dE9 mice resulted in specific deficits in spatial working memory in female but not male mice. These sex-specific deficits were abolished by pharmacological inhibition of COX-2 activity. Importantly, COX-2-associated deficits were dependent on co-expression of all three transgenes since COX-2 single transgenic and APPswe-PS1dE9 bigenic mice showed normal memory. Quantification of amyloid plaque load and total Abeta 40 and 42 peptides did not reveal significant differences in trigenic versus bigenic mice treated with either vehicle or celecoxib. Taken together, these data indicate an interaction between the effects of COX-2 and Abeta peptides on cognition that occurs in a sex-specific manner in the absence of significant changes in amyloid burden. These findings suggest that pathological activation of COX-2 may potentiate the toxicity of Abeta peptides, particularly in females, without significantly affecting Abeta accumulation.

Age-dependent cognitive deficits and neuronal apoptosis in cyclooxygenase-2 transgenic mice.

The cyclooxygenases catalyze the rate-limiting step in the formation of prostaglandins from arachidonic acid and are the pharmacological targets of (NSAIDs). In brain, cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, is selectively expressed in neurons of the cerebral cortex, hippocampus, and amygdala. As an immediate-early gene, COX-2 is dramatically and transiently induced in these neurons in response to NMDA receptor activation. In models of acute excitotoxic neuronal injury, elevated and sustained levels of COX-2 have been shown to promote neuronal apoptosis, indicating that upregulated COX-2 activity is injurious to neurons. COX-2 may also contribute to the development of Alzheimer's disease, for which early administration of NSAIDs is protective against development of the disease. To test the effect of constitutively elevated neuronal COX-2, transgenic mice were generated that overexpressed COX-2 in neurons and produced elevated levels of prostaglandins in brain. In cross-sectional behavioral studies, COX-2 transgenic mice developed an age-dependent deficit in spatial memory at 12 and 20 months but not at 7 months and a deficit in aversive behavior at 20 months of age. These behavioral changes were associated with a parallel age-dependent increase in neuronal apoptosis occurring at 14 and 22 months but not at 8 months of age and astrocytic activation at 24 months of age. These findings suggest that neuronal COX-2 may contribute to the pathophysiology of age-related diseases such as Alzheimer's disease by promoting memory dysfunction, neuronal apoptosis, and astrocytic activation in an age-dependent manner.

Medical and ethical challenges in the case of a prenatally undiagnosed massive congenital brain tumor.

Fetal and neonatal brain tumors are rare. Prenatal ultrasound aids early tumor detection. Nonetheless, we encountered a preterm neonate born at 32 weeks gestation with a massive supratentorial glioma, which was undetected on ultrasound at 19-6/7 weeks gestation. The patient presented at birth with unanticipated massive macrocephaly. Resuscitation and stabilization were difficult, but the medical team felt that futility of care was not established and opted to transfer the baby to an academic center for further imaging and specialist consultations. Diagnosis of an extensive, inoperable tumor was confirmed and support withdrawn. Postmortem histologic examination and immunohistochemical stains identified the majority of tumor cells as glial in origin. This case report illustrates well how a severe and potentially fatal anomaly, which remained undetected prenatally, presented the medical team and family with multiple medical, ethical and emotional challenges at birth; decisions regarding futility of care in the neonatal transport setting are difficult.

Regional cortical white matter reductions in velocardiofacial syndrome: a volumetric MRI analysis.

BACKGROUND: Velocardiofacial syndrome, caused by a microdeletion on chromosome 22q.11, is associated with craniofacial anomalies, cardiac defects, learning disabilities, and psychiatric disorders. To understand how the 22q.11 deletion affects brain development, this study examined gray and white matter volumes in major lobar brain regions of children with velocardiofacial syndrome relative to control subjects. METHODS: Subjects were ten children with velocardiofacial syndrome and ten age- and gender-matched unaffected children. Coronal images were acquired with a 3-D spoiled gradient echo series and partitioned into 124, 1.5-mm contiguous slices. A stereotaxic grid was used to subdivide brain tissue into cerebral lobes, which were segmented into gray, white, and CSF compartments using an algorithm based on intensity values and tissue boundaries. Nonparametric statistics were used to compare lobar volumes of gray and white matter. RESULTS: Analyses indicated that children with velocardiofacial syndrome had significantly smaller volumes in nonfrontal, but not frontal, lobar brain regions. Volume reductions affected nonfrontal white matter to a greater extent than nonfrontal gray matter. CONCLUSIONS: The presence of white matter reductions may be related to disturbances in myelination or axonal integrity in velocardiofacial syndrome. Further work is required to delineate the nature and extent of white matter anomalies, and to link them to variation in the neurocognitive and neuropsychiatric phenotype of velocardiofacial syndrome.

Cerebrocortical microdysgenesis in neurologically normal subjects: a histopathologic study.

Previous studies showed that few foci of cerebro-cortical microdysgenesis (molecular layer neuronal ectopias and focal laminar dysplasia) are present in up to 26% of variably processed normal brains; they are more common in the right inferior frontal region. Brains of male developmental dyslexics processed in serial histologic sections showed 30 to 140 foci of these types of anomalies, predominantly in left perisylvian cortex. Here, we present the results of a detailed analysis of ten normal brains also processed in serial sections. The ages ranged from 3.5 to 87 years, all male. Three brains showed abnormalities similar in type to those of the dyslexic, but in far smaller numbers and in different locations: two showed a single cingulate focus--one right, one left; the third brain showed two right supratemporal foci. We conclude that the present form of developmental anomaly is rare in normal brains, and that the findings in the dyslexic brains may be significant.

Evidence for neuronal degeneration and dendritic plasticity in cortical pyramidal neurons of Huntington's disease: a quantitative Golgi study.

Aided by a computer microscope, the Eutectic Neuron Tracing System, we performed a quantitative analysis of 59 rapid Golgi-impregnated pyramidal neurons from the third and fifth prefrontal cortical layers (P III and P V neurons) in tissue sections obtained from seven autopsied Huntington's disease (HD) patients (grades 2 through 4) and 59 corresponding cells from eight age-matched control cases. Relative to controls, P III HD neurons had a significant increase in the number of primary dendritic segments arising from soma, total dendritic length, and total surface area. The HD cells also had significantly more dendritic branches at three intervals of measurement in a Sholl diagram (100 microns, 200 microns, and 400 microns from the soma) and a significant increase in the number of dendritic branching points. The dendritic spine density in P III HD neurons was comparable to that of control subjects and significantly lower than that in P V HD cells. The total number and the total density of dendritic swellings were significantly increased in both P III and P V neurons, being most numerous in grades 2 and 3 cases. Rare withered cells with shrunken dendritic trees, harboring few spines and numerous varicosities on their dendritic shafts, were present in HD but not in control cases. Thus, while a small fraction of prefrontal cortical pyramidals degenerates in HD, the plasticity of the remaining pyramidal neurons, evidenced as an orderly augmentation of the dendritic tree, may represent a compensatory response sufficient to maintain relatively normal metabolic function of the cortex in most adult-onset cases.

Quantitative 1H MR spectroscopic imaging in early Rett syndrome.

OBJECTIVE: To determine cerebral regional concentrations of N-acetyl aspartate (NAA), total choline (Cho), and total creatine (Cr) in Rett syndrome (RS) using 1H magnetic resonance spectroscopic imaging (MRSI). BACKGROUND: The biochemical defect underlying RS is unknown. Because in vivo MRSI can detect important cerebral metabolites, MRSI has a potential to reveal impairment of regional cerebral metabolism in RS noninvasively. METHODS: High-resolution, multislice 1H MRSI was carried out in 17 girls with RS. The control group consisted of nine healthy children. RESULTS: In patients with RS, average Cho concentration was 12% higher (p < 0.005) and average NAA concentration 11% lower (p < 0.0001) compared with the control group. Regional metabolic differences included significantly lower NAA concentration in the frontal gray and white matter, insula, and hippocampus in RS; no difference in regional Cho and Cr concentrations were found. A 20 to 38% higher Cho:NAA ratio in frontal and parietal gray and white matter, insular gray matter, and hippocampus (p < 0.05) and a 14 to 47% lower NAA:Cr ratio in frontal cortical gray matter, parietal and temporal white matter, insula, and putamen (p < 0.05) were found in subjects with RS compared with controls. Patients with seizures had higher average concentrations of Cho, Cr, and NAA compared with those without seizures (8-19%, p < 0.05). CONCLUSION: Metabolic impairment in RS involves both gray and white matter and particularly involves frontal and parietal lobes and the insular cortex. Loss of NAA most likely reflects reduced neuronal and dendritic tree size; increased Cho concentration may result from gliosis.

Megalencephaly in NF1: predominantly white matter contribution and mitigation by ADHD.

BACKGROUND: Megalencephaly is a frequent CNS manifestation in neurofibromatosis type 1 (NF1); however, its tissue composition, modification by attention deficit hyperactivity disorder (ADHD), and relationship with unidentified bright objects (UBO) remain controversial. METHODS: Eighteen male patients with NF1, seven of whom had ADHD (NF1+ADHD), were compared with 18 age- and sex-matched controls in terms of MRI-, Talairach-based brain, cerebral, lobar, and sublobar gray and white matter volumes. Twelve subjects with NF1 had UBO in the centrencephalic region, whereas six had no UBO or exclusively infratentorial lesions. RESULTS: Patients with NF1 without ADHD (NF1-pure) had the largest total cerebral, gray, and white matter volumes with larger parietal/somatosensory white matter volumes than controls, particularly if UBO were present in the basal ganglia. All subjects with NF1 (including NF1+ADHD) had larger total and frontal white matter volumes than controls. Smaller frontal/right prefrontal gray matter volumes were found in NF1+ADHD when compared with NF1-pure patients. CONCLUSIONS: The increase in frontal and parietal white matter volumes in male patients with NF1, including the preferential centrencephalic distribution, supports the hypothesis that NF1's white matter pathology encompasses but is not limited to visible UBO. Male patients with NF1+ADHD, as compared with NF1-pure patients, showed frontal reductions that are largely consistent with those found in idiopathic ADHD.

Disproportionate increases of white matter in right frontal lobe in Tourette syndrome.

OBJECTIVE: Based on previous findings implicating abnormalities of cortico-striatal-thalamo-cortical circuitry in Tourette syndrome (TS), the authors performed a volumetric analysis of frontal and nonfrontal tissue (gray + white matter) in boys with TS, with and without attention deficit hyperactivity disorder (ADHD). METHODS: Frontal and nonfrontal gray and white matter compartment volumes, obtained by a MRI protocol, were analyzed with a 2 x 2 factorial multivariate analysis of variance approach for associations with a TS or ADHD factor in 11 boys with TS only, 14 with TS + ADHD, 12 with ADHD only, and 26 healthy boys. RESULTS: In subjects with TS, the right frontal lobe showed a larger proportion of white matter. In addition, results were consistent with previous reports of reduced frontal lobe volumes associated with ADHD. Our analyses suggested these reductions to be mainly the consequence of smaller gray matter volumes, particularly on the left. CONCLUSIONS: These findings, suggesting the volumetric composition of frontal lobe tissue to be different in TS, support the hypothesis proposing frontostriatal pathway involvement in the pathophysiology of the disorder. Differences in composition of right frontal lobe attributable to white matter do not definitively implicate the hypothesized fiber pathways; however, considered in the context of the unilateral directionality of frontal-striatal circuitry, these results suggest the white matter connections as one explanation for basal ganglia anomalies (loss of normal left > right asymmetry) in TS.

Expression of agrin in the developing and adult rat brain.

Agrin, a synaptic basal lamina protein, is essential for the formation of the vertebrate neuromuscular junction. Agrin's role in synaptogenesis in the central nervous system has, however, not been elucidated. Therefore, we performed immunohistochemical analysis of agrin localization in adult rat brain using agrin-specific polyclonal antibodies. Our results show that agrin immunoreactivity is detected in neuronal cells throughout the brain, and that agrin is expressed in many morphologically and neurochemically distinct neuronal populations. Within neurons, agrin-immunoreactive material is present in dendrites. To determine agrin isoform expression in the central nervous system, we analysed the pattern of expression of several isoforms during development of the rat brain. Our results indicate that alternative splicing of agrin is specifically regulated in the nervous system; isoforms of the Y=4 (i.e. Ag x,4,0, Ag x,4,8 and Ag x,4,19), Z=8 and Z=19 type are expressed exclusively in the nervous system. Agrin expression precedes synaptogenesis and is developmentally regulated in neural tissues. To evaluate stimuli that may be involved in the regulation of agrin expression, we monitored the patterns of isoform expression following a depolarizing stimulus. Our results show that agrin expression in the adult hippocampus is regulated in an activity-dependent manner, with kinetics of induction resembling a delayed early response gene.

Methyl-CpG-binding protein 2 is localized in the postsynaptic compartment: an immunochemical study of subcellular fractions.

Methyl-CpG-binding protein 2 is a characteristic member of the methyl-CpG-binding protein family of transcription regulators. In conjunction with Sin3, MeCP2 recruits class I histone deacetylases to methyl-CpG regions to suppress transcription. Rett syndrome, a disorder characterized by mental retardation and autistic features, is associated in a majority of cases with mutations within the coding region of the MeCP2 gene. Considering that defective MeCP2 has mainly been related to Rett syndrome and other neurologic manifestations, we examined methyl-CpG-binding protein 2 cellular and subcellular compartmentalization in normal brain by immunochemical methods. Methyl-CpG-binding protein 2 immunoreactivity is present mainly in neurons; while the few immunostained glia show label confined to nuclei, many neurons also show slight perikaryal staining. Using well-characterized tissue fractions, we found that methyl-CpG-binding protein 2 but not Sin3 is found in both nuclear and postsynaptic compartments. This novel extranuclear localization is not unique to methyl-CpG-binding protein 2, since it has been previously reported for other transcription regulators such as c-Fos. These findings support the concept that methyl-CpG-binding protein 2 may link synaptic activity and transcriptional regulation in neurons.

Annexin-1 is abnormally expressed in fragile X syndrome: two-dimensional electrophoresis study in lymphocytes.

The search for targets of FMRP (the product of FMR1, the mutated gene in Fragile X syndrome) has predominantly focused on identifying transcripts that are regulated by this RNA-binding protein. This study introduces the use of two-dimensional gel electrophoresis (2D PAGE) as a novel approach for demonstrating changes in protein synthesis secondary to FMRP deficit. By a standardized 2D PAGE protocol, we studied leukocyte homogenates from 30 males with different patterns of FMR1 mutation and different levels of FMRP. Samples from these subjects were compared to those of 12 normal control males and eight subjects with other mental retardation-associated conditions (i.e., Rett and Down syndromes). We found an abnormal pattern of a major leukocytic protein, identified by 2D PAGE datasets and immunoblotting as annexin-1 (Anx-1). Anx-1 appeared in subjects with Fragile X as multiple rather than 1-2 spots, at approximately 37 kd, in the pI 5-7 range. The presence and intensity of this Anx-1 pattern was relatively independent of Anx-1 levels and inversely related to total and high MW FMRP immunoreactivities. Based on the 2D PAGE pattern, without obvious MW change, and on dephosphorylation assays, we concluded that Anx-1's abnormality represents an aberrant posttranslational modification other than phosphorylation. Comparisons of our data with published cytoskeletal protein 2D profiles suggest that Anx-1 may be abnormally acetylated and, consequently, incapable of establishing appropriate N-terminal protein-protein interactions. In addition to its peripheral anti-inflammatory function, Anx-1 mediates glucocorticoid inhibition of the hypothalamo-pituitary-adrenal axis. As the latter seems to be disrupted in Fragile X syndrome, the reported Anx-1 abnormality could be responsible for some aspects of the Fragile X neurobehavioral phenotype. Our data also emphasize the feasibility of using 2D PAGE for disclosing molecular abnormalities in Fragile X and other genetic disorders.

Genotype, molecular phenotype, and cognitive phenotype: correlations in fragile X syndrome.

The study of the neurobehavioral consequences of mutations of FMR1, the gene responsible for fragile X syndrome (FraX), has been based largely on correlations between mutation patterns and cognitive profile. Following the characterization of FMRP, the FMR1 gene product, preliminary correlations between FMRP levels, and neurologic phenotype have been established. However, most of these investigations have focused on individuals at both ends of the genetic and cognitive spectra of FraX, subjects with normal or premutation (PM) alleles or males with the FMR1 full mutation (FM). The present study is designed to characterize FMRP expression and to correlate it with IQ, in a sample representing a wide spectrum of FMR1 mutations. For this purpose we developed a highly sensitive immunoblotting assay using peripheral leukocytes. Three distinct patterns of FMRP immunoreactivity (-ir) emerged. Individuals with normal (n = 28) and PM (n = 8) alleles as well as most females with the FM (n = 14) showed the highest levels with multiple approximately 70-80 kDa FMRP-ir bands. Males with the FM (n = 10) demonstrated only a 70 kDa FMRP-ir band, and had significantly lower levels when compared with any previous groups. Males with mosaicism and three of 14 females with FM displayed a doublet with equal amounts of the highest and lowest molecular weight FMRP-ir bands. Multiple regression models that adjust for the effect of parental IQ indicated that both activation ratio and FMRP-ir are significantly correlated to subject IQ. We conclude that FMRP-ir offers promise as an indicator of the impact of FMR1 mutations upon neurologic function. Furthermore, our unexpected finding of FMRP-ir in all males with FM suggests that most of them are not transcriptionally silent.

Diagnoses of neuronal ceroid-lipofuscinosis by immunochemical methods.

The neuronal ceroid-lipofuscinoses (NCL), also known as Batten disease, are a not uncommon group of disorders affecting infants, children, and young adults. The abnormal ultrastructural profiles seen in NCL are used for standard diagnosis; however, they can be missed, and are also found in other neurodegenerative conditions. Furthermore, there is an overlap between the types of inclusion profiles among the different forms of NCL. Therefore, a more specific and biochemically-based marker is necessary to confirm the diagnosis of NCL. Antibodies raised against the storage material from the ovine form of NCL (mitochondrial ATP synthase subunit c) were utilized to determine whether NCL could be distinguished from other metabolic-neurodegenerative disorders. By immunoblotting and immunohistochemistry, several brain samples of well-evaluated NCL cases confirmed increased accumulations in all NCL cases except in the brain of an infantile-onset NCL patient. The immunoblot studies of skin fibroblasts and brain were sensitive but not highly specific to NCL, due to the recognition of this material in normal controls as well as in other neurogenetic diseases. Immunocytochemistry of skin fibroblasts clearly distinguished LINCL and JNCL cases from controls, and with further refinement has the potential for becoming a diagnostic tool.