The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children's Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community.

Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.

BACKGROUND: Cognitive status in females with mutations in the FMR1 (fragile X mental retardation 1) gene is highly variable. A biomarker would be of value for predicting which individuals were liable to develop cognitive impairment and could benefit from early intervention. A detailed analysis of CpG sites bridging exon 1 and intron 1 of FMR1, known as fragile X-related epigenetic element 2 (FREE2), suggests that a simple blood test could identify these individuals. METHODS: Study participants included 74 control females (<40 CGG repeats), 62 premutation (PM) females (55-200 CGG repeats), and 18 full-mutation (FM) females assessed with Wechsler intelligence quotient (IQ) tests. We used MALDI-TOF mass spectrometry to determine the methylation status of FREE2 CpG sites that best identified low-functioning (IQ <70) FM females (>200 CGG repeats), compared the results with those for Southern blot FMR1 activation ratios, and related these assessments to the level of production of the FMR1 protein product in blood. RESULTS: A methylation analysis of intron 1 CpG sites 10-12 showed the highest diagnostic sensitivity (100%) and specificity (98%) of all the molecular measures tested for detecting females with a standardized verbal IQ of <70 among the study participants. In the group consisting of only FM females, methylation of these sites was significantly correlated with full-scale IQ, verbal IQ, and performance IQ. Several verbal subtest scores showed strong correlation with the methylation of these sites (P = 1.2 × 10(-5)) after adjustment for multiple measures. CONCLUSIONS: The data suggest that hypermethylation of the FMR1 intron 1 sites in blood is predictive of cognitive impairment in FM females, with implications for improved fragile X syndrome diagnostics in young children and screening of the newborn population.

FMR1 intron 1 methylation predicts FMRP expression in blood of female carriers of expanded FMR1 alleles.

Fragile X syndrome (FXS) is caused by loss of the fragile X mental retardation gene protein product (FMRP) through promoter hypermethylation, which is usually associated with CGG expansion to full mutation size (>200 CGG repeats). Methylation-sensitive Southern blotting is the current gold standard for the molecular diagnosis of FXS. For females, Southern blotting provides the activation ratio (AR), which is the proportion of unmethylated alleles on the active X chromosome. Herein, we examine the relationship of FMRP expression with methylation patterns of two fragile X-related epigenetic elements (FREE) analyzed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and the AR. We showed that the differential methylation of the FREE2 sequence within fragile X mental retardation gene intron 1 was related to depletion of FMRP expression. We also show that, using the combined cohort of 12 females with premutation (55 to 200 CGG repeats) and 22 females with full mutation alleles, FREE2 methylation analysis was superior to the AR as a predictor of the proportion of FMRP-positive cells in blood. Because matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is amenable to high-throughput processing and requires minimal DNA, these findings have implications for routine FXS testing and population screening.

Early onset of neurological symptoms in fragile X premutation carriers exposed to neurotoxins.

We present four cases of fragile X premutation carriers with early neurological symptoms, including symptoms consistent with multiple sclerosis (MS) and fragile X-associated tremor/ataxia syndrome (FXTAS). Each patient had significant exposure to one or more environmental neurotoxicants that have documented neurotoxicity (i.e. hexachlorocyclopentadiene or C56, Agent Orange, and 2,4- or 2,6-toluene diisocyanate and dichlormate). We hypothesize that premutation carriers are a vulnerable group to neurotoxins because elevated mRNA in the premutation can lead to early cell death and brain disease, leading to neuropsychiatric and neurological symptoms consistent with FXTAS.

Advances in the treatment of fragile X syndrome.

The FMR1 mutations can cause a variety of disabilities, including cognitive deficits, attention-deficit/hyperactivity disorder, autism, and other socioemotional problems, in individuals with the full mutation form (fragile X syndrome) and distinct difficulties, including primary ovarian insufficiency, neuropathy and the fragile X-associated tremor/ataxia syndrome, in some older premutation carriers. Therefore, multigenerational family involvement is commonly encountered when a proband is identified with a FMR1 mutation. Studies of metabotropic glutamate receptor 5 pathway antagonists in animal models of fragile X syndrome have demonstrated benefits in reducing seizures, improving behavior, and enhancing cognition. Trials of metabotropic glutamate receptor 5 antagonists are beginning with individuals with fragile X syndrome. Targeted treatments, medical and behavioral interventions, genetic counseling, and family supports are reviewed here.

Secondary medical diagnosis in fragile X syndrome with and without autism spectrum disorder.

This study examined whether secondary medical diagnoses that affect CNS function (i.e., seizures, malformations, or genetic disorders), are more likely to occur in individuals with fragile X syndrome (FXS) and autism spectrum disorder (FXS + ASD) or FXS alone. Ninety males (3-25 years) with FXS or FXS + ASD were evaluated for secondary medical diagnoses by medical history and examination. A significant difference in the incidence of medical problems was found between patients with FXS + ASD (38.6%) and FXS alone (18.2%, P < 0.05). Medical problems that affect the CNS are more likely to occur in those with FXS + ASD and it is probable that additional brain dysfunction associated with these medical problems enhance the risk of autism in those with FXS.

The effectiveness of parent-child interaction therapy for families of children on the autism spectrum.

We report the results of a pilot trial of an evidence-based treatment-Parent-Child Interaction Therapy (PCIT; Eyberg et al. Psychopharmacology Bulletin, 31(1), 83-91, 1995) for boys aged 5-12 with high functioning autism spectrum disorders and clinically significant behavioral problems. The study also included an investigation of the role of shared positive affect during the course of therapy on child and parent outcomes. The intervention group showed reductions in parent perceptions of child problem behaviors and child atypicality, as well as an increase in child adaptability. Shared positive affect in parent child dyads and parent positive affect increased between the initial and final phases of the therapy. Parent positive affect after the first phase was related to perceptions of improvement in problem behaviors and adaptive functioning.

Cognitive, anxiety and mood disorders in the fragile X-associated tremor/ataxia syndrome.

OBJECTIVE: We evaluated patients with fragile X-associated tremor/ataxia syndrome (FXTAS), a neurodegenerative disorder associated with a CGG repeat expansion in the premutation range in the fragile X mental retardation 1 (FMR1) gene. METHODS: Neurological, psychiatric and neuropsychological evaluations were performed on 15 male patients with FXTAS. RESULTS: Seven cases were diagnosed with dementia, and seven others were diagnosed with mood and/or anxiety disorders. Twelve subjects demonstrated deficits on neuropsychological testing. CONCLUSIONS: Physicians assessing dementia patients are urged to consider this newly described syndrome, especially in patients with dementia associated with a movement disorder and in those with a family history of mental retardation. If FXTAS is a possible diagnosis, physicians may carry out FMR1 DNA testing; patients who test positive on DNA testing should undergo magnetic resonance imaging, be referred to neurology and receive genetic counseling.

The Prader-Willi phenotype of fragile X syndrome.

The Prader-Willi phenotype (PWP) of fragile X syndrome (FXS) is associated with obesity and hyperphagia similar to Prader-Willi syndrome (PWS), but without cytogenetic or methylation abnormalities at 15q11-13. Thirteen cases of PWP and FXS are reported here that were identified by obesity and hyperphagia. Delayed puberty was seen in 5 of 9 cases who had entered puberty, a small penis or testicles in seven of 13 cases, and infant hypotonia and/or a poor suck in seven of 13 cases. Autism spectrum disorder occurred in 10 of 13 cases, and autism was diagnosed in seven of 13 cases. We investigated cytoplasmic interacting FMR1 protein (CYFIP) expression, which is a protein that interacts with FMR1 protein (FMRP) because the gene for CYFIP is located at 15q11-13. CYFIP mRNA levels were significantly reduced in our patients with the PWP and FXS compared to individuals without FXS (p < .001) and also individuals with FXS without PWP (p = .03).

In vitro and in vivo characterization of [3H]CNS-5161--a use-dependent ligand for the N-methyl-D-aspartate receptor in rat brain.

Glutamate is the major excitatory neurotransmitter in the brain. Glutamate activation of the N-methyl-D-aspartate (NMDA) receptor subtype is thought to mediate important physiological and pathological processes, including memory formation and excitotoxicity. The goal of the present work was to characterize and validate a candidate agent for noninvasive positron emission tomography (PET) imaging of this receptor. [(3)H]-labeled N-[3-(3)H]-methyl-3-(thiomethylphenyl)cyanamide (CNS-5161) was incubated with rat brain homogenates at increasing concentrations, temperatures, and times to establish the binding kinetics and affinity of the ligand in vitro. Nonspecific binding was measured with 100 microM MK-801. The compound was also injected i.v. in rats pretreated with saline, NMDA, MK801, or a combination, and organ and brain regional uptake was assessed at various times after injection by autoradiography or dissection. Blood and brain samples were assayed for metabolites by high-performance liquid chromatography. CNS-5161 binds brain membranes with high affinity (K(d) < 4 nM) and fast association and dissociation kinetics. Specific binding increased in the presence of glutamate and glycine. Intravenous administration in control rats resulted in a heterogeneous brain distribution with hippocampus and cortex > thalamus > striatum > cerebellum, and a cortex/cerebellum ratio of 1.4. Pretreatment with NMDA increased the hippocampus-to-cerebellum ratio to 1.6-1.9 while MK801 abolished this increase, resulting in ratios close to 1. Thus, CNS-5161 binds preferentially to the activated state of the NMDA receptor channel in vitro and in vivo. The high affinity and fast kinetics make it compatible with PET imaging of a carbon-11 labeled CNS-5161.

Anilinodialkoxyquinazolines: screening epidermal growth factor receptor tyrosine kinase inhibitors for potential tumor imaging probes.

The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogues, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A new EGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC50 = 0.4-51 nM) for the EGFR tyrosine kinase. All of the analogues inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 = 0.8-20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2 to 5.5. Four compounds, 4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3'-chloroanilino)- and 4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice.

Recent advances in fragile X: a model for autism and neurodegeneration.

PURPOSE OF REVIEW: This review will describe recent developments in the neurobiology of fragile X syndrome (FXS), the association between FXS and autism, and involvement in premutation carriers. RECENT FINDINGS: Metabotropic glutamate receptor 5 (mGluR5)-coupled pathways are dysregulated in individuals with FXS and this is thought to relate to the FXS phenotype. The mGluR5 model suggests that mGluR5 antagonists, including downstream effectors such as lithium, could be useful for treating FXS. Two forms of clinical involvement associated with the fragile X mental retardation 1 (FMR1) gene, autism and fragile X-associated tremor/ataxia syndrome (FXTAS), have received additional attention during the past year. One study has found that approximately 30% of individuals with FXS have autism; those with autism have lowered cognitive abilities, language problems, and behavioral difficulties compared to those with FXS alone. Furthermore, evidence is mounting that autism also occurs in some young males who have premutation alleles. Finally, males and occasional females with premutation alleles may develop a neurological syndrome with aging that consists of tremor, ataxia, peripheral neuropathy, and cognitive deficits. Significant brain atrophy and white-matter disease is usually seen. This new disorder (FXTAS) is thought to be related to elevated levels of abnormal FMR1 mRNA. SUMMARY: Full-mutation forms of the gene (> 200 repeats) can cause autism, learning disabilities, anxiety disorders, and mental retardation. Disorders associated with premutation forms of the gene (55-200 repeats) include, in addition to autism, FXTAS in older males and females, and premature ovarian failure.