Toward Fulfilling the Promise of Molecular Medicine in Fragile X

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and a leading known cause of autism. It is caused by loss of expression of the fragile X mental retardation protein (FMRP), an RNA-binding protein that negatively regulates protein synthesis. In neurons, multiple lines of evidence suggest that protein synthesis at synapses is triggered by activation of group 1 metabotropic glutamate receptors (Gp1 mGluRs) and that many functional consequences of activating these receptors are altered in the absence of FMRP. These observations have led to the theory that exaggerated protein synthesis downstream of Gp1 mGluRs is a core pathogenic mechanism in FXS. This excess can be corrected by reducing signaling by Gp1 mGluRs, and numerous studies have shown that inhibition of mGluR5, in particular, can ameliorate multiple mutant phenotypes in animal models of FXS. Clinical trials based on this therapeutic strategy are currently under way. FXS is therefore poised to be the first neurobehavioral disorder in which corrective treatments have been developed from the bottom up: from gene identification to pathophysiology in animals to novel therapeutics in humans. The insights gained from FXS and other autism-related single-gene disorders may also assist in identifying molecular mechanisms and potential treatment approaches for idiopathic autism.Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.)National Institute of Mental Health (U.S.)FRAXA Research Foundatio

Arbaclofen in fragile X syndrome: results of phase 3 trials

Background: Arbaclofen improved multiple abnormal phenotypes in animal models of fragile X syndrome (FXS) and showed promising results in a phase 2 clinical study. The objective of the study is to determine safety and efficacy of arbaclofen for social avoidance in FXS. Methods: Two phase 3 placebo-controlled trials were conducted, a flexible dose trial in subjects age 12-50 (209FX301, adolescent/adult study) and a fixed dose trial in subjects age 5-11 (209FX302, child study). The primary endpoint for both trials was the Social Avoidance subscale of the Aberrant Behavior Checklist-Community Edition, FXS-specific (ABC-C FX ). Secondary outcomes included other ABC-C FX subscale scores, Clinical Global Impression-Improvement (CGI-I), Clinical Global Impression-Severity (CGI-S), and Vineland Adaptive Behavior Scales, Second Edition (Vineland-II) Socialization domain score. Results: A total 119 of 125 randomized subjects completed the adolescent/adult study (n = 57 arbaclofen, 62 placebo) and 159/172 completed the child study (arbaclofen 5 BID n = 38; 10 BID n = 39; 10 TID n = 38; placebo n = 44). There were no serious adverse events (AEs); the most common AEs included somatic (headache, vomiting, nausea), neurobehavioral (irritability/agitation, anxiety, hyperactivity), decreased appetite, and infectious conditions, many of which were also common on placebo. In the combined studies, there were 13 discontinuations (n = 12 arbaclofen, 1 placebo) due to AEs (all neurobehavioral). The adolescent/adult study did not show benefit for arbaclofen over placebo for any measure. In the child study, the highest dose group showed benefit over placebo on the ABC-C FX Irritability subscale (p = 0.03) and Parenting Stress Index (PSI, p = 0.03) and trends toward benefit on the ABC-C FX Social Avoidance and Hyperactivity subscales (both p < 0.1) and CGI-I (p = 0.119). Effect size in the highest dose group was similar to effect sizes for FDA-approved serotonin reuptake inhibitors (SSRIs). Conclusions: Arbaclofen did not meet the primary outcome of improved social avoidance in FXS in either study. Data from secondary measures in the child study suggests younger patients may derive benefit, but additional studies with a larger cohort on higher doses would be required to confirm this finding. The reported studies illustrate the challenges but represent a significant step forward in translating targeted treatments from preclinical models to clinical trials in humans with FXS

The challenges of clinical trials in fragile X syndrome

RATIONALE: Advances in understanding the underlying mechanisms of conditions such as fragile X syndrome (FXS) and autism spectrum disorders have revealed heterogeneous populations. Recent trials of novel FXS therapies have highlighted several challenges including subpopulations with possibly differential therapeutic responses, the lack of specific outcome measures capturing the full range of improvements of patients with FXS, and a lack of biomarkers that can track whether a specific mechanism is responsive to a new drug and whether the response correlates with clinical improvement. OBJECTIVES: We review the phenotypic heterogeneity of FXS and the implications for clinical research in FXS and other neurodevelopmental disorders. RESULTS: Residual levels of fragile X mental retardation protein (FMRP) expression explain in part the heterogeneity in the FXS phenotype; studies indicate a correlation with both cognitive and behavioral deficits. However, this does not fully explain the extent of phenotypic variance observed or the variability of drug response. Post hoc analyses of studies involving the selective mGluR5 antagonist mavoglurant and the GABAB agonist arbaclofen have uncovered significant therapeutic responses following patient stratification according to FMR1 promoter methylation patterns or baseline severity of social withdrawal, respectively. Future studies designed to quantify disease modification will need to develop new strategies to track changes effectively over time and in multiple symptom domains. CONCLUSION: Appropriate selection of patients and outcome measures is central to optimizing future clinical investigations of these complex disorders

Open-label add-on treatment trial of minocycline in fragile X syndrome

<p>Abstract</p> <p>Background</p> <p>Fragile X syndrome (FXS) is a disorder characterized by a variety of disabilities, including cognitive deficits, attention-deficit/hyperactivity disorder, autism, and other socio-emotional problems. It is hypothesized that the absence of the fragile X mental retardation protein (FMRP) leads to higher levels of matrix metallo-proteinase-9 activity (MMP-9) in the brain. Minocycline inhibits MMP-9 activity, and alleviates behavioural and synapse abnormalities in <it>fmr1 </it>knockout mice, an established model for FXS. This open-label add-on pilot trial was conducted to evaluate safety and efficacy of minocycline in treating behavioural abnormalities that occur in humans with FXS.</p> <p>Methods</p> <p>Twenty individuals with FXS, ages 13-32, were randomly assigned to receive 100 mg or 200 mg of minocycline daily. Behavioural evaluations were made prior to treatment (baseline) and again 8 weeks after daily minocycline treatment. The primary outcome measure was the Aberrant Behaviour Checklist-Community Edition (ABC-C) Irritability Subscale, and the secondary outcome measures were the other ABC-C subscales, clinical global improvement scale (CGI), and the visual analog scale for behaviour (VAS). Side effects were assessed using an adverse events checklist, a complete blood count (CBC), hepatic and renal function tests, and antinuclear antibody screen (ANA), done at baseline and at 8 weeks.</p> <p>Results</p> <p>The ABC-C Irritability Subscale scores showed significant improvement (p < 0.001), as did the VAS (p = 0.003) and the CGI (p < 0.001). The only significant treatment-related side effects were minor diarrhea (n = 3) and seroconversion to a positive ANA (n = 2).</p> <p>Conclusions</p> <p>Results from this study demonstrate that minocycline provides significant functional benefits to FXS patients and that it is well-tolerated. These findings are consistent with the <it>fmr1 </it>knockout mouse model results, suggesting that minocycline modifies underlying neural defects that account for behavioural abnormalities. A placebo-controlled trial of minocycline in FXS is warranted.</p> <p>Trial registration</p> <p>ClinicalTrials.gov Open-Label Trial NCT00858689.</p

Developing a utility index for the Aberrant Behavior Checklist (ABC-C) for fragile X syndrome

Purpose This study aimed to develop a utility index (the ABC-UI) from the Aberrant Behavior Checklist-Community (ABC-C), for use in quantifying the benefit of emerging treatments for fragile X syndrome (FXS). Methods The ABC-C is a proxy-completed assessment of behaviour and is a widely used measure in FXS. A subset of ABC-C items across seven dimensions was identified to include in health state descriptions. This item reduction process was based on item performance, factor analysis and Rasch analysis performed on an observational study dataset, and consultation with five clinical experts and a methodological expert. Dimensions were combined into health states using an orthogonal design and valued using time trade-off (TTO), with lead-time TTO methods used where TTO indicated a state valued as worse than dead. Preference weights were estimated using mean, individual level, ordinary least squares and random-effects maximum likelihood estimation [RE (MLE)] regression models. Results A representative sample of the UK general public (n = 349; mean age 35.8 years, 58.2 % female) each valued 12 health states. Mean observed values ranged from 0.92 to 0.16 for best to worst health states. The RE (MLE) model performed best based on number of significant coefficients and mean absolute error of 0.018. Mean utilities predicted by the model covered a similar range to that observed. Conclusions The ABC-UI estimates a wide range of utilities from patient-level FXS ABC-C data, allowing estimation of FXS health-related quality of life impact for economic evaluation from an established FXS clinical trial instrument

Rapid-Onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD): Exome sequencing of trios, monozygotic twins and tumours

BACKGROUND: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is thought to be a genetic disease caused by de novo mutations, though causative mutations have yet to be identified. We searched for de novo coding mutations among a carefully-diagnosed and clinically homogeneous cohort of 35 ROHHAD patients. METHODS: We sequenced the exomes of seven ROHHAD trios, plus tumours from four of these patients and the unaffected monozygotic (MZ) twin of one (discovery cohort), to identify constitutional and somatic de novo sequence variants. We further analyzed this exome data to search for candidate genes under autosomal dominant and recessive models, and to identify structural variations. Candidate genes were tested by exome or Sanger sequencing in a replication cohort of 28 ROHHAD singletons. RESULTS: The analysis of the trio-based exomes found 13 de novo variants. However, no two patients had de novo variants in the same gene, and additional patient exomes and mutation analysis in the replication cohort did not provide strong genetic evidence to implicate any of these sequence variants in ROHHAD. Somatic comparisons revealed no coding differences between any blood and tumour samples, or between the two discordant MZ twins. Neither autosomal dominant nor recessive analysis yielded candidate genes for ROHHAD, and we did not identify any potentially causative structural variations. CONCLUSIONS: Clinical exome sequencing is highly unlikely to be a useful diagnostic test in patients with true ROHHAD. As ROHHAD has a high risk for fatality if not properly managed, it remains imperative to expand the search for non-exomic genetic risk factors, as well as to investigate other possible mechanisms of disease. In so doing, we will be able to confirm objectively the ROHHAD diagnosis and to contribute to our understanding of obesity, respiratory control, hypothalamic function, and autonomic regulation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13023-015-0314-x) contains supplementary material, which is available to authorized users

Mutations in GPAA1, Encoding a GPI Transamidase Complex Protein, Cause Developmental Delay, Epilepsy, Cerebellar Atrophy, and Osteopenia.

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system

The Cyclic AMP Cascade Is Altered in the Fragile X Nervous System

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3′, 5′-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling