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The FMR1 premutation (PM:55-199 CGG) is associated with fragile X-associated tremor/ataxia syndrome (FXTAS) and when maternally transmitted is at risk of expansion to a hypermethylated full mutation (FM: ≥ 200 CGG) that causes fragile X syndrome (FXS). We describe a maternally transmitted PM (77 CGG) that was passed to a son (103 CGG), and to a daughter (220-1822 CGG), who were affected with FXTAS and FXS, respectively. The male with the PM showed low-level mosaicism for normal size of 30 and 37 CGG. This male had two offspring: one female mosaic for PM and FM (56, 157, >200 CGG) and another with only a 37 CGG allele detected in multiple tissues, neither with a clinical phenotype. The female with the 37 CGG allele showed normal levels of FMR1 methylation and mRNA and passed this 37 CGG allele to one of her daughters, who was also unaffected. These findings show that post-zygotic paternal retraction can lead to low-level mosaicism for normal size alleles, with these normal alleles being functional when passed over two generations.

The study characterised differences in costs associated with raising a child between four rare disorders and examined the associations between these costs with clinical severity. Caregivers of 108 individuals with Prader-Willi, Angelman (AS), Chromosome 15q Duplication and fragile X (FXS) syndromes completed a modified Client Services Receipt Inventory and participants completed intellectual/developmental functioning and autism assessments. AS incurred the highest yearly costs per individual ($AUD96,994), while FXS had the lowest costs ($AUD33,221). Intellectual functioning negatively predicted total costs, after controlling for diagnosis. The effect of intellectual functioning on total costs for those with AS was significantly different to the other syndromes. The study highlights the significant costs associated with these syndromes, particularly AS, linked with severity of intellectual functioning.

Fragile X syndrome (FXS) is caused by CGG expansions of ≥200 repeats (full mutation: FM). Typically, FM causes abnormal methylation of the FMR1 promoter and silencing of FMR1, leading to reduction of FMRP, a protein essential for normal neurodevelopment. However, if unmethylated, these alleles cause over-expression of FMR1 mRNA which has been associated with Fragile X Tremor and Ataxia Syndrome (FXTAS), a late onset disorder. This report details the molecular and clinical profile of an asymptomatic male (29 years) identified as a result of cascade testing who was found to have a rare unmethylated FM (UFM) allele, as well as premutation (PM: 55-199 CGG) size alleles in multiple tissues. Full-scale IQ was within the normal range and minimal features of autism were observed. Southern blot analysis identified FM smears in blood (220-380 CGG) and saliva (212-378 CGG). A PM of 159 CGG was identified in blood and saliva. FMR1 promoter methylation analysis showed all alleles to be unmethylated. FMR1 mRNA levels were greater than fivefold of median levels in typically developing controls and males with FXS mosaic for PM and FM alleles. Issues raised during genetic counseling related to risk for FXTAS associated with UFM and elevated FMR1 mRNA levels, as well as, reproductive options, with implications for future practice.

Often included within ‘high-risk sibling’ studies, the Autism Observation Scale for Infants (AOSI) has only one independent replication study and no evaluation with community-ascertained cohorts. We administered the AOSI and established clinical measures with 103 infants (68% male) at ‘high autism likelihood’ on the Social Attention and Communication Surveillance - Revised (SACS-R) tool, at 9–14 months of age and again 6 months later. AOSI Total scores showed adequate internal consistency and strong inter-rater agreement (live- or video-coded) and were approximately normally distributed at each visit. Modest significant associations presented between Time 1 AOSI scores and concurrent developmental/adaptive skills measures. Concurrent associations were stronger at Time 2, particularly between AOSI Total and Autism Diagnostic Observation Schedule (ADOS) Social Affect scores. AOSI scores were only moderately associated across Time 1 and 2 assessments, as were Time 1 AOSI with Time 2 ADOS scores. These data from a clinically indicated cohort broadly replicate previous AOSI validity accounts from ‘high-risk sibling’ studies, particularly beyond the first year. Strong inter-rater agreement indicates viable AOSI inclusion within protocols necessitating blinded evaluation (e.g. intervention trials). Moderate within-participant stability suggests that, like ‘high-risk siblings,’ community-ascertained infants experience variable early trajectories. Lay Abstract We investigated whether a commonly used research assessment – the Autism Observation Scale for Infants (AOSI) – accurately measures autism behaviours among infants showing early signs of autism identified within the community. The AOSI is often included in studies tracking the development of infants at increased likelihood of autism, such as the infant siblings of diagnosed children. However, the suitability of this measure has not previously been tested with community-referred infants. We administered the AOSI with infants when aged 9 to 14 months and again 6 months later. Our researchers – independent of the AOSI development team and newly trained on this measure – were able to administer the brief interactive assessment and score it accurately. The infants’ AOSI scores were linked to their scores on other established and validated clinical assessments, particularly at the second visit when average age was 18 months. Stronger correspondence of AOSI and other scores at this second visit suggests early autism behaviours are better established and more consistent by 18 months of age, even though these infants showed clear enough signs of possible autism to prompt referral to our study around 12 months of age. However, the moderate association of AOSI scores over time suggests that, like infant siblings – who mostly do not develop autism – community-identified infants showing early signs may also have variable developmental pathways in early life.

BackgroundFragile X syndrome (FXS) is a common cause of intellectual disability and autism spectrum disorder (ASD) usually associated with a CGG expansion, termed full mutation (FM: CGG ≥ 200), increased DNA methylation of theFMR1promoter and silencing of the gene. Mosaicism for presence of cells with either methylated FM or smaller unmethylated pre-mutation (PM: CGG 55–199) alleles in the same individual have been associated with better cognitive functioning. This study compares age- and sex-matched FM-only and PM/FM mosaic individuals on intellectual functioning, ASD features and maladaptive behaviours.MethodsThis study comprised a large international cohort of 126 male and female participants with FXS (aged 1.15 to 43.17 years) separated into FM-only and PM/FM mosaic groups (90 males, 77.8% FM-only; 36 females, 77.8% FM-only). Intellectual functioning was assessed with age appropriate developmental or intelligence tests. The Autism Diagnostic Observation Schedule-2nd Edition was used to examine ASD features while the Aberrant Behavior Checklist-Community assessed maladaptive behaviours.ResultsComparing males and females (FM-only + PM/FM mosaic), males had poorer intellectual functioning on all domains (p< 0.0001). Although females had less ASD features and less parent-reported maladaptive behaviours, these differences were no longer significant after controlling for intellectual functioning. Participants with PM/FM mosaicism, regardless of sex, presented with better intellectual functioning and less maladaptive behaviours compared with their age- and sex-matched FM-only counterparts (p< 0.05). ASD features were similar between FM-only and PM/FM mosaics within each sex, after controlling for overall intellectual functioning.ConclusionsMales with FXS had significantly lower intellectual functioning than females with FXS. However, there were no significant differences in ASD features and maladaptive behaviours, after controlling for intellectual functioning, independent of the presence or absence of mosaicism. This suggests that interventions that primarily target cognitive abilities may in turn reduce the severity of maladaptive behaviours including ASD features in FXS.

Background Fragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the FMR1 product (FMRP), mosaicism for active and inactive FMR1 alleles, including alleles termed premutation (PM: 55–199 CGGs), is not uncommon. Importantly, both PM and active full mutation (FM: ≥ 200 CGGs) alleles often express elevated levels of mRNA that are thought to be toxic. This study determined if complete FMR1 mRNA silencing from FM alleles and/or levels of FMR1 mRNA (if present) in blood are associated with intellectual functioning and autism features in FXS. Methods The study cohort included 98 participants (70.4% male) with FXS (FM-only and PM/FM mosaic) aged 1–43 years. A control group of 14 females were used to establish control FMR1 mRNA reference range. Intellectual functioning and autism features were assessed using the Mullen Scales of Early Learning or an age-appropriate Wechsler Scale and the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2), respectively. FMR1 mRNA was analysed in venous blood collected at the time of assessments, using the real-time PCR relative standard curve method. Results Females with FXS had significantly higher levels of FMR1 mRNA (p

BackgroundAngelman syndrome (AS) is a rare genetic condition characterised by global developmental delay, including severe to profound intellectual disability. The parents of persons with AS experience increased stress, anxiety and depression. This impacts parents’ career choices and productivity.AimsTo estimate, for the first time, the total productivity lost by the parents of persons with AS over a 10-year period in Australia and the corresponding cost to society.Methods and proceduresA cost-of-illness model with simulated follow-up over a 10-year period was developed, with 2019 as the baseline year, facilitated by a Markov chain of life tables. The prevalence of persons with AS and their parents, the productivity-adjusted life years (PALYs) lost by parents, and the cost to society were estimated. Key data were obtained from a prospective cohort of AS families, peer-reviewed literature, and publicly available sources.Outcomes and resultsThe base-case productivity burden borne by the estimated 330 living parents of the 428 prevalent-persons with AS totalled AUD$45.30 million, corresponding to a loss of 38.42% of PALYs per-parent.Conclusions and implicationsCaring for a child with AS has a significant impact on the productivity of affected parents, with a large associated impact on the broader Australian economy.What this paper adds?Persons with AS require lifelong care and support. Consequently, AS results in a significant socioeconomic impact, borne both by the healthcare system and affected families. This is the first known study to estimate the total impact of caring for a child with AS on parental productivity, as well as the first study known to estimate the PALYs lost by a parental or caregiver population. This study found that caring for a child with AS has a significant impact on the productivity of affected parents, with a large associated impact on the broader Australian economy. At present, the supports available to persons with AS and their families include sleep aids and behavioural therapy. In future, specific therapeutic treatments for AS may become available, with trials underway at present investigating the efficacy and effectiveness of gene therapies for AS. As such, evidence regarding the total socioeconomic impact, including the parental productivity burden, attributable to AS is needed to inform future funding decisions.

Chromosome 15 (C15) imprinting disorders including Prader–Willi (PWS), Angelman (AS) and chromosome 15 duplication (Dup15q) syndromes are severe neurodevelopmental disorders caused by abnormal expression of genes from the 15q11–q13 region, associated with abnormal DNA methylation and/or copy number changes. This study compared changes in mRNA levels of UBE3A and SNORD116 located within the 15q11–q13 region between these disorders and their subtypes and related these to the clinical phenotypes. The study cohort included 58 participants affected with a C15 imprinting disorder (PWS = 27, AS = 21, Dup15q = 10) and 20 typically developing controls. Semi-quantitative analysis of mRNA from peripheral blood mononuclear cells (PBMCs) was performed using reverse transcription droplet digital polymerase chain reaction (PCR) for UBE3A and SNORD116 normalised to a panel of internal control genes determined using the geNorm approach. Participants completed an intellectual/developmental functioning assessment and the Autism Diagnostic Observation Schedule-2nd Edition. The Dup15q group was the only condition with significantly increased UBE3A mRNA levels when compared to the control group (p SNORD116 mRNA levels compared to controls (AS: p p = 0.002). Both UBE3A and SNORD116 mRNA levels were positively correlated with all developmental functioning scores in the deletion AS group (p p UBE3A and SNORD116 in PBMCs and brain specific processes underlying motor and language impairments and autism features in these disorders.

Fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥ 200 CGG repeats, and a decrease in FMR1 mRNA and its protein. However, incomplete silencing from FM alleles has been associated with more severe autism features in FXS males. This study compared scores on the Aberrant Behavior Checklist-Community-FXS version (ABC-CFX) in 62 males affected with FXS (3 to 32 years) stratified based on presence or absence of mosaicism and/or FMR1 mRNA silencing. Associations between ABC-CFX subscales and FMR1 mRNA levels, assessed using real-time PCR relative standard curve method, were also examined. The FXS group mosaic for premutation (PM: 55–199 CGGs) and FM alleles had lower irritability (p = 0.014) and inappropriate speech (p FMR1 mRNA. The PM/FM mosaic group also showed lower inappropriate speech scores compared to the incomplete silencing (p = 0.002) group. Increased FMR1 mRNA levels were associated with greater irritability (p FMR1 mRNA levels may be warranted in future research and clinical trials utilising ABC-CFX subscales as outcome measures.

Both sleep problems and unemployment are common in adults with autism spectrum disorder; however, little research has explored this relationship in this population. This study aimed to explore factors that may be associated with the presence of an International Classification of Sleep Disorders–Third Edition defined sleep disorder in adults with autism spectrum disorder (IQ > 80). A total of 36 adults with autism spectrum disorder and 36 controls were included in the study. Participants completed a 14-day actigraphy assessment and questionnaire battery. Overall, 20 adults with autism spectrum disorder met the International Classification of Sleep Disorders–Third Edition criteria for insomnia and/or a circadian rhythm sleep-wake disorder, while only 4 controls met criteria for these disorders. Adults with autism spectrum disorder and an International Classification of Sleep Disorders–Third Edition sleep disorder had higher scores on the Pittsburgh Sleep Quality Index and were more likely to be unemployed compared to adults with autism spectrum disorder and no sleep disorder. The findings demonstrate, for the first time, that sleep problems are associated with unemployment in adults with autism spectrum disorder. Further research exploring the direction of this effect is required; sleep problems that have developed during adolescence make attainment of employment for those with autism spectrum disorder difficult, or unemployment results in less restrictions required for optimal and appropriate sleep timing.

Increased intragenic DNA methylation of the Fragile X Related Epigenetic Element 2 (FREE2) in blood has been correlated with lower intellectual functioning in females with fragile X syndrome (FXS). This study explored these relationships in a paediatric cohort of males with FXS using Buccal Epithelial Cells (BEC). BEC were collected from 25 males with FXS, aged 3 to 17 years and 19 age-matched male controls without FXS. Methylation of 9 CpG sites within the FREE2 region was examined using the EpiTYPER approach. Full Scale IQ (FSIQ) scores of males with FXS were corrected for floor effect using the Whitaker and Gordon (WG) extrapolation method. Compared to controls, children with FXS had significant higher methylation levels for all CpG sites examined (p −7), and within the FXS group, lower FSIQ (WG corrected) was associated with higher levels of DNA methylation, with the strongest relationship found for CpG sites within FMR1 intron 1 (p −5). Applying the WG method to the FXS cohort unmasked significant epi-genotype-phenotype relationships. These results extend previous evidence in blood to BEC and demonstrate FREE2 DNA methylation to be a sensitive epigenetic biomarker significantly associated with the variability in intellectual functioning in FXS.

Rationale: Raised serum C-Reactive Protein (CRP) 14 days after a COPD exacerbation predicts a second exacerbation, presumably due to persistent inflammation or bacterial infection. We examined if a further exacerbation could be prevented by re-treating incompletely resolved COPD exacerbations using Ciprofloxacin. Methods: This multi-centre randomized double-blind placebo-controlled study assessed retreatment with twice daily oral ciprofloxacin 500mg vs placebo for 7 days in patients whose symptoms and/or CRP had not normalised ( Results: Of 826 COPD patients screened at 4 centres, 144 eligible participants with incomplete recovery at day 12-16 post exacerbation were randomised to ciprofloxacin (n=72) or placebo (n=72). Patients had baseline mean age 69.0, 63.2% male, FEV1 49.5% predicted and CAT score 20.5. Median time to the next exacerbation was 32.5 days (IQR 13-50) in the placebo arm and 34 days (IQR 17-62) in the treatment arm. After pre-specified adjustments for previous exacerbations and site there were no significant differences between the groups (p=0.76) (fig 1). No significant differences were seen in CAT, SGRQ or lung function between treatment groups. Conclusion: In patients with incomplete recovery after a treated COPD exacerbation, an additional course of ciprofloxacin provided no additional benefit relative to placebo.

This study describes monozygotic (MZ) male twins with fragile X syndrome (FXS), mosaic for normal size (NS: &lt, 44 CGGs), premutation (PM: 55&ndash, 199 CGG) and full mutation (FM alleles &ge, 200) alleles, with autism. At 4 years of age chromosomal microarray confirmed monozygosity with both twins showing an XY sex complement. Normal size (30 CGG), PM (99 CGG) and FM (388&ndash, 1632 CGGs) alleles were detected in Twin 1 (T1) by standard polymerase chain reaction (PCR) and Southern blot testing, while only PM (99 CGG) and FM (672&ndash, 1025) alleles were identified in Twin 2 (T2). At ~5 years, T2 had greater intellectual impairments with a full scale IQ (FSIQ) of 55 and verbal IQ (VIQ) of 59, compared to FSIQ of 62 and VIQ of 78 for T1. This was consistent with the quantitative FMR1 methylation testing, revealing 10% higher methylation at 80% for T2, suggesting that less active unmethylated alleles were present in T2 as compared to T1. AmplideX methylation PCR also identified partial methylation, including an unmethylated NS allele in T2, undetected by standard testing. In conclusion, this report demonstrates significant differences in intellectual functioning between the MZ twins mosaic for NS, PM and FM alleles with partial FMR1 promoter methylation.

People with autism spectrum disorder (ASD) commonly experience other comorbidities. Studies indicate that between 50% and 83% of individuals with ASD have sleep problems or disorders. The most commonly reported sleep problems are: (a) insomnia symptoms including the inability to get to sleep or stay asleep; and (b) circadian rhythm sleep-wake disorders, defined as a misalignment between the timing of endogenous circadian rhythms and the external environment. The circadian system provides timing information for the sleep-wake cycle that is regulated by the interaction of an endogenous processes (circadian - Process C, and homeostatic - Process S) and synchronizing agents (neurohormones and neurotransmitters), which produce somnogenic activity. A clinical priority in ASD is understanding the cause of these sleep problems in order to improve treatment outcomes. This review approaches sleep in autism from several perspectives: Sleep-wake mechanisms and problems, and brain areas and molecules controlling sleep (e.g., GABA and melatonin) and wake maintenance (e.g., serotonin, acetylcholine and glutamate). Specifically, this review examines how altered sleep structure could be related to neurobiological alterations or genetic mutations and the implications this may have for potential pharmacological treatments in individuals with ASD.

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2&ndash, 17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene (FMR1) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

Children with developmental disabilities experience a high degree of sleep disturbance. Insomnia symptoms are the most commonly reported problems, though circadian rhythm sleep-wake disorders may also be common. There is significant overlap between insomnia symptoms and several symptoms associated with circadian rhythm sleep-wake disorders (CRSWDs), suggesting that such disorders should be considered. A small body of literature has demonstrated that children with developmental disorders can present with circadian rhythm sleep-wake disorders. In particular irregular and free-running rhythms are commonly reported. Supporting the presence of CRSWDs, melatonin abnormalities have also been reported. This chapter demonstrates that CRSWDs may be common in children with developmental disorders and intellectual impairment and that consideration should be given to such diagnoses as these can inform the most appropriate treatment for each individual.

This study aimed to investigate the relationship between sleep, psychopathology (anxiety, depression and presleep arousal) symptoms, and cortisol in adults with autism spectrum disorder (ASD). The sample composed of 29 adults with ASD (51.7% males) and 29 control adults (51.7% males) aged 21-44 years. Thirteen adults with ASD were medicated for a comorbid diagnosis of anxiety and/or depression (ASD-Med), while the remaining 16 adults with ASD were not medicated for such diagnoses (ASD-Only). Participants completed a questionnaire battery, 14-day sleep/wake diary and 14-day actigraphy assessment. On one day during the data collection period, participants collected five saliva samples, hourly, prior to sleep and two morning samples; immediately upon waking and 30 min thereafter for the analysis of cortisol. Cortisol 1 hr prior to habitual sleep onset time was associated with poorer sleep efficiency in both ASD groups and increased wake after sleep onset duration (ASD-Only). Higher subjective somatic arousal was also associated with increased sleep onset latency, regardless of group, and poorer sleep efficiency in the ASD-Only group. ASD-Only participants had significantly greater reductions in evening cortisol concentrations compared to both ASD-Med and control participants. No significant group differences were found for the cortisol awakening response. Findings suggest a hyperarousal hypothesis of insomnia in adults with ASD. Moreover, the low cortisol levels observed in ASD-Only adults suggest dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Longitudinal studies exploring the interplay between insomnia, anxiety and HPA axis regulation across the lifespan in those with ASD are warranted. Autism Res 2019, 12: 897-910. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Both objective (cortisol) and subjective (somatic) physiological arousal were associated with poor sleep quality in adults on the autism spectrum. Adults with autism spectrum disorder (ASD) who were not medicated for a comorbid diagnosis of anxiety and/or depression also had dampened cortisol secretion, suggesting a dysregulation of the hypothalamic pituitary axis. Longitudinal studies investigating the relationship between sleep, psychopathology symptoms and physiological arousal in autistic individuals are warranted. Autism Res 2019. © 2019 International Society for Autism Research, Wiley Periodicals, Inc.

Background Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders that are caused by abnormal expression of imprinted genes in the 15q11-13 region. Dysregulation of genes located in this region has been proposed as a susceptibility factor for autism spectrum disorder (ASD) in both disorders. Methods This study aimed to explore symptoms of ASD in 25 PWS and 19 AS individuals aged between 1 and 39 years via objective assessment. Participants completed the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2) and a developmentally or age-appropriate intellectual functioning assessment. All participants had their genetic diagnosis confirmed using DNA methylation analysis and microarray testing of copy number changes within the 15q11-13 region. Results Participants with PWS had significantly higher overall and social affect calibrated severity scores (CSS) on the ADOS-2 compared to AS participants (p = .0055 and .0015, respectively), but the two groups did not differ significantly on CSS for the repetitive and restricted behaviour domain. Conclusions PWS cases presented with greater symptoms associated with ASD compared to individuals with AS. Mental health issues associated with PWS may contribute to elevated symptoms of ASD, particularly in adolescents and adults with PWS. Electronic supplementary material The online version of this article (10.1186/s11689-018-9242-0) contains supplementary material, which is available to authorized users.

Study objectives To examine sleep patterns and sleep problems and their relationship with daytime functioning in adults with a diagnosis of an autism spectrum disorder and no comorbid intellectual disability (high-functioning autism spectrum disorder [HFASD]) compared to neurotypical (NT) adults. Design Cross-sectional. Setting Home-based study. Participants 36 adults with HFASD and 36 age-, intelligence quotient- and sex-matched NT adults. Measurements Participants completed an online questionnaire battery including the Pittsburgh Sleep Quality Index (PSQI), a 14-d sleep wake diary and 14-d actigraphy data collection. Results Adults with HFASD had significantly more general sleep disturbances and higher scores on the PSQI, longer sleep onset latencies (actigraphy), and poorer sleep efficiency (diary) and these results remained significant after accounting for the False Discovery Rate. Those adults with HFASD who did not have a comorbid diagnosis of anxiety/depression had significantly shorter total sleep time (diary and actigraphy) compared to NT adults. Compared to NT adults, the HFASD group self-reported significantly poorer refreshment scores upon waking in the morning and higher scores on the daytime dysfunction due to sleepiness subscale of the PSQI. Conclusions These findings support the notion that problems related to sleep, in particular insomnia, continue into adulthood in individuals with high-functioning autism spectrum disorder.

Background There is variability in the language of children with autism, even those who are high functioning. However, little is known about how they process language structures in real time, including how they handle potential ambiguity, and whether they follow referential constraints. Previous research with older autism spectrum disorder (ASD) participants has shown that these individuals can use context to access rapidly the meaning of ambiguous words. The severity of autism has also been shown to influence the speed in which children with ASD access lexical information. Aims To understand more about how children with ASD process language in real time (i.e., as it unfolds). The focus was the integration of information and use of referential constraints to identify a referent named in a sentence. Methods & Procedures We used an eye-tracking task to compare performance between young, high-functioning children with autism (HFA) and children with typical development (TD). A large sample of 5–9-year-old children (mean age = 6;8 years), 48 with HFA and 56 with TD participated; all were attending mainstream schools. For each item participants were shown a display of four images that differed in two dimensions. Each sentence contained an adjective and noun that restricted the choice from four to two (the target and competitor), followed by a prepositional phrase (e.g., the blue square with dots); this added modifying information to provide a unique description of the target. We calculated looking time at the target, the competitor and the two distractors for each 200 ms time interval as children processed the sentence and looked at the display. Generalized estimating equations were used to carry out repeated-measures analyses on the proportion of looking time to target and competitor and time to fixate to target. Outcomes & Results Children in both groups (HFA and TD) looked at the target and competitor more than at the distractors following the adjective and noun and following the modifying information in the prepositional phrase more at the target. However, the HFA group was significantly slower in both phases and looked proportionally less at the target. Across the sample, IQ and language did not affect the results; however, age and attention had an impact. The older children showed an advantage in processing the information as did the children with higher attention scores. Conclusions & Implications The HFA group took longer than the TD group to integrate the disambiguating information provided in the course of processing a sentence and integrate it with the visual information, indicating that for the ASD group incremental processing was not as advanced as for children with ASD, and they were less sensitive to referential conventions. Training for young children with ASD on the use of referential conventions and available contextual clues may be of benefit to them in understanding the language they hear.

This study assessed melatonin levels and the dim light melatonin onset (DLMO) in adults with Autism Spectrum Disorder (ASD) and also investigated the relationships between melatonin and objectively measured sleep parameters. Sixteen adults with ASD (ASD-Only), 12 adults with ASD medicated for comorbid diagnoses of anxiety and/or depression (ASD-Med) and 32 controls participated in the study. Although, the timing of the DLMO did not differ between the two groups, advances and delays of the melatonin rhythm were observed in individual profiles. Overall mean melatonin levels were lower in the ASD-Med group compared to the two other groups. Lastly, greater increases in melatonin in the hour prior to sleep were associated with greater sleep efficiency in the ASD groups.

This study aimed to examine the behavioural sleep-wake rhythm in 36 adults with autism spectrum disorder (ASD) and to determine the prevalence of circadian sleep-wake rhythm disorders compared to age- and sex-matched controls. Participants completed an online questionnaire battery, a 14-day sleep-wake diary and 14-day actigraphy assessment. The results indicated that a higher proportion of adults with ASD met criteria for a circadian rhythm sleep-wake disorder compared to control adults. In particular, delayed sleep-wake phase disorder was particularly common in adults with ASD. Overall the findings suggest that individuals with ASD have sleep patterns that may be associated with circadian rhythm disturbance; however factors such as employment status and co-morbid anxiety and depression appear to influence their sleep patterns.

Problems in language processing have been associated with autism spectrum disorder (ASD), with some research attributing the problems to overall language skills rather than a diagnosis of ASD. Lexical access was assessed in a looking-while-listening task in three groups of 5- to 7-year-old children; two had high-functioning ASD (HFA), an ASD severe (ASD-S) group (n = 16) and an ASD moderate (ASD-M) group (n = 21). The third group were typically developing (TD) (n = 48). Participants heard sentences of the form “Where's the x?” and their eye movements to targets (e.g., train), phonological competitors (e.g., tree), and distractors were recorded. Proportions of looking time at target were analyzed within 200 ms intervals. Significant group differences were found between the ASD-S and TD groups only, at time intervals 1000–1200 and 1200–1400 ms postonset. The TD group was more likely to be fixated on target. These differences were maintained after adjusting for language, verbal and nonverbal IQ, and attention scores. An analysis using parent report of autistic-like behaviors showed higher scores to be associated with lower proportions of looking time at target, regardless of group. Further analysis showed fixation for the TD group to be significantly faster than for the ASD-S. In addition, incremental processing was found for all groups. The study findings suggest that severity of autistic behaviors will impact significantly on children's language processing in real life situations when exposed to syntactically complex material. They also show the value of using online methods for understanding how young children with ASD process language. Autism Res 2014, 7: 687–694. © 2014 International Society for Autism Research, Wiley Periodicals, Inc.

Objectives: Sleep disturbance and psychopathology are common during adolescence and are highly prevalentin individuals diagnosed with autism spectrum disorder (ASD). The aim of this study was to investigate relationships between sleep disturbance, psychopathology symptoms, and daytime functioning in adolescents with high-functioning autism spectrum disorder (HFASD) compared to typically developing(TD) adolescents. Conclusions: Both sleep disturbance and psychopathology are more prevalent in adolescents with HFASD and are major contributors to poor daytime functioning in these individuals. Methods: Twenty-seven adolescents with HFASD and 27 age- and sex-matched TD adolescents completed questionnaires related to sleep, psychopathology and daytime functioning. Participants also completed a 7-day sleep/wake diary. A subsample of HFASD adolescents (55%) and all the TD adolescents wore an actigraphy monitor concurrently with the sleep diary. Results: Adolescents with HFASD had significantly higher scores for depressed mood, anxiety and presleep arousal compared with TD adolescents and poorer daytime functioning. There were more significant correlations between sleep variables and psychopathology variables, and sleep variables and daytime functioning, in the HFASD group than in the TD group. Standard regression found that sleep variables significantly accounted for 57% of the variance in daytime functioning symptoms of insufficient sleep in the HFASD group, while psychopathology variables accounted for 63% of the variance in daytime functioning. Refereed/Peer-reviewed

Sleep problems are a commonly reported complaint for individuals with an intellectual or developmental disability (DD), with prevalence generally exceeding that reported for individuals in the general population. Nevertheless, while there has been an increase in interest in sleep problems in children with autism spectrum disorder (ASD), sleep research in very young children and older adolescents and adults with ASD and sleep research in other DDs remains sparse. This review examines articles investigating sleep in DDs, including behavioural and melatonin interventions, published during 2012 and most of 2013, evaluating their contribution. Overall, the majority of publications concern sleep in ASD, and there remains a lack of research examining aetiology, impact and intervention for sleep difficulties in populations of individuals with other DDs. This is disappointing, given the high prevalence of sleep difficulties in DD populations and the negative impact they are known to have on individuals and their families.

Although fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥200 cytosine-guanine-guanine (CGG) repeats, and a decrease in FMR1 mRNA and its protein (FMRP), incomplete silencing has been associated with more severe autism features in FXS males. This study reports on brothers (B1 and B2), aged 5 and 2 years, with autistic features and language delay, but a higher non-verbal IQ in comparison to typical FXS. CGG sizing using AmplideX PCR only identified premutation (PM: 55−199 CGGs) alleles in blood. Similarly, follow-up in B1 only revealed PM alleles in saliva and skin fibroblasts; whereas, an FM expansion was detected in both saliva and buccal DNA of B2. While Southern blot analysis of blood detected an unmethylated FM, methylation analysis with a more sensitive methodology showed that B1 had partially methylated PM alleles in blood and fibroblasts, which were completely unmethylated in buccal and saliva cells. In contrast, B2 was partially methylated in all tested tissues. Moreover, both brothers had FMR1 mRNA ~5 fold higher values than those of controls, FXS and PM cohorts. In conclusion, the presence of unmethylated FM and/or PM in both brothers may lead to an overexpression of toxic expanded mRNA in some cells, which may contribute to neurodevelopmental problems, including elevated autism features.

Background & Aim Background: Advances in neonatal intensive care have been associated with improved survival of preterm infants. But along with survival, the morbidity bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is also increasing. BPD is associated with adverse respiratory outcomes into adulthood and greater risk of adverse neurodevelopment. Infants born ≤ 28 weeks’ gestation, during the canalicular phase of lung development prior to alveolar and distal capillary development, are at greatest risk of BPD. Disruption of lung maturation by the pro-inflammatory environment created by lifesaving neonatal intensive care interventions results in the BPD we see today – a disease characterised by arrested development with simplified alveoli and abnormal angiogenesis. As the role of inflammation in BPD comes into focus, cell therapy presents a promising intervention for its prevention. Human amnion epithelial cells (hAECs), stem-like cells derived from placental tissues, have multi-lineage potential, are immune privileged and, in small animal models are non-teratogenic. Preclinical work demonstrates the cells capacity to modulate the inflammatory environment to both prevent injury and aid repair of established injury in diverse models of lung disease. A first-in-human study recently demonstrated the safety of low dose hAECs in preterm infants with established BPD. However, hAECs are likely to have greater therapeutic potential when used to prevent lung injury thus, the next step in translation is to evaluate the safety of hAECs in less mature infants at high risk of developing BPD. Aim To evaluate the safety of escalating doses of intravenous hAECs in infants born ≤ 28 weeks’ gestation at high risk of BPD. Methods, Results & Conclusion Method In this phase 1 study, 24 infants born at ≤28 weeks’ gestation at high risk of BPD will each receive intravenous hAECs from Day 14 of life. The infusion dose will escalate from 2 million cells/kg to 10 million cells/kg. Further dose escalation will be achieved by repeat infusions at 5 day intervals to achieve a maximum cumulative dose of 30 million cells/kg. Safety is the primary outcome. Infants will be followed until 2 years of age corrected for prematurity. Results The first dose cohort, comprising of 4 infants, has been recruited. Infants received an intravenous infusion of 2 million hAECs/kg. Infusions have been well tolerated with no short term adverse events observed.

Research with adults and older children indicates that verb biases are strong influences on listeners’ interpretations when processing sentences, but they can be overruled. In this paper, we ask two questions: (i) are children with Autism Spectrum Disorder (ASD) who are high functioning sensitive to verb biases like their same age typically developing peers?, and (ii) do young children with ASD and young children with typical development (TD) override strong verb biases to consider alternative interpretations of ambiguous sentences? Participants were aged 5–9 years (mean age 6.65 years): children with ASD who were high functioning and children with TD. In task 1, biasing and neutral verbs were included (e.g., eat cake versus move cake). In task 2, the focus was on whether the prepositional phrase occurring with an instrument biasing verb (e.g., ‘Chop the tree with the axe’) was interpreted as an instrument even if the named item was an implausible instrument (e.g., candle in ‘Cut the cake with the candle’). Overall, the results showed similarities between groups but the ASD group was generally slower. In task 1, both groups looked at the named object faster in the biasing than the non-biasing condition, and in the biasing condition the ASD group looked away from the target more quickly than the TD group. In task 2, both groups identified the target in the prepositional phrase. They were more likely to override the verb instrument bias and consider the alternative (modification) interpretation in the implausible condition (e.g., looking at the picture of a cake with a candle on it’). Our findings indicate that children of age 5 years and above can use context to override verb biases. Additionally, an important component of the sentence processing mechanism is largely intact for young children with ASD who are high functioning. Like children with TD, they draw on verb semantics and plausibility in integrating information. However, they are likely to be slower in processing the language they hear. Based on previous findings of associations between processing speed and cognitive functioning, the implication is that their understanding will be negatively affected, as will their academic outcomes.

In two studies we developed and tested a new self-report measure of restricted and repetitive behaviours (RRB) suitable for adults. In Study 1, The Repetitive Behaviours Questionnaire-2 for adults (RBQ-2A) was completed by a sample of 163 neurotypical adults. Principal components analysis revealed two components: Repetitive Motor Behaviours and Insistence on Sameness. In Study 2, the mean RBQ-2A scores of a group of adults with autism spectrum disorder (ASD; N = 29) were compared to an adult neurotypical group (N = 37). The ASD sample had significantly higher total and subscale scores. These results indicate that the RBQ-2A has utility as a self-report questionnaire measure of RRBs suitable for adults, with potential clinical application.

Purpose: Osteosarcoma is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of osteosarcoma, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for osteosarcoma patients. Experimental Design: We have used genetically engineered murine models of human osteosarcoma in a systematic, genome-wide screen to identify new candidate therapeutic targets. We performed a genome-wide siRNA screen, with or without doxorubicin. In parallel, a screen of therapeutically relevant small molecules was conducted on primary murine– and primary human osteosarcoma–derived cell cultures. All results were validated across independent cell cultures and across human and mouse osteosarcoma. Results: The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary osteosarcoma cell cultures in vitro in both human and mouse osteosarcoma, whereas specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on osteosarcoma cell cultures. Conclusions: The approaches described here have identified dual inhibition of the PI3K–mTOR pathway as a sensitive, druggable target in osteosarcoma, and provide rationale for translational studies with these agents. Clin Cancer Res; 21(14); 3216–29. ©2015 AACR.

Ohdo syndrome (MIM 249620) is a multiple malformation syndrome characterized by blepharophimosis, ptosis, dental hypoplasia, hearing impairment and intellectual disability. A wide range of dysmorphic features and congenital abnormalities have been described in cases reported as Ohdo and Ohdo-like syndromes. We report a further two cases of Ohdo syndrome, one with mild features and the other more severely affected, illustrating the phenotypic variability of the condition. A review of the literature highlights the severe phenotype associated with distinctive facial features, as seen in Case 2 in this report. All cases with the severe phenotype have been sporadic. Subtelomeric FISH studies of all chromosome arms on the two cases showed no abnormality. We propose clinical criteria for the diagnosis of Ohdo syndrome and delineate features of the severe phenotype.

Skeletal-related events resulting from accelerated bone loss are common complications in patients treated for a range of cancers. However, the mechanisms and rate of bone loss after myelosuppression are unclear. We, therefore, investigated this in mice and humans. We treated mice with different myelosuppressive therapies (chemotherapy or irradiation with or without transplantation) and studied their effects on bone structure. Myelosuppression of mice rapidly caused an increase in bone resorption that was not matched by bone formation. The resultant significant and persistent bone loss early after therapy was associated with increased inflammatory cytokines, in particular, monocyte chemoattractant protein 1 (MCP1). Therapy-induced bone loss was prevented with a single dose of the bisphosphonate zoledronic acid (ZA), administered before myelosuppression. Importantly, ZA treatment of mice did not impair hematopoiesis, including hematopoietic stem cell function. Furthermore, examination of serum from patients before and after autologous or allogeneic stem cell transplantion (SCT) revealed altered levels of bone turnover markers and elevated inflammatory cytokines. MCP1 levels in serum obtained between days 7 and 14 post-SCT positively correlated with bone loss observed at 100 days after allogeneic SCT. Similar to that observed in our studies in mice, the bone loss was long term, persisting at 12 months post-SCT. Furthermore, patients who received chemotherapy less than 100 days before SCT had significantly more bone loss at the hip. In these patients, serum levels of MCP1, but not routine biomarkers of bone turnover, including C-terminal cross-linking telopeptide of type-1 collagen (β-CTx), positively correlated with their bone loss. Hence, myelosuppressive therapies increase inflammation and directly contribute to bone loss. Administration of an osteoclast inhibitor before the initiation of cancer therapy is likely to have the best outcome in preventing bone loss in patients with cancer.

Objective: To investigate the sleep patterns and disturbances in a pure sample of adolescents with high-functioning autism spectrum disorder (HFASD). Method: Adolescents completed a sleep questionnaire battery and a 7 d sleep diary. Actigraphic data were collected from a sub-sample of participants (55%) with HFASD and all typically developing (TD) adolescents. Conclusion: The findings suggest that adolescents with HFASD show a continuation of the maladaptive sleep patterns as seen in children with an autism spectrum disorder and these sleep disturbances are associated with increased daytime sleepiness. Results: Adolescents with HFASD were three times more likely to report a sleep problem than their TD peers (46.2% vs. 14.8%). Adolescents with HFASD had decreased sleep efficiency (diary) (p=0.04, n2=0.10), and more fatigue (p=0.002, n2=0.18) compared with TD adolescents. While TD adolescents generally experienced one symptom of insomnia, adolescents with HFASD were likely to experience two or three symptoms of insomnia (p=0.02, V=0.36). Refereed/Peer-reviewed

Osteosarcoma is the most common primary cancer of bone and one that predominantly affects children and adolescents. Osteoblastic osteosarcoma represents the major subtype of this tumor, with approximately equal representation of fibroblastic and chondroblastic subtypes. We and others have previously described murine models of osteosarcoma based on osteoblast-restricted Cre:lox deletion of Trp53 (p53) and Rb1 (Rb), resulting in a phenotype most similar to fibroblastic osteosarcoma in humans. We now report a model of the most prevalent form of human osteosarcoma, the osteoblastic subtype. In contrast to other osteosarcoma models that have used Cre:lox mediated gene deletion, this model was generated through shRNA-based knockdown of p53. As is the case with the human disease the shRNA tumors most frequently present in the long bones and preferentially disseminate to the lungs; feature less consistently modeled using Cre:lox approaches. Our approach allowed direct comparison of the in vivo consequences of targeting the same genetic drivers using two different technologies, Cre:lox and shRNA. This demonstrated that the effects of Cre:lox and shRNA mediated knock-down are qualitatively different, at least in the context of osteosarcoma, and yielded distinct subtypes of osteosarcoma. Through the use of complementary genetic modification strategies we have established a model of the most common clinical subtype of osteosarcoma that was not previously represented and more fully recapitulated the clinical spectrum of this cancer.

Autism spectrum disorder (ASD) is a perplexing and pervasive developmental disorder characterized by social difficulties, communicative deficits, and repetitive behavior. The increased rate of ASD diagnosis has raised questions concerning the genetic and environmental factors contributing to the development of this disorder; meanwhile, the cause of ASD remains unknown. This study surveyed mothers of ASD and non-ASD children to determine possible effects of labor and delivery (L&D) drugs on the development of ASD. The survey was administered to mothers; however, the results were analyzed by child, as the study focused on the development of autism. Furthermore, an independent ASD dataset from the Southwest Autism Research and Resource Center was analyzed and compared. Indeed, L&D drugs are associated with ASD ( p = .039). Moreover, the Southwest Autism Research and Resource Center dataset shows that the labor induction drug, Pitocin, is significantly associated with ASD ( p = .004). We also observed a synergistic effect between administrations of L&D drugs and experiencing a birth complication, in which both obstetrics factors occurring together increased the likelihood of the fetus developing ASD later in life ( p = .0003). The present study shows the possible effects of L&D drugs, such as Pitocin labor-inducing and analgesic drugs, on children and ASD.

In recent years, the different classes of drugs and regimens used clinically have provided an improvement in tumour management. However, treatment is often palliative for the majority of cancer patients. Transformed cells respond poorly to chemotherapy mainly due to the development of the multidrug resistance (MDR) phenotype. Response to treatment does not generally result in complete remission and disease cure is uncommon for patients presenting with advanced stage cancer. Successful treatment of cancer requires a clearer understanding of chemotherapeutic resistance. Here, we examine what is known of one of the most extensively studied mechanisms of cellular drug resistance. The human multidrug resistance gene 1 (MDR1) is associated with expression of p-glycoprotein (Pgp). A transmembrane protein, Pgp acts as an efflux pump and reduces intracellular drug levels and thus its effectiveness as an antitumor agent. The precise mechanism of transcriptional regulation has been unclear due to the complex regulatory nature of the gene. It has become increasingly apparent that trans-activation or genetic amplification is by no means the only mechanism of activation. Consequently, alternative pathways have received more attention in the area of epigenetics to help explain transcriptional competence at a higher level of organization. The goal of this article is to highlight important findings in the field of methylation and explain how they impinge on MDR1 gene regulation. In this review, we cover the current information and postulate that epigenetic modification of MDR1 chromatin influences gene transcription in leukaemia. Finally, we explore transcriptional regulation and highlight recent progress with engineered ZFP's (zinc finger proteins).

Objectives. To investigate short-term effects of Multi-Sensory Stimulation (MSS) on behaviour, mood and cognition of older adults with dementia, the generalization of effects to day hospital and home environments and the endurance of any effects over time. Design. A randomized controlled trial comparing MSS with a credible control of one-to-one activities. Methods. Fifty patients with diagnoses of moderate to severe dementia were randomized to either MSS or Activity groups. Patients participated in eight 30-minute sessions over a 4-week period. Ratings of behaviour and mood were taken before, during and after sessions to investigate immediate effects. Pre, mid, post-trial, and follow-up assessments were taken to investigate any generalization of effects on cognition, behaviour at the day hospital and behaviour and mood at home and endurance of effects once sessions had ceased. Results. Immediately after MSS and Activity sessions patients talked more spontaneously, related better to others, did more from their own initiative, were less bored/inactive, and were more happy, active or alert. Both groups were more attentive to their environment than before, with a significantly greater improvement from the MSS group. At the day hospital, patients in the Activity group improved on their 'speech skills' (amount of speech; initiation of speech), whereas the MSS group remained unchanged during the trial. The MSS group showed a significant improvement in mood and behaviour at home compared to the Activity group whose behaviour deteriorated. No longer-term benefits were shown; indeed, behaviour declined sharply during the month follow-up period. Conclusions. Both MSS and Activity sessions appear to be effective and appropriate therapies for people with dementia.

Abstract 1447 Poster Board I-470 Patients with a range of blood cell diseases are usually treated with myeloablative therapies such as chemotherapy or irradiation accompanied by bone marrow (BM) transplants. These therapies have severe effects on the hematopoietic system and cause prolonged pancytopenia, which is life-threatening to the patient. The effects of myeloablative therapies on hematopoietic cells are well-documented but their impacts on the cells of the BM microenvironment, which have important roles in regulating hematopoiesis, are largely unknown. Using the mouse model, we have analyzed the effects of 11 Gy irradiation and transplantation of 5 × 106 BM cells/mouse on hematopoietic and BM microenvironment cells: osteoblasts (Obs), endothelial cells (ECs) (both which have been shown to be important positive regulators of hematopoiesis) and adipocytes (known negative regulators of hematopoiesis). Mature and immature hematopoietic cell contents in BM, peripheral blood and spleen were assessed by cell counts together with fluorescence-activated cell-sorting (FACS) analysis and colony-forming cell potential. BM microenvironment Ob and adipocyte cell content were examined by standard histomorphometry methods on non-decalcified plastic-embedded tibiae. BM microenvironment ECs were measured by immunohistochemical staining of paraffin-embedded sections accompanied by FACS-analysis of lineage/CD45/CD48-negative, CD31-positive cells. Quantitative real-time PCR of BM provided further insight into changes in transcripts specific for immature populations of BM microenvironment cells post-transplant. Cohorts of male mice were analyzed at numerous time-points corresponding to: age-matched non-transplanted male mice (day 0); periods of severe pancytopenia (days 2-7); early recovery of hematopoiesis (days 10-35) and the time-point at which stable hematopoiesis was re-established (day 84). The earliest change in the BM microenvironment was to ECs, as massive hemorrhaging was observed, with damaged vasculature throughout the BM cavity occurring as early as day 2 post-transplant. Furthermore, the numbers of ECs in the BM markedly dropped at this time-point, and this was accompanied by 2-fold reductions in transcripts for the BM vascular-specific markers VEGFR2 and VEGFR3. In contrast, the expression of VE-cadherin, a molecule important for endothelial cell-cell interactions, was significantly up-regulated (6-fold, P Disclosures No relevant conflicts of interest to declare.

Osteosarcoma is the most common form of bone cancer. Pivotal insight into the genes involved in human osteosarcoma has been provided by the study of rare familial cancer predisposition syndromes. Three kindreds stand out as predisposing to the development of osteosarcoma: Li-Fraumeni syndrome, familial retinoblastoma and RecQ helicase disorders, which include Rothmund-Thomson Syndrome in particular. These disorders have highlighted the important roles of P53 and RB respectively, in the development of osteosarcoma. The association of OS with RECQL4 mutations is apparent but the relevance of this to OS is uncertain as mutations in RECQL4 are not found in sporadic OS. Application of the knowledge or mutations of P53 and RB in familial and sporadic OS has enabled the development of tractable, highly penetrant murine models of OS. These models share many of the cardinal features associated with human osteosarcoma including, importantly, a high incidence of spontaneous metastasis. The recent development of these models has been a significant advance for efforts to improve our understanding of the genetics of human OS and, more critically, to provide a high-throughput genetically modifiable platform for preclinical evaluation of new therapeutics.