Race, ethnicity, and ancestry reporting in genetic counseling research: A focused mapping review and synthesis.

Studies on the use of Race, Ethnicity, and Ancestry (REA) concepts and terms in genetic research are limited. We aimed to describe the collection, reporting, and use of REA data in genetic counseling research. We undertook a focused mapping review and synthesis of the Journal of Genetic Counseling 2021 publications. We used a mapping proforma based on the Race, Ethnicity, And Culture in Health checklist to extract data. Of the 177 screened articles, 132 met our inclusion criteria of reporting primary data about participants. The sample REA characteristics were described in 80 (61%) articles, with 6% providing a definition or conceptualization of the REA term/s used and 23% including a rationale for their study in terms of REA factors. Group labels were most often reported using population descriptors, such as "race," "ethnicity," "race/ethnicity," and "ancestry." Several group labels were used under different population descriptors. For instance, the group labels "White" and "Asian" were used under all population descriptors. Most studies (79%) ascertained REA characteristics by participants' self-report. Three (15%) of the 20 qualitative studies mentioned the relevance of the interviewers' REA characteristics in relation to the participants' REA characteristics. Of the 55 quantitative studies, 19 (35%) used REA factors in the data analysis. Of the 80 articles describing the sample REA characteristics, 20% referred moderately or a great deal to any REA factors in the results interpretation, 46% acknowledged the REA factors in the study limitations, and 15% discussed the implications of REA reporting for genetic counseling practice. Our review documents extensive variation in how sample REA characteristics are described and used in genetic counseling research. Our findings provide a baseline against which to evaluate the effects of guidelines and recommendations for the collection, responsible use, and report of participants' REA characteristics in genetic counseling research.

Effectiveness of a Novel Tablet Application in Reducing Guideline Deviations During Pediatric Cardiac Arrest: A Randomized Clinical Trial.

Importance: Deviations from international resuscitation guidelines during the management of pediatric cardiac arrest are frequent and affect clinical outcomes. An interactive tablet application (app), PediAppRREST, was developed to reduce guideline deviations during pediatric cardiac arrest. Objective: To assess the effectiveness of PediAppRREST in improving the management of simulated in-hospital pediatric cardiac arrest. Design, Setting, and Participants: This multicenter 3-group simulation-based randomized clinical trial was conducted from September 2020 to December 2021 at 4 Italian university hospitals (Padua, Florence, Rome, Novara). Participants included residents in pediatrics, emergency medicine, and anesthesiology. Analyses were conducted as intention-to-treat. Data were analyzed from January to June 2022. Interventions: Teams were randomized to 1 of 3 study groups: an intervention group that used the PediAppRREST app; a control group that used a paper-based cognitive aid, the Pediatric Advanced Life Support (PALS) pocket card; and a control group that used no cognitive aids. All the teams managed the same standardized simulated scenario of nonshockable pediatric cardiac arrest. Main Outcomes and Measures: The primary outcome was the number of deviations from guidelines, measured by a 15-item checklist based on guideline recommendations. The main secondary outcomes were quality of chest compressions, team clinical performance (measured by the Clinical Performance Tool), and perceived team leader's workload. Study outcomes were assessed via video reviews of the scenarios. Results: Overall 100 teams of 300 participants (mean [SD] age, 29.0 [2.2] years; 195 [65%] female) were analyzed by intention-to-treat, including 32 teams randomized to the PediAppRREST group, 35 teams randomized to the PALS control group, and 33 teams randomized to the null control group. Participant characteristics (210 pediatric residents [70%]; 48 anesthesiology residents [16%]; 42 emergency medicine residents [14%]) were not statistically different among the study groups. The number of deviations from guidelines was significantly lower in the PediAppRREST group than in the control groups (mean difference vs PALS control, -3.0; 95% CI, -4.0 to -1.9; P < .001; mean difference vs null control, -2.6; 95% CI, -3.6 to -1.5; P < .001). Clinical Performance Tool scores were significantly higher in the PediAppRREST group than control groups (mean difference vs PALS control, 1.4; 95% CI, 0.4 to 2.3; P = .002; mean difference vs null control, 1.1; 95% CI, 0.2 to 2.1; P = .01). The other secondary outcomes did not significantly differ among the study groups. Conclusions and Relevance: In this randomized clinical trial, the use of the PediAppRREST app resulted in fewer deviations from guidelines and a better team clinical performance during the management of pediatric cardiac arrest. Trial Registration: ClinicalTrials.gov Identifier: NCT04619498.

Concussion Knowledge and Self-Reported Behaviors in Youth Rugby Players and Their Coaches: A Population-Wide Cross-Sectional Survey.

OBJECTIVE: To investigate concussion knowledge and self-reported behaviors in Italian youth rugby players and their coaches. To investigate predictors of lower concussion knowledge and association between athletes' self-reported knowledge and behavior. DESIGN: Cross-sectional, population-wide, survey study. SETTING: All rugby clubs (n = 52) of the Veneto region (Italy). PARTICIPANTS: Players and coaches of all under 15, 17 and 19 teams. Overall, 1719 athlete surveys (92.2% male; response rate, 71.1%) and 235 coach surveys (93.6% male; response rate, 93.2%) were eligible for analysis. INTERVENTION: Surveys circulated from September 20 to December 13, 2021. MAIN OUTCOME MEASURES: Knowledge scores were reported as a percentage of correct answers. Descriptive statistics were reported for all answers. The primary outcomes were concussion knowledge and self-reported behaviors. The secondary outcomes were the association between knowledge and participant individual factors and self-reported behaviors. RESULTS: Median knowledge score for athletes was 55% (IQR: 44-67) and for coaches was 60% (IQR: 52.5-69). Only 33.3% of athletes and 40% of coaches were aware of an increased risk of a second concussion after sustaining one. Athletes who had never heard of the word concussion (effect: -9.31; SE: 1.35, 95% CI: -12.0 to -6.7; P < 0.0001) and coaches with longer coaching experience (effect: -4.35; SE: 2.0, 95% CI: -8.29 to -0.41; P < 0.03) reported lower knowledge scores. There was no statistical association between knowledge scores and athlete self-reported behavior. CONCLUSION: Athletes and coaches had a similar level of concussion knowledge. Knowledge score of athletes did not predict self-reported behaviors. Although enhanced concussion education should be undertaken, interventions to ensure appropriate concussion reporting behaviors are also required.

Tissue mosaicism, FMR1 expression and intellectual functioning in males with fragile X syndrome.

Fragile X syndrome (FXS) is caused by hypermethylation of the FMR1 promoter due to the full mutation expansion (full mutation [FM]: CGG ≥ 200 repeats) and silencing of FMR1. Assessment of mosaicism for active-unmethylated alleles has prognostic utility. This study examined relationships between FMR1 methylation in different tissues with FMR1 messenger ribonucleic acid (mRNA) and intellectual functioning in 87 males with FXS (1.89-43.17 years of age). Methylation sensitive Southern blot (mSB) and Methylation Specific-Quantitative Melt Aanalysis (MS-QMA) were used to examine FMR1 methylation. FMR1 mRNA levels in blood showed strong relationships with FMR1 methylation assessed using MS-QMA in blood (n = 68; R2  = 0.597; p = 1.4 × 10-10 ) and buccal epithelial cells (BEC) (n = 62; R2  = 0.24; p = 0.003), with these measures also showing relationships with intellectual functioning scores (p < 0.01). However, these relationships were not as strong for mSB, with ~40% of males with only FM alleles that were 100% methylated and non-mosaic by mSB, showing methylation mosaicism by MS-QMA. This was confirmed through presence of detectable levels of FMR1 mRNA in blood. In summary, FMR1 methylation levels in blood and BEC examined by MS-QMA were significantly associated with FMR1 mRNA levels and intellectual functioning in males with FXS. These relationships were not as strong for mSB, which underestimated prevalence of mosaicism.

Agreement between parents' and clinical researchers' ratings of behavioral problems in children with fragile X syndrome and chromosome 15 imprinting disorders.

BACKGROUND: Despite the increasing number of clinical trials involving children with neurodevelopmental disorders, appropriate and objective outcome measures for behavioral symptoms are still required. AIM: This study assessed the agreement between parents' and clinical researchers' ratings of behavioral problem severity in children with fragile X syndrome (FXS) and chromosome 15 imprinting disorders. METHODS AND PROCEDURES: The cohort comprised 123 children (64% males), aged 3-17 years, with FXS (n = 79), Prader-Willi (PWS; n = 19), Angelman (AS; n = 15), and Chromosome 15q duplication (n = 10) syndromes. Specific items from the Autism Diagnostic Observation Schedule-Second Edition and Aberrant Behavior Checklist-Community Edition mapping to corresponding behavioral domains were selected ad-hoc, to assess behavioral problems. OUTCOMES AND RESULTS: Inter-rater agreement for the cohort was slight for self-injury (Intraclass Correlation Coefficient (ICC) = 0.12), fair for tantrums/aggression (0.24) and mannerisms/stereotypies (0.25), and moderate for hyperactivity (0.48). When stratified by diagnosis, ICC ranged from poor (0; self-injury, AS and PWS) to substantial (0.48; hyperactivity, females with FXS). CONCLUSIONS AND IMPLICATIONS: The high level of inter-rater disagreement across most domains suggests that parents' and researchers' assessments led to discrepant appraisal of behavioral problem severity. These findings have implications for treatment targets and outcome measure selection in clinical trials, supporting a multi-informant approach.

The impact of cognitive aids on resuscitation performance in in-hospital cardiac arrest scenarios: a systematic review and meta-analysis.

Different cognitive aids have been recently developed to support the management of cardiac arrest, however, their effectiveness remains barely investigated. We aimed to assess whether clinicians using any cognitive aids compared to no or alternative cognitive aids for in-hospital cardiac arrest (IHCA) scenarios achieve improved resuscitation performance. PubMed, EMBASE, the Cochrane Library, CINAHL and ClinicalTrials.gov were systematically searched to identify studies comparing the management of adult/paediatric IHCA simulated scenarios by health professionals using different or no cognitive aids. Our primary outcomes were adherence to guideline recommendations (overall team performance) and time to critical resuscitation actions. Random-effects model meta-analyses were performed. Of the 4.830 screened studies, 16 (14 adult, 2 paediatric) met inclusion criteria. Meta-analyses of eight eligible adult studies indicated that the use of electronic/paper-based cognitive aids, in comparison with no aid, was significantly associated with better overall resuscitation performance [standard mean difference (SMD) 1.16; 95% confidence interval (CI) 0.64; 1.69; I2 = 79%]. Meta-analyses of the two paediatric studies, showed non-significant improvement of critical actions for resuscitation (adherence to guideline recommended sequence of actions, time to defibrillation, rate of errors in defibrillation, time to start chest compressions), except for significant shorter time to amiodarone administration (SMD - 0.78; 95% CI - 1.39; - 0.18; I2 = 0). To conclude, the use of cognitive aids appears to have benefits in improving the management of simulated adult IHCA scenarios, with potential positive impact on clinical practice. Further paediatric studies are necessary to better assess the impact of cognitive aids on the management of IHCA scenarios.

PediAppRREST: effectiveness of an interactive cognitive support tablet app in reducing deviations from guidelines in the management of paediatric cardiac arrest: protocol for a simulation-based randomised controlled trial.

INTRODUCTION: Paediatric cardiac arrest (PCA), despite its low incidence, has a high mortality. Its management is complex and deviations from guideline recommendations occur frequently. We developed a new interactive tablet app, named PediAppRREST, to support the management of PCA. The app received a good usability evaluation in a previous pilot trial. The aim of the study is to evaluate the effectiveness of the PediAppRREST app in reducing deviations from guideline recommendations in PCA management. METHODS AND ANALYSIS: This is a multicentre, simulation-based, randomised controlled, three-parallel-arm study. Participants are residents in Paediatric, Emergency Medicine, and Anaesthesiology programmes in Italy. All 105 teams (315 participants) manage the same scenario of in-hospital PCA. Teams are randomised by the study statistician into one of three study arms for the management of the PCA scenario: (1) an intervention group using the PediAppRREST app or (2) a control group Paediatric Advanced Life Support (CtrlPALS+) using the PALS pocket reference card; or (3) a control group (CtrlPALS-) not allowed to use any PALS-related cognitive aid. The primary outcome of the study is the number of deviations (delays and errors) in PCA management from PALS guideline recommendations, according to a novel checklist, named c-DEV15plus. The c-DEV15plus scores will be compared between groups with a one-way analysis of variance model, followed by the Tukey-Kramer multiple comparisons adjustment procedure in case of statistical significance. ETHICS AND DISSEMINATION: The Ethics Committee of the University Hospital of Padova, coordinating centre of the trial, deemed the project to be a negligible risk study and approved it through an expedited review process. The results of the study will be disseminated in peer-reviewed journals, and at national and international scientific conferences. Based on the study results, the PediAppRREST app will be further refined and will be available for download by institutions/healthcare professionals. TRIAL REGISTRATION NUMBER: NCT04619498; Pre-results.

Missed intracranial injuries are rare in emergency departments using the PECARN head injury decision rules.

PURPOSE: The PECARN head trauma (HT) prediction rules have been developed to guide computed tomography-related decision-making for children with minor HT (mHT). There are currently limited data on the rate of unscheduled revisits to emergency departments (EDs), and initially missed intracranial injuries, in children with mHT initially assessed using the PECARN rules. This study aimed to fill this gap in knowledge. METHODS: Clinical charts of children assessed for mHT over a 5-year period at two EDs that implemented the PECARN rules in Italy and France were reviewed retrospectively. Children who returned to EDs for mHT-related, or potentially related complaints, within 1 month of initial assessment were included. RESULTS: The total number of children with mHT presenting for the first time to the EDs of both sites was 11,749. Overall, 180 (1.5%) unscheduled revisits to the EDs occurred for mHT-related or potentially related complaints. Twenty-three of these 180 patients underwent neuroimaging, and seven had an intracranial injury (including one ischemic stroke). Of these, three were hospitalized and none needed neurosurgery or intensive care. CONCLUSION: Unscheduled revisits for mHT in EDs using the PECARN rules were very uncommon. Initially missed intracranial injuries were rare, and none needed neurosurgery or intensive care.

DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome.

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 (~2-17 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.

External validation of the DIVA and DIVA3 clinical predictive rules to identify difficult intravenous access in paediatric patients.

BACKGROUND: Intravenous (IV) peripheral access is often a difficult procedure in the paediatric ED, causing pain and significant distress. Clinical prediction tools including reproducible variables have been developed to help clinicians identify children at risk of difficult IV access, likely to need additional resources/interventions to maximise success at first attempt. We aimed to externally validate the Difficult IntraVenous Access (DIVA) and DIVA3 scores developed for this purpose. METHODS: Cross-sectional study of children undergoing IV cannulation by nurses in a tertiary-care paediatric ED. Data were collected at the time of the procedure in a clinical report form. RESULTS: Of 440 children included (56.8% males; median age 4.7 years (IQR 1.5-9.5)), 23.4% had a difficult IV access (defined as requiring >1 attempt). Diagnostic accuracy measures for a DIVA cut-off ≥4 and their 95% CIs were sensitivity 24.3% (16.4% to 33.7%), specificity 92.6% (89.2% to 95.1%), positive and negative predictive value 50.0% (35.3% to 64.5%) and 80.0% (75.7% to 83.9%), respectively. The same measures for the DIVA3 were 22.3% (14.7% to 31.6%), 93.5% (90.3% to 95.9%), 51.1% (35.8% to 66.3%) and 79.8% (75.4% to 83.6%). The area under the receiver operating characteristic curve was 0.652 (95% CI 0.591 to 0.712) for the DIVA and 0.649 (95% CI 0.589 to 0.709) for the DIVA3 score. In patients with DIVA and DIVA3 <4, nurses' prediction of greater difficulty in IV placement and moderate/severe dehydration were common independent predictors of difficult IV at multivariate analysis. Only nurses' prediction of greater difficulty in IV placement were associated with higher odds of difficult cannulation for both DIVA/DIVA3 scores ≥4. CONCLUSION: We externally validated the DIVA and DIVA3 showing a similar accuracy compared with the DIVA derivation cohort and between DIVA and DIVA3. We identified factors that can help refine further the risk of difficult IV access and support decision making on the best strategy to maximise the chances of cannulation success on first attempt.

Cognitive, behavioral, and social functioning in children and adults with Dravet syndrome.

AIM: The objective of the study was to delineate the cognitive, behavioral, psychological, and social functioning of individuals with Dravet syndrome. METHOD: Cognitive, behavioral, and social functioning were assessed in patients with Dravet syndrome by comprehensive, age-appropriate standardized neuropsychological testing. Primary caregivers completed standardized measures regarding participants' behavior, psychological status, adaptive functioning, and social skills, including their involvement with intervention services. RESULTS: The cohort comprised 45 patients, aged 2-30 years. Intellectual functioning ranged from average intellect to profound intellectual disability, with a decrease in cognitive and adaptive functioning with age. Only 6 children were able to complete the entire neuropsychological battery and showed a range of cognitive profiles. Five of 6 participants scored within the average range on Affect Recognition and 5/6 on Motor Free Visual Perception tests. Twenty-one (58%) participants had deficits in social skills and 18/27 (67%) in social communication, with 10 participants, who did not yet have a diagnosis of autism spectrum disorder (ASD), screening positive for social communication deficits. Behavioral problems were frequently reported, with attention problems in 24 (65%) and atypicality in 25 (70%). Despite this, parents reported that psychological services were the least utilized health interventions. CONCLUSIONS: Cognitive functioning varies markedly in individuals with Dravet syndrome, with some patients demonstrating global impairment while others have a discordant neuropsychological profile. Behavioral, psychological, social problems, and ASD are common. Social deficits should be reviewed to identify those who warrant ASD assessment. Early identification of behavioral and psychological disorders and targeted use of psychological intervention are essential components of holistic care in Dravet syndrome.

Development and Usability of a Novel Interactive Tablet App (PediAppRREST) to Support the Management of Pediatric Cardiac Arrest: Pilot High-Fidelity Simulation-Based Study.

BACKGROUND: Pediatric cardiac arrest (PCA), although rare, is associated with high mortality. Deviations from international management guidelines are frequent and associated with poorer outcomes. Different strategies/devices have been developed to improve the management of cardiac arrest, including cognitive aids. However, there is very limited experience on the usefulness of interactive cognitive aids in the format of an app in PCA. No app has so far been tested for its usability and effectiveness in guiding the management of PCA. OBJECTIVE: To develop a new audiovisual interactive app for tablets, named PediAppRREST, to support the management of PCA and to test its usability in a high-fidelity simulation-based setting. METHODS: A research team at the University of Padova (Italy) and human-machine interface designers, as well as app developers, from an Italian company (RE:Lab S.r.l.) developed the app between March and October 2019, by applying an iterative design approach (ie, design-prototyping-evaluation iterative loops). In October-November 2019, a single-center nonrandomized controlled simulation-based pilot study was conducted including 48 pediatric residents divided into teams of 3. The same nonshockable PCA scenario was managed by 11 teams with and 5 without the app. The app user's experience and interaction patterns were documented through video recording of scenarios, debriefing sessions, and questionnaires. App usability was evaluated with the User Experience Questionnaire (UEQ) (scores range from -3 to +3 for each scale) and open-ended questions, whereas participants' workload was measured using the NASA Raw-Task Load Index (NASA RTLX). RESULTS: Users' difficulties in interacting with the app during the simulations were identified using a structured framework. The app usability, in terms of mean UEQ scores, was as follows: attractiveness 1.71 (SD 1.43), perspicuity 1.75 (SD 0.88), efficiency 1.93 (SD 0.93), dependability 1.57 (SD 1.10), stimulation 1.60 (SD 1.33), and novelty 2.21 (SD 0.74). Team leaders' perceived workload was comparable (P=.57) between the 2 groups; median NASA RTLX score was 67.5 (interquartile range [IQR] 65.0-81.7) for the control group and 66.7 (IQR 54.2-76.7) for the intervention group. A preliminary evaluation of the effectiveness of the app in reducing deviations from guidelines showed that median time to epinephrine administration was significantly longer in the group that used the app compared with the control group (254 seconds versus 165 seconds; P=.015). CONCLUSIONS: The PediAppRREST app received a good usability evaluation and did not appear to increase team leaders' workload. Based on the feedback collected from the participants and the preliminary results of the evaluation of its effects on the management of the simulated scenario, the app has been further refined. The effectiveness of the new version of the app in reducing deviations from guidelines recommendations in the management of PCA and its impact on time to critical actions will be evaluated in an upcoming multicenter simulation-based randomized controlled trial.

FMR1 mRNA from full mutation alleles is associated with ABC-CFX scores in males with fragile X syndrome.

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 < 0.001) scores compared to males with only FM alleles and complete loss of 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 < 0.001), and lower health-related quality of life scores (p = 0.004), but only in the incomplete silencing FM-only group. The findings suggest that stratification based on CGG sizing and FMR1 mRNA levels may be warranted in future research and clinical trials utilising ABC-CFX subscales as outcome measures.

Intellectual functioning and behavioural features associated with mosaicism in fragile X syndrome.

BACKGROUND: Fragile 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 the FMR1 promoter 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. METHODS: This 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. RESULTS: Comparing 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. CONCLUSIONS: Males 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.

Significantly Elevated FMR1 mRNA and Mosaicism for Methylated Premutation and Full Mutation Alleles in Two Brothers with Autism Features Referred for Fragile X Testing.

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.

Incomplete silencing of full mutation alleles in males with fragile X syndrome is associated with autistic features.

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 < 0.001) than males. FMR1 mRNA levels were positively associated with age (p < 0.001), but not with intellectual functioning and autistic features in females. FM-only males (aged < 19 years) expressing FM FMR1 mRNA had significantly higher ADOS calibrated severity scores compared to FM-only males with completely silenced FMR1 (p = 0.011). However, there were no significant differences between these subgroups on intellectual functioning. In contrast, decreased levels of FMR1 mRNA were associated with decreased intellectual functioning in FXS males (p = 0.029), but not autism features, when combined with the PM/FM mosaic group. Conclusion: Incomplete silencing of toxic FM RNA may be associated with autistic features, but not intellectual functioning in FXS males. While decreased levels of mRNA may be more predictive of intellectual functioning than autism features. If confirmed in future studies, these findings may have implications for patient stratification, outcome measure development, and design of clinical and pre-clinical trials in FXS.

Clinical and Molecular Differences between 4-Year-Old Monozygous Male Twins Mosaic for Normal, Premutation and Fragile X Full Mutation Alleles.

: This study describes monozygotic (MZ) male twins with fragile X syndrome (FXS), mosaic for normal size (NS: <44 CGGs), premutation (PM: 55­199 CGG) and full mutation (FM alleles ≥ 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 (388­1632 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 (672­1025) 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.

Accuracy of NEXUS II head injury decision rule in children: a prospective PREDICT cohort study.

OBJECTIVE: The National Emergency X-Radiography Utilisation Study II (NEXUS II) clinical decision rule (CDR) can be used to optimise the use of CT in children with head trauma. We set out to externally validate this CDR in a large cohort. METHODS: We performed a prospective observational study of patients aged <18 years presenting with head trauma of any severity to 10 Australian/New Zealand EDs. In a planned secondary analysis, we assessed the accuracy of the NEXUS II CDR (with 95% CI) to detect clinically important intracranial injury (ICI). We also assessed clinician accuracy without the rule. RESULTS: Of 20 137 total patients, we excluded 28 with suspected penetrating injury. Median age was 4.2 years. CTs were obtained in ED for 1962 (9.8%), of whom 377 (19.2%) had ICI as defined by NEXUS II. 74 (19.6% of ICI) patients underwent neurosurgery.Sensitivity for ICI based on the NEXUS II CDR was 379/383 (99.0 (95% CI 97.3% to 99.7%)) and specificity was 9320/19 726 (47.2% (95% CI 46.5% to 47.9%)) for the total cohort. Sensitivity in the CT-only cohort was similar. Of the 18 022 children without CT in ED, 49.4% had at least one NEXUS II risk criterion. Sensitivity for ICI by the clinicians without the rule was 377/377 (100.0% (95% CI 99.0% to 100.0%)) and specificity was 18 147/19 732 (92.0% (95% CI 91.6% to 92.3%)). CONCLUSIONS: NEXUS II had high sensitivity, similar to the derivation study. However, approximately half of unimaged patients were positive for NEXUS II risk criteria; this may result in an increased CT rate in a setting with high clinician accuracy.

Penetrating head injuries in children presenting to the emergency department in Australia and New Zealand: A PREDICT prospective study.

AIM: Penetrating head injuries (pHIs) are associated with high morbidity and mortality. Data on pHIs in children outside North America are limited. We describe the mechanism of injuries, neuroimaging findings, neurosurgery and mortality for pHIs in Australia and New Zealand. METHODS: This was a planned secondary analysis of a prospective observational study of children <18 years who presented with a head injury of any severity at any of 10 predominantly paediatric Australian/New Zealand emergency departments (EDs) between 2011 and 2014. We reviewed all cases where clinicians had clinically suspected pHI as well as all cases of clinically important traumatic brain injuries (death, neurosurgery, intubation >24 h, admission >2 days and abnormal computed tomography). RESULTS: Of 20 137 evaluable patients with a head injury, 21 (0.1%) were identified to have sustained a pHI. All injuries were of non-intentional nature, and there were no gunshot wounds. The mechanisms of injuries varied from falls, animal attack, motor vehicle crashes and impact with objects. Mean Glasgow Coma Scale on ED arrival was 10; 10 (48%) had a history of loss of consciousness, and 7 (33%) children were intubated pre-hospital or in the ED. Fourteen (67%) children underwent neurosurgery, two (10%) craniofacial surgery, and five (24%) were treated conservatively; four (19%) patients died. CONCLUSIONS: Paediatric pHIs are very rare in EDs in Australia and New Zealand but are associated with high morbidity and mortality. The absence of firearm-related injuries compared to North America is striking and may reflect Australian and New Zealand firearm regulations.

Intragenic DNA methylation in buccal epithelial cells and intellectual functioning in a paediatric cohort of males with fragile X.

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 < 3.3 × 10-7), 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 < 5.6 × 10-5). 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.