Electroencephalographic Correlates of Clinical Severity in the Natural history study of RTT and Related Disorders.

OBJECTIVE: This study was undertaken to characterize quantitative electroencephalographic (EEG) features in participants from the Natural history study of RTT and Related Disorders and to assess the potential for these features to act as objective measures of cortical function for Rett syndrome (RTT). METHODS: EEG amplitude and power features were derived from the resting EEG of 60 females with RTT (median age = 10.7 years) and 26 neurotypical females (median age = 10.6 years). Analyses focus on group differences and within the RTT group, associations between the EEG parameters and clinical severity. For a subset of participants (n = 20), follow-up data were available for assessing the reproducibility of the results and the stability in the parameters over 1 year. RESULTS: Compared to neurotypical participants, participants with RTT had greater amplitude variability and greater low-frequency activity as reflected by greater delta power, more negative 1/f slope, and lower theta/delta, alpha/delta, beta/delta, alpha/theta, and beta/theta ratios. Greater delta power, more negative 1/f slope, and lower power ratios were associated with greater severity. Analyses of year 1 data replicated the associations between 1/f slope and power ratios and clinical severity and demonstrated good within-subject consistency in these measures. INTERPRETATION: Overall, group comparisons reflected a greater predominance of lower versus higher frequency activity in participants with RTT, which is consistent with prior clinical interpretations of resting EEG in this population. The observed associations between the EEG power measures and clinical assessments and the repeatability of these measures underscore the potential for EEG to provide an objective measure of cortical function and clinical severity for RTT. ANN NEUROL 2024.

Rett syndrome diagnostic odyssey: Limitations of NextGen sequencing.

Typical (or classic) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by a period of regression, partial or complete loss of purposeful hand movements, and acquired speech, impaired gait, and stereotyped hand movements. In over 95% of typical RTT, a pathogenic variant is found in the methyl-CPG binding protein 2 gene (MECP2). Here, we describe a young woman with clinically diagnosed typical RTT syndrome who lacked a genetic diagnosis despite 20 years of investigation and multiple rounds of sequencing the MECP2 gene. Recently, additional genetic testing using next-generation sequencing was completed, which revealed a partial insertion of the BCL11A gene within exon 4 of MECP2, resulting in a small deletion in MECP2, causing likely disruption of MeCP2 function due to a frameshift. This case demonstrates the ever-changing limitations of genetic testing, as well as the importance of continual pursuit of a diagnosis as technologies improve and are more widely utilized.

Psychometric Assessment of the Rett Syndrome Caregiver Assessment of Symptom Severity (RCASS).

Rett syndrome is a severe neurodevelopmental disorder that affects about 1 in 10,000 females. Clinical trials of disease modifying therapies are on the rise, but there are few psychometrically sound caregiver-reported outcome measures available to assess treatment benefit. We report on a new caregiver-reported outcome measure, the Rett Caregiver Assessment of Symptom Severity (RCASS). Using data from the Rett Natural History Study (n = 649), we examined the factor structure, using both exploratory and confirmatory factor analysis, and the reliability and validity of the RCASS. The four-factor model had the best overall fit, which covered movement, communication, behavior, and Rett-specific symptoms. The RCASS had moderate internal consistency. Strong face validity was found with age and mutation type, and convergent validity was established with other similar measures, including the Revised Motor-Behavior Assessment Scale, Clinical Severity Scale, Clinical Global Impression Scale, and the Child Health Questionnaire. These data provide initial evidence that the RCASS is a viable caregiver-outcome measure for use in clinical trials in Rett syndrome. Future work to assess sensitivity to change and other measures of reliability, such as test-retest and inter-rater agreement, are needed.

Challenges in developing therapies in fragile X syndrome: how the FXLEARN trial can guide research.

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and the single-gene cause of autism, is caused by decreased expression of the fragile X messenger ribonucleoprotein protein (FMRP), a ribosomal-associated RNA-binding protein involved in translational repression. Extensive preclinical work in several FXS animal models supported the therapeutic potential of decreasing metabotropic glutamate receptor (mGluR) signaling to correct translation of proteins related to synaptic plasticity; however, multiple clinical trials failed to show conclusive evidence of efficacy. In this issue of the JCI, Berry-Kravis and colleagues conducted the FXLEARN clinical trial to address experimental design concerns from previous trials. Unfortunately, despite treatment of young children with combined pharmacological and learning interventions for a prolonged period, no efficacy of blocking mGluR activity was observed. Future systematic evaluation of potential therapeutic approaches should evaluate consistency between human and animal pathophysiological mechanisms, utilize innovative clinical trial design from FXLEARN, and incorporate translatable biomarkers.

Trofinetide Treatment Demonstrates a Benefit Over Placebo for the Ability to Communicate in Rett Syndrome.

BACKGROUND: Trofinetide was approved by the US Food and Drug Administration for the treatment of Rett syndrome (RTT) in March 2023. Benefiting the ability to communicate in RTT is often identified as the most important caregiver goal for new therapies. This analysis reports the communication-related end points from the phase 3 LAVENDER study of trofinetide in RTT. METHODS: Females with RTT, aged five to 20 years, were randomized 1:1 to trofinetide or placebo for 12 weeks. Secondary efficacy end points related to communication were based on change from baseline to week 12 and included the caregiver-rated Communication and Symbolic Behavior Scales Developmental Profile™ Infant-Toddler Checklist (CSBS-DP-IT) Social Composite score (key secondary end point; scores ranged from 0 to 26 [higher scores indicated better communication]) and novel clinician rating scales (0 [normal] to 7 [severe impairment]) measuring the ability to communicate choices nonverbally (RTT-COMC) and verbally (RTT-VCOM). RESULTS: Trofinetide demonstrated a statistically significant difference versus placebo for the CSBS-DP-IT Social Composite score (least squares mean [LSM] difference = 1.0; 95% confidence interval [CI], 0.3 to 1.7; P = 0.0064; Cohen's d effect size = 0.43) and a nominally significant difference for the RTT-COMC (LSM difference: -0.3; 95% CI, -0.6 to -0.0; P = 0.0257; Cohen's d effect size = 0.36). As expected, there was no difference for the RTT-VCOM. CONCLUSIONS: Significant treatment benefit for trofinetide versus placebo was observed in scales measuring the ability to communicate. These scales may be appropriate for future clinical studies in RTT and other neurodevelopmental disorders.

Parental age effects and Rett syndrome.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder, and pathogenic Methyl-CpG-binding Protein 2 (MECP2) variants are identified in >95% of individuals with typical RTT. Most of RTT-causing variants in MECP2 are de novo and usually on the paternally inherited X chromosome. While paternal age has been reported to be associated with increased risk of genetic disorders, it is unknown whether parental age contributes to the risk of the development of RTT. Clinical data including parental age, RTT diagnostic status, and clinical severity are collected from 1226 participants with RTT and confirmed MECP2 variants. Statistical analyses are performed using Student t-test, single factor analysis of variance (ANOVA), and multi-factor regression. No significant difference is observed in parental ages of RTT probands compared to that of the general population. A small increase in parental ages is observed in participants with missense variants compared to those with nonsense variants. When we evaluate the association between clinical severity and parental ages by multiple regression analysis, there is no clear association between clinical severity and parental ages. Advanced parental ages do not appear to be a risk factor for RTT, and do not contribute to the clinical severity in individuals with RTT.

Real-world clinical management of individuals with Rett syndrome: a physician survey.

BACKGROUND: Rett syndrome (RTT) is a severe neurodevelopmental disorder. Management strategies are heterogeneous with no clear definition of success. This study describes physician decision-making regarding diagnosis, therapeutic goals, and management strategies to better understand RTT clinical management in the US. METHODS: This study was conducted among practicing physicians, specifically neurologists and pediatricians in the US with experience treating ≥2 individuals with RTT, including ≥1 individuals within the past two years. In-depth interviews with five physicians informed survey development. A cross-sectional survey was then conducted among 100 physicians. RESULTS: Neurologists had treated more individuals with RTT (median: 12 vs. 5, p < 0.001) than pediatricians throughout their career and were more likely to report being "very comfortable" managing RTT (31 vs. 4%, p < 0.001). Among physicians with experience diagnosing RTT (93%), most evaluated symptoms (91%) or used genetic testing (86%) for RTT diagnoses; neurologists used the 2010 consensus diagnostic criteria more than pediatricians (54 vs. 29%; p = 0.012). Improving the quality of life (QOL) of individuals with RTT was the most important therapeutic goal among physicians, followed by improving caregivers' QOL. Most physicians used clinical practice guidelines to monitor the progress of individuals with RTT, although neurologists relied more on clinical scales than pediatricians. Among all physicians, the most commonly treated symptoms included behavioral issues, epilepsy/seizures, and feeding issues. Management strategies varied by symptom, with referral to appropriate specialists being common across symptoms. A large proportion of physicians (37%) identified the lack of novel therapies and reliance on symptom-specific management as an unmet need. CONCLUSION: Although most physicians had experience and were comfortable diagnosing and treating individuals with RTT, better education and support among pediatricians is warranted. Additionally, novel treatments that target multiple symptoms associated with RTT could reduce the burden and improve the QOL of individuals with RTT and their caregivers.

Top caregiver concerns in Rett syndrome and related disorders: data from the US natural history study.

OBJECTIVE: Recent advances in the understanding of neurodevelopmental disorders such as Rett syndrome (RTT) have enabled the discovery of novel therapeutic approaches that require formal clinical evaluation of efficacy. Clinical trial success depends on outcome measures that assess clinical features that are most impactful for affected individuals. To determine the top concerns in RTT and RTT-related disorders we asked caregivers to list the top caregiver concerns to guide the development and selection of appropriate clinical trial outcome measures for these disorders. METHODS: Caregivers of participants enrolled in the US Natural History Study of RTT and RTT-related disorders (n = 925) were asked to identify the top 3 concerning problems impacting the affected participant. We generated a weighted list of top caregiver concerns for each of the diagnostic categories and compared results between the disorders. Further, for classic RTT, caregiver concerns were analyzed by age, clinical severity, and common RTT-causing mutations in MECP2. RESULTS: The top caregiver concerns for classic RTT were effective communication, seizures, walking/balance issues, lack of hand use, and constipation. The frequency of the top caregiver concerns for classic RTT varied by age, clinical severity, and specific mutations, consistent with known variation in the frequency of clinical features across these domains. Caregivers of participants with increased seizure severity often ranked seizures as the first concern, whereas caregivers of participants without active seizures often ranked hand use or communication as the top concern. Comparison across disorders found commonalities in the top caregiver concerns between classic RTT, atypical RTT, MECP2 duplication syndrome, CDKL5 deficiency disorder, and FOXG1 syndrome; however, distinct differences in caregiver concerns between these disorders are consistent with the relative prevalence and impact of specific clinical features. CONCLUSION: The top caregiver concerns for individuals with RTT and RTT-related disorders reflect the impact of the primary clinical symptoms of these disorders. This work is critical in the development of meaningful therapies, as optimal therapy should address these concerns. Further, outcome measures to be utilized in clinical trials should assess these clinical issues identified as most concerning by caregivers.

A review of the Rett Syndrome Behaviour Questionnaire and its utilization in the assessment of symptoms associated with Rett syndrome.

The Rett Syndrome Behaviour Questionnaire (RSBQ), which is completed by the caregiver, is one of the most widely used efficacy measures in clinical studies of Rett syndrome (RTT) due to its specificity to the core features of RTT. As healthcare providers participate in routine healthcare assessments of individuals with RTT in clinical practice, there is a need for these providers to understand the psychometric properties of the RSBQ and how it relates to the core clinical features of RTT. Here, we describe the characteristics of the RSBQ, review the literature on its validity and reliability as well as its performance in a phase 2 study and the recent phase 3 LAVENDER study. The RSBQ was first shown to discriminate RTT from other intellectual disorders with good inter-rater and test-retest reliability scores. It was subsequently validated as an appropriate instrument for measuring behavior in females with RTT and adopted as a clinical trial outcome. In LAVENDER, the FDA-approved drug trofinetide significantly improved the RSBQ total score over placebo in girls and women with RTT and change from baseline for all RSBQ subscores were directionally in favor of trofinetide. The change in RSBQ was aligned with the Clinical Global Impression-Improvement scale, suggesting that improvement in behavioral components may be related to overall clinical status. Given its validity and ubiquity in RTT clinical studies, it is important that the interplay of the domains and the psychometric profile of the RSBQ are understood.

Trofinetide for the treatment of Rett syndrome: a randomized phase 3 study.

Rett syndrome is a rare, genetic neurodevelopmental disorder. Trofinetide is a synthetic analog of glycine-proline-glutamate, the N-terminal tripeptide of the insulin-like growth factor 1 protein, and has demonstrated clinical benefit in phase 2 studies in Rett syndrome. In this phase 3 study ( https://clinicaltrials.gov identifier NCT04181723 ), females with Rett syndrome received twice-daily oral trofinetide (n = 93) or placebo (n = 94) for 12 weeks. For the coprimary efficacy endpoints, least squares mean (LSM) change from baseline to week 12 in the Rett Syndrome Behaviour Questionnaire for trofinetide versus placebo was -4.9 versus -1.7 (P = 0.0175; Cohen's d effect size, 0.37), and LSM Clinical Global Impression-Improvement at week 12 was 3.5 versus 3.8 (P = 0.0030; effect size, 0.47). For the key secondary efficacy endpoint, LSM change from baseline to week 12 in the Communication and Symbolic Behavior Scales Developmental Profile Infant-Toddler Checklist Social Composite score was -0.1 versus -1.1 (P = 0.0064; effect size, 0.43). Common treatment-emergent adverse events included diarrhea (80.6% for trofinetide versus 19.1% for placebo), which was mostly mild to moderate in severity. Significant improvement for trofinetide compared with placebo was observed for the coprimary efficacy endpoints, suggesting that trofinetide provides benefit in treating the core symptoms of Rett syndrome.

Learning about neurodiversity from parents - Auditory gestalt perception of prelinguistic vocalisations.

BACKGROUND: Infants with Rett syndrome (RTT) may have subtle anomalies in their prelinguistic vocalisations but the detection of these is difficult, since their conspicuous vocalisations are often interspersed with inconspicuous ones. AIMS AND METHODS: Extending a previous study with predominantly non-parents, the present study sampled parents of children with RTT and aimed to examine their gestalt perception of prelinguistic vocalisations. METHODS AND PROCEDURE: Parents (n = 76) of female children with RTT listened to vocalisation recordings from RTT and typically developing (TD) infants, including an inconspicuous vocalisation from a RTT girl. For each recording, parents indicated if the vocalisation was produced by a RTT or a TD child. RESULTS: Overall correct to incorrect identification rate was 2:1, which was comparable to that of the previous study. Intriguingly, parents of RTT children seemed to be sensitive to features characterising the vocalisations of RTT infants, which has especially influenced their perception of the inconspicuous vocalisation from a RTT girl. CONCLUSIONS AND IMPLICATIONS: These results invite further research on the potential characterising differences between vocalisations from TD infants and infants with divergent neurodevelopment.

Comparison of evoked potentials across four related developmental encephalopathies.

BACKGROUND: Developing biomarkers is a priority for drug development for all conditions, but vital in the rare neurodevelopmental disorders where sensitive outcome measures are lacking. We have previously demonstrated the feasibility and tracking of evoked potentials to disease severity in Rett syndrome and CDKL5 deficiency disorder. The aim of the current study is to characterize evoked potentials in two related developmental encephalopathies, MECP2 duplication syndrome and FOXG1 syndrome, and compare across all four groups to better understand the potential of these measures to serve as biomarkers of clinical severity for the developmental encephalopathies. METHODS: Visual and auditory evoked potentials were acquired from participants with MECP2 duplication syndrome and FOXG1 syndrome across five sites of the Rett Syndrome and Rett-Related Disorders Natural History Study. A group of age-matched individuals (mean = 7.8 years; range = 1-17) with Rett syndrome, CDKL5 deficiency disorder, and typically-developing participants served as a comparison group. The analysis focused on group-level differences as well as associations between the evoked potentials and measures of clinical severity from the Natural History Study. RESULTS: As reported previously, group-level comparisons revealed attenuated visual evoked potentials (VEPs) in participants with Rett syndrome (n = 43) and CDKL5 deficiency disorder (n = 16) compared to typically-developing participants. VEP amplitude was also attenuated in participants with MECP2 duplication syndrome (n = 15) compared to the typically-developing group. VEP amplitude correlated with clinical severity for Rett syndrome and FOXG1 syndrome (n = 5). Auditory evoked potential (AEP) amplitude did not differ between groups, but AEP latency was prolonged in individuals with MECP2 duplication syndrome (n = 14) and FOXG1 syndrome (n = 6) compared to individuals with Rett syndrome (n = 51) and CDKL5 deficiency disorder (n = 14). AEP amplitude correlated with severity in Rett syndrome and CDKL5 deficiency disorder. AEP latency correlated with severity in CDKL5 deficiency disorder, MECP2 duplication syndrome, and FOXG1 syndrome. CONCLUSIONS: There are consistent abnormalities in the evoked potentials in four developmental encephalopathies some of which correlate with clinical severity. While there are consistent changes amongst these four disorders, there are also condition specific findings that need to be further refined and validated. Overall, these results provide a foundation for further refinement of these measures for use in future clinical trials for these conditions.

Distribution of hand function by age in individuals with Rett syndrome.

Objective: To determine the longitudinal distribution of hand function skills in individuals with classic Rett Syndrome (RTT), an X-linked dominant neurodevelopmental disorder, and correlate with MECP2 variants. Method: We conducted a longitudinal study of 946 girls and young women with typical RTT seen between 2006 and 2021 in the US Natural History Study (NHS) featuring a structured clinical evaluation to assess the level of hand function skills. The specific focus in this study was to assess longitudinal variation of hand skills from age 2 through age 18 years in relation to specific MECP2 variant groups. Results: Following the initial regression period, hand function continues to decline across the age spectrum in individuals with RTT. Specific differences are noted with steeper declines in hand function among those with milder variants (Group A: R133C, R294X, R306C, and C-terminal truncations) compared to groups composed of individuals with more severe variants. Conclusions: These temporal variations in hand use represent specific considerations which could influence the design of clinical trials that test therapies aiming to ameliorate specific functional limitations in individuals with RTT. Furthermore, the distinct impact of specific MECP2 variants on clinical severity, especially related to hand use, should be considered in such interventional trials.

Development of trofinetide for the treatment of Rett syndrome: from bench to bedside.

Rett syndrome (RTT) is rare neurodevelopmental disorder caused by mutations in the MECP2 gene that encodes methyl-CpG-binding protein 2 (MeCP2), a DNA-binding protein with roles in epigenetic regulation of gene expression. Functional loss of MeCP2 results in abnormal neuronal maturation and plasticity, characterized by loss of verbal communication and loss of fine and gross motor function, among others. Trofinetide, a synthetic analog of glycine-proline-glutamate, was approved by the US Food and Drug Administration for the treatment of RTT in adult and pediatric patients aged 2 years and older. Here, we present the development of trofinetide from bench research to clinical studies and emphasize how the collaboration between academia, the pharmaceutical industry, and patient advocacy led to the recent approval. The bench-to-bedside development of trofinetide underscores the value of collaboration between these groups in the development and approval of treatments for rare diseases.

Rett Syndrome and MECP2 Duplication Syndrome: Disorders of MeCP2 Dosage.

Rett syndrome (RTT) is a neurodevelopmental disorder caused predominantly by loss-of-function mutations in the gene Methyl-CpG-binding protein 2 (MECP2), which encodes the MeCP2 protein. RTT is a MECP2-related disorder, along with MECP2 duplication syndrome (MDS), caused by gain-of-function duplications of MECP2. Nearly two decades of research have advanced our knowledge of MeCP2 function in health and disease. The following review will discuss MeCP2 protein function and its dysregulation in the MECP2-related disorders RTT and MDS. This will include a discussion of the genetic underpinnings of these disorders, specifically how sporadic X-chromosome mutations arise and manifest in specific populations. We will then review current diagnostic guidelines and clinical manifestations of RTT and MDS. Next, we will delve into MeCP2 biology, describing the dual landscapes of methylated DNA and its reader MeCP2 across the neuronal genome as well as the function of MeCP2 as a transcriptional modulator. Following this, we will outline common MECP2 mutations and genotype-phenotype correlations in both diseases, with particular focus on mutations associated with relatively mild disease in RTT. We will also summarize decades of disease modeling and resulting molecular, synaptic, and behavioral phenotypes associated with RTT and MDS. Finally, we list several therapeutics in the development pipeline for RTT and MDS and available evidence of their safety and efficacy.

Behavioral and brain anatomical analysis of Foxg1 heterozygous mice.

FOXG1 Syndrome (FS) is a devastating neurodevelopmental disorder that is caused by a heterozygous loss-of-function (LOF) mutation of the FOXG1 gene, which encodes a transcriptional regulator important for telencephalic brain development. People with FS have marked developmental delays, impaired ambulation, movement disorders, seizures, and behavior abnormalities including autistic features. Current therapeutic approaches are entirely symptomatic, however the ability to rescue phenotypes in mouse models of other genetic neurodevelopmental disorders such as Rett syndrome, Angelman syndrome, and Phelan-McDermid syndrome by postnatal expression of gene products has led to hope that similar approaches could help modify the disease course in other neurodevelopmental disorders such as FS. While FoxG1 protein function plays a critical role in embryonic brain development, the ongoing adult expression of FoxG1 and behavioral phenotypes that present when FoxG1 function is removed postnatally provides support for opportunity for improvement with postnatal treatment. Here we generated a new mouse allele of Foxg1 that disrupts protein expression and characterized the behavioral and structural brain phenotypes in heterozygous mutant animals. These mutant animals display changes in locomotor behavior, gait, anxiety, social interaction, aggression, and learning and memory compared to littermate controls. Additionally, they have structural brain abnormalities reminiscent of people with FS. This information provides a framework for future studies to evaluate the potential for post-natal expression of FoxG1 to modify the disease course in this severe neurodevelopmental disorder.

Electrophysiological biomarkers of brain function in CDKL5 deficiency disorder.

CDKL5 deficiency disorder is a debilitating developmental and epileptic encephalopathy for which no targeted treatment exists. A number of promising therapeutics are under development for CDKL5 deficiency disorder but a lack of validated biomarkers of brain function and clinical severity may limit the ability to objectively assess the efficacy of new treatments as they become available. To address this need, the current study quantified electrophysiological measures in individuals with CDKL5 deficiency disorder and the association between these parameters and clinical severity. Visual and auditory evoked potentials, as well as resting EEG, were acquired across 5 clinical sites from 26 individuals with CDKL5 deficiency disorder. Evoked potential and quantitative EEG features were calculated and compared with typically developing individuals in an age- and sex-matched cohort. Baseline and Year 1 data, when available, were analysed and the repeatability of the results was tested. Two clinician-completed severity scales were used for evaluating the clinical relevance of the electrophysiological parameters. Group-level comparisons revealed reduced visual evoked potential amplitude in CDKL5 deficiency disorder individuals versus typically developing individuals. There were no group differences in the latency of the visual evoked potentials or in the latency or amplitude of the auditory evoked potentials. Within the CDKL5 deficiency disorder group, auditory evoked potential amplitude correlated with disease severity at baseline as well as Year 1. Multiple quantitative EEG features differed between CDKL5 deficiency disorder and typically developing participants, including amplitude standard deviation, 1/f slope and global delta, theta, alpha and beta power. Several quantitative EEG features correlated with clinical severity, including amplitude skewness, theta/delta ratio and alpha/delta ratio. The theta/delta ratio was the overall strongest predictor of severity and also among the most repeatable qEEG measures from baseline to Year 1. Together, the present findings point to the utility of evoked potentials and quantitative EEG parameters as objective measures of brain function and disease severity in future clinical trials for CDKL5 deficiency disorder. The results also underscore the utility of the current methods, which could be similarly applied to the identification and validation of electrophysiological biomarkers of brain function for other developmental encephalopathies.

Anxiety-like behavior and anxiolytic treatment in the Rett syndrome natural history study.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder most often related to a pathogenic variant in the X-linked MECP2 gene. Internalizing behaviors appear to be common, but standard methods of diagnosing anxiety are not readily applied in this population which typically has cognitive impairment and limited expressive language. This study aims to describe the frequency of anxiety-like behavior and anxiolytic treatments along with associated clinical features in individuals with RTT. METHODS: Parental reports and medication logs provided data from 1380 females with RTT participating in two iterations of the multicenter U.S. RTT Natural History Study (RNHS) from 2006 to 2019. RESULTS: Most participants with RTT (77.5%) had at least occasional anxious or nervous behavior. Anxiety was reported to be the most troublesome concern for 2.6%, and within the top 3 concerns for 10.0%, of participants in the second iteration. Parents directly reported treatment for anxious or nervous behavior in 16.6% of participants in the second iteration with most reporting good control of the behavior (71.6%). In the medication logs of both RNHS iterations, the indication of anxiety was listed for a similar number of participants (15% and 14.5%, respectively). Increased use of anxiolytics and selective serotonin reuptake inhibitors (SSRIs) was related to more frequent anxiety-like behaviors (P < 0.001), older age (P < 0.001), and mild MECP2 variants (P = 0.002). CONCLUSION: Anxiety-like behavior is frequent at all ages and is a significant parental concern in RTT. Older individuals and those with mild MECP2 variants are more likely to be treated with medications. Better diagnosis and treatment of anxiety in RTT should be a goal of both future studies and clinical care. TRIAL REGISTRATION: NCT00299312 and NCT02738281.

Analysis of X-inactivation status in a Rett syndrome natural history study cohort.

BACKGROUND: Rett syndrome (RTT) is a rare neurodevelopmental disorder associated with pathogenic MECP2 variants. Because the MECP2 gene is subject to X-chromosome inactivation (XCI), factors including MECP2 genotypic variation, tissue differences in XCI, and skewing of XCI all likely contribute to the clinical severity of individuals with RTT. METHODS: We analyzed the XCI patterns from blood samples of 320 individuals and their mothers. It includes individuals with RTT (n = 287) and other syndromes sharing overlapping phenotypes with RTT (such as CDKL5 Deficiency Disorder [CDD, n = 16]). XCI status in each proband/mother duo and the parental origin of the preferentially inactivated X chromosome were analyzed. RESULTS: The average XCI ratio in probands was slightly increased compared to their unaffected mothers (73% vs. 69%, p = .0006). Among the duos with informative XCI data, the majority of individuals with classic RTT had their paternal allele preferentially inactivated (n = 180/220, 82%). In sharp contrast, individuals with CDD had their maternal allele preferentially inactivated (n = 10/12, 83%). Our data indicate a weak positive correlation between XCI skewing ratio and clinical severity scale (CSS) scores in classic RTT patients with maternal allele preferentially inactivated XCI (rs  = 0.35, n = 40), but not in those with paternal allele preferentially inactivated XCI (rs  = -0.06, n = 180). The most frequent MECP2 pathogenic variants were enriched in individuals with highly/moderately skewed XCI patterns, suggesting an association with higher levels of XCI skewing. CONCLUSION: These results extend our understanding of the pathogenesis of RTT and other syndromes with overlapping clinical features by providing insight into the both XCI and the preferential XCI of parental alleles.

Design and outcome measures of LAVENDER, a phase 3 study of trofinetide for Rett syndrome.

INTRODUCTION: Rett syndrome (RTT) is a debilitating neurodevelopmental disorder with no approved treatments. Trofinetide is a synthetic analog of glycine-proline-glutamate, the N-terminal tripeptide of insulin-like growth factor 1. In a phase 2, placebo-controlled trial in 82 females with RTT aged 5-15 years, a significant (p ≤ 0.042) improvement over placebo was observed with the highest trofinetide dose (200 mg/kg twice daily [BID]) on three measures: Rett Syndrome Behaviour Questionnaire (RSBQ), Clinical Global Impression-Improvement (CGI-I), and RTT-Clinician Domain Specific Concerns-Visual Analog Scale (RTT-DSC-VAS). Trofinetide was well tolerated at all doses (50, 100, and 200 mg/kg BID). A phase 3 trial utilizing disease-specific and novel scales was designed to investigate the efficacy and safety of trofinetide in girls and women with RTT. METHODS: This 12-week, double-blind, randomized, placebo-controlled study (LAVENDER; NCT04181723) will evaluate trofinetide in 187 females, aged 5-20 years, with RTT. Co-primary endpoints are the RSBQ and CGI-I scales. Clinical domains of the CGI-I include communication, ambulation, hand use, seizures, attentiveness, and social (eye contact) and autonomic (breathing) aspects. Secondary endpoints will leverage four novel RTT-specific clinician ratings (derived from the RTT-DSC-VAS) of hand function, ambulation, ability to communicate, and verbal communication, and existing scales, to evaluate other core symptoms of RTT, quality of life and caregiver burden. A 40-week, open-label extension study will follow. DISCUSSION: This study was designed using disease-specific scales optimized to demonstrate changes in core symptoms of RTT and may provide the first phase 3 data demonstrating drug efficacy in individuals with RTT. TRIAL REGISTRATION: Clinicaltrials.govNCT04181723.