Mark E.S. Bailey

Rett Syndrome: Revised Diagnostic Criteria and Nomenclature

Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in Methyl‐CpG‐binding protein 2 (MECP2). Despite distinct clinical features, the accumulation of clinical and molecular information in recent years has generated considerable confusion regarding the diagnosis of RTT. The purpose of this work was to revise and clarify 2002 consensus criteria for the diagnosis of RTT in anticipation of treatment trials.

Objective vs. Self-Reported Physical Activity and Sedentary Time: Effects of Measurement Method on Relationships with Risk Biomarkers

Purpose Imprecise measurement of physical activity variables might attenuate estimates of the beneficial effects of activity on health-related outcomes. We aimed to compare the cardiometabolic risk factor dose-response relationships for physical activity and sedentary behaviour between accelerometer- and questionnaire-based activity measures. Methods Physical activity and sedentary behaviour were assessed in 317 adults by 7-day accelerometry and International Physical Activity Questionnaire (IPAQ). Fasting blood was taken to determine insulin, glucose, triglyceride and total, LDL and HDL cholesterol concentrations and homeostasis model-estimated insulin resistance (HOMAIR). Waist circumference, BMI, body fat percentage and blood pressure were also measured. Results For both accelerometer-derived sedentary time (<100 counts.min−1) and IPAQ-reported sitting time significant positive (negative for HDL cholesterol) relationships were observed with all measured risk factors – i.e. increased sedentary behaviour was associated with increased risk (all p≤0.01). However, for HOMAIR and insulin the regression coefficients were >50% lower for the IPAQ-reported compared to the accelerometer-derived measure (p<0.0001 for both interactions). The relationships for moderate-to-vigorous physical activity (MVPA) and risk factors were less strong than those observed for sedentary behaviours, but significant negative relationships were observed for both accelerometer and IPAQ MVPA measures with glucose, and insulin and HOMAIR values (all p<0.05). For accelerometer-derived MVPA only, additional negative relationships were seen with triglyceride, total cholesterol and LDL cholesterol concentrations, BMI, waist circumference and percentage body fat, and a positive relationship was evident with HDL cholesterol (p = 0.0002). Regression coefficients for HOMAIR, insulin and triglyceride were 43–50% lower for the IPAQ-reported compared to the accelerometer-derived MVPA measure (all p≤0.01). Conclusion Using the IPAQ to determine sitting time and MVPA reveals some, but not all, relationships between these activity measures and metabolic and vascular disease risk factors. Using this self-report method to quantify activity can therefore underestimate the strength of some relationships with risk factors.

Association analysis of the ACTN3 R577X polymorphism and complex quantitative body composition and performance phenotypes in adolescent Greeks.

The functional allele (577R) of ACTN3, which encodes human α-actinin-3, has been reported to be associated with elite athletic status and with response to resistance training, while the nonfunctional allele (577X) has been proposed as a candidate metabolically thrifty allele. In a study of 992 adolescent Greeks, we show that there is a significant association (P=0.003) between the ACTN3 R577X polymorphism and 40 m sprint time in males that accounts for 2.3% of phenotypic variance, with the 577R allele contributing to faster times in an additive manner. The R577X polymorphism is not associated with other power phenotypes related to 40 m sprint, nor with an endurance phenotype. Furthermore, the polymorphism is not associated with obesity-related phenotypes in our population, suggesting that the 577X allele is not a thrifty allele, and thus the persistence of this null allele must be explained in other terms.

Mutation screening in Rett syndrome patients

Rett syndrome (RTT) was first described in 1966. Its biological and genetic foundations were not clear until recently when Amiret al reported that mutations in the MECP2 gene were detected in around 50% of RTT patients. In this study, we have screened theMECP2 gene for mutations in our RTT material, including nine familial cases (19 Rett girls) and 59 sporadic cases. A total of 27 sporadic RTT patients were found to have mutations in the MECP2 gene, but no mutations were identified in our RTT families. In order to address the possibility of further X chromosomal or autosomal genetic factors in RTT, we evaluated six candidate genes for RTT selected on clinical, pathological, and genetic grounds:UBE1 (human ubiquitin activating enzyme E1, located in chromosome Xp11.23),UBE2I (ubiquitin conjugating enzyme E2I, homologous to yeast UBC9, chromosome 16p13.3),GdX (ubiquitin-like protein, chromosome Xq28), SOX3 (SRY related HMG box gene 3, chromosome Xq26-q27),GABRA3 (γ-aminobutyric acid type A receptor α3 subunit, chromosome Xq28), andCDR2 (cerebellar degeneration related autoantigen 2, chromosome 16p12-p13.1). No mutations were detected in the coding regions of these six genes in 10 affected subjects and, therefore, alterations in the amino acid sequences of the encoded proteins can be excluded as having a causative role in RTT. Furthermore, gene expression ofMECP2, GdX, GABRA3, and L1CAM(L1 cell adhesion molecule) was also investigated by in situ hybridisation. No gross differences were observed in neurones of several brain regions between normal controls and Rett patients.

MeCP2 and Rett syndrome: reversibility and potential avenues for therapy

Mutations in the X-linked gene MECP2 (methyl CpG-binding protein 2) are the primary cause of the neurodevelopmental disorder RTT (Rett syndrome), and are also implicated in other neurological conditions. The expression product of this gene, MeCP2, is a widely expressed nuclear protein, especially abundant in mature neurons of the CNS (central nervous system). The major recognized consequences of MECP2 mutation occur in the CNS, but there is growing awareness of peripheral effects contributing to the full RTT phenotype. MeCP2 is classically considered to act as a DNA methylation-dependent transcriptional repressor, but may have additional roles in regulating gene expression and chromatin structure. Knocking out Mecp2 function in mice recapitulates many of the overt neurological features seen in RTT patients, and the characteristic postnatally delayed onset of symptoms is accompanied by aberrant neuronal morphology and deficits in synaptic physiology. Evidence that reactivation of endogenous Mecp2 in mutant mice, even at adult stages, can reverse aspects of RTT-like pathology and result in apparently functionally mature neurons has provided renewed hope for patients, but has also provoked discussion about traditional boundaries between neurodevelopmental disorders and those involving dysfunction at later stages. In the present paper we review the neurobiology of MeCP2 and consider the various genetic (including gene therapy), pharmacological and environmental interventions that have been, and could be, developed to attempt phenotypic rescue in RTT. Such approaches are already providing valuable insights into the potential tractability of RTT and related conditions, and are useful pointers for the development of future therapeutic strategies.

Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation

Introduction: Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder that is usually associated with mutations in the MECP2 gene. The most common mutations in the gene are p.R168X and p.T158M. The influence of X-chromosome inactivation (XCI) on clinical severity in patients with RTT with these mutations was investigated, taking into account the extent and direction of skewing. Methods: Female patients and their parents were recruited from the UK and Australia. Clinical severity was measured by the Pineda Severity and Kerr profile scores. The degree of XCI and its direction relative to the X chromosome parent of origin were measured in DNA prepared from peripheral blood leucocytes, and allele-specific polymerase chain reaction was used to determine the parental origin of mutation. Combining these, the percentage of cells expected to express the mutant allele was calculated. Results: Linear regression analysis was undertaken for fully informative cases with p.R168X (n = 23) and p.T158M (n = 20) mutations. A statistically significant increase in clinical severity with increase in the proportion of active mutated allele was shown for both the p.R168X and p.T158M mutations. Conclusions: XCI may vary in neurological and haematological tissues. However, these data are the first to show a relationship between the degree and direction of XCI in leucocytes and clinical severity in RTT, although the clinical utility of this in giving a prognosis for individual patients is unclear.

Genome-wide analysis of over 106 000 individuals identifies 9 neuroticism-associated loci

Neuroticism is a personality trait of fundamental importance for psychological well-being and public health. It is strongly associated with major depressive disorder (MDD) and several other psychiatric conditions. Although neuroticism is heritable, attempts to identify the alleles involved in previous studies have been limited by relatively small sample sizes. Here we report a combined meta-analysis of genome-wide association study (GWAS) of neuroticism that includes 91 370 participants from the UK Biobank cohort, 6659 participants from the Generation Scotland: Scottish Family Health Study (GS:SFHS) and 8687 participants from a QIMR (Queensland Institute of Medical Research) Berghofer Medical Research Institute (QIMR) cohort. All participants were assessed using the same neuroticism instrument, the Eysenck Personality Questionnaire-Revised (EPQ-R-S) Short Form’s Neuroticism scale. We found a single-nucleotide polymorphism-based heritability estimate for neuroticism of ∼15% (s.e.=0.7%). Meta-analysis identified nine novel loci associated with neuroticism. The strongest evidence for association was at a locus on chromosome 8 (P=1.5 × 10−15) spanning 4 Mb and containing at least 36 genes. Other associated loci included interesting candidate genes on chromosome 1 (GRIK3 (glutamate receptor ionotropic kainate 3)), chromosome 4 (KLHL2 (Kelch-like protein 2)), chromosome 17 (CRHR1 (corticotropin-releasing hormone receptor 1) and MAPT (microtubule-associated protein Tau)) and on chromosome 18 (CELF4 (CUGBP elav-like family member 4)). We found no evidence for genetic differences in the common allelic architecture of neuroticism by sex. By comparing our findings with those of the Psychiatric Genetics Consortia, we identified a strong genetic correlation between neuroticism and MDD and a less strong but significant genetic correlation with schizophrenia, although not with bipolar disorder. Polygenic risk scores derived from the primary UK Biobank sample captured ∼1% of the variance in neuroticism in the GS:SFHS and QIMR samples, although most of the genome-wide significant alleles identified within a UK Biobank-only GWAS of neuroticism were not independently replicated within these cohorts. The identification of nine novel neuroticism-associated loci will drive forward future work on the neurobiology of neuroticism and related phenotypes.

Improved Survival and Reduced Phenotypic Severity Following AAV9/MECP2 Gene Transfer to Neonatal and Juvenile Male Mecp2 Knockout Mice

Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/chicken β-actin (CBA)-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for green fluorescent protein (GFP)-treated mice. ssAAV9/CBA-MECP2-treated mice also showed significant improvement in the phenotype severity score, in locomotor function, and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type (WT) mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary (sc) adeno-associated virus (AAV) vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously (IV) into juvenile (4-5 weeks old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2-4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT.

FTO genotype and adiposity in children: physical activity levels influence the effect of the risk genotype in adolescent males

Studies of the fat mass and obesity-associated (FTO) gene provide compelling evidence of genetic variation in the general population that influences fat levels and obesity risk. Studies of the interaction between genetic and environmental factors such as physical activity (PA) will promote the understanding of how lifestyle can modulate genetic contributions to obesity. In this study, we investigated the effect of FTO genotype, and interactions with PA or energy intake, in young children and adolescents. In all, 1–5-year-old children from the Growth, Exercise and Nutrition Epidemiological Study in preSchoolers (GENESIS) study (N=1980) and 11–18-year-old Greek adolescents (N=949) were measured for adiposity-related phenotypes and genotyped at the FTO single-nucleotide polymorphism (SNP) marker, rs17817449. Adolescents were classified as physically active or inactive based on self-reported levels of PA. In adolescents, FTO genotype influenced weight (P=0.001) and BMI (P=0.007). There was also a significant SNP*PA*gender interaction (P=0.028) on BMI, which reflected the association between FTO genotype and BMI in males (P=0.016), but not females (P=0.15), and significant SNP*PA interaction in males (P=0.007), but not females (P=0.74). The FTO genotype effect was more pronounced in inactive than active males. Inactive males homozygous for the G allele had a mean BMI 3 kg/m2 higher than T carriers (P=0.008). In the GENESIS study, no significant association between FTO genotype and adiposity was found. The present findings highlight PA as an important factor modifying the effect of FTO genotype.

Dimensional phenotypic analysis and functional categorisation of mutations reveal novel genotype-phenotype associations in Rett syndrome.

We aimed to improve the understanding of genotype–phenotype correlations in Rett syndrome (RS) by adopting a novel approach to categorising phenotypic dimensions – separating typicality of presentation, outcome severity and age of onset – and by classifying MECP2 mutations strictly by predicted functional attributes. MECP2 mutation screening results were available on 190 patients with a clinical diagnosis of RS (140 cases with classic RS, 50 with atypical RS). 135 cases had identified mutations. Of the 140 patients, 116 with classic RS (82.9%) had an identified mutation compared with 19 of 50 patients (38%) with an atypical presentation. Cases with early onset of regression and seizures, and those with clinical features that might indicate alternative aetiologies, were less likely to have mutations. Individuals with late truncating mutations had a less typical presentation than cases with missense and early truncating mutations, presumably reflecting greater residual function of MECP2 protein. Individuals with early truncating mutations had a more severe outcome than cases with missense and late truncating mutations. These findings held when restricting the analysis to cases over 15 years of age and classic cases only. Previous findings of variation in severity among the common mutations were confirmed. The approach to phenotypic and genotypic classification adopted here allowed us to identify genotype–phenotype associations in RS that may aid our understanding of pathogenesis and also contribute to clinical knowledge on the impact of different types of mutations.