Workshop report

An update on clinically applicable diagnostic criteria in Rett syndrome: Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting Baden Baden, Germany, 11 September 2001

Methyl-CpG-binding protein 2 (MeCP2) is of utmost importance in neuronal function. We aim to characterize phenotypic traits in the sleep of individuals with Rett Syndrome (RTT, OMIM # 312750), a rare disorder predominantly caused by mutations of the MECP2 gene.

An overnight polysomnographic recording was performed. Outcomes investigated were parameters of nocturnal sleep macrostructure, and sample stratification per genetic and clinical characteristics, and six key features of clinical severity was applied.

The sleep of our 21 RTT female subjects with a mutant MECP2 gene, aged 8.8 ± 5.4 years, showed no significant differences within strata. However, compared to a normative dataset, we found longer duration of wake time after sleep onset and total sleep time (TST) but shorter sleep onset latency, in RTT. Regarding the proportion of sleep stages per TST, higher stage N3 (%) with lower stage N2 (%) and REM (%) were generally seen. Such abnormalities became more uniformly expressed at the severe level of clinical features, particularly for hand functioning and walking.

RTT girls with MECP2 mutations in our study demonstrated an increased deep sleep and reduced rapid eye movement sleep proportion, which is mostly allied with their hand dysfunction severity. Poor sleep-on/off switching in RTT since embryogenesis is possibly linked to (psycho)motor impairment in the cases with MECP2 mutations.

Eukaryotic gene expression is controlled at multiple steps that work in harmony to ensure proper maintenance of cellular morphology and function. Such regulatory mechanisms would include transcriptional gene regulation, which is in turn controlled by chromatin remodeling, distinct topologically associating domains of the chromatin structure, cis-regulatory elements such as enhancers and promoters, action of trans-acting factors, DNA methylation, RNA modifications, and post-translational modification of histones. These guiding mechanisms of gene expression play critical roles in the epigenetic setting of individual cells within the eukaryotic systems. Some epigenetic factors may play multiple functional roles in guarding the accurate gene expression program of the eukaryotic cells, especially within the central nervous system. A well-studied example of such multi-functional factors is the methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that is encoded by the X-linked MECP2 gene. Here, we aim to provide an overview of eukaryotic gene regulation, the three-dimensional chromatin organization, standard techniques to study newly synthesized RNA transcripts, and the role of MeCP2 as an important transcriptional regulator in eukaryotes.

Although CRISPR/Cas9-mediated gene editing is widely applied to mimic human disorders, whether acute manipulation of disease-causing genes in the brain leads to behavioral abnormalities in non-human primates remains to be determined. Here we induced genetic mutations in MECP2, a critical gene linked to Rett syndrome (RTT) and autism spectrum disorders (ASD), in the hippocampus (DG and CA1–4) of adolescent rhesus monkeys (Macaca mulatta) in vivo via adeno-associated virus (AAV)-delivered Staphylococcus aureus Cas9 with small guide RNAs (sgRNAs) targeting MECP2. In comparison to monkeys injected with AAV-SaCas9 alone (n = 4), numerous autistic-like behavioral abnormalities were identified in the AAV-SaCas9-sgMECP2-injected monkeys (n = 7), including social interaction deficits, abnormal sleep patterns, insensitivity to aversive stimuli, abnormal hand motions, and defective social reward behaviors. Furthermore, some aspects of ASD and RTT, such as stereotypic behaviors, did not appear in the MECP2 gene-edited monkeys, suggesting that different brain areas likely contribute to distinct ASD symptoms. This study showed that acute manipulation of disease-causing genes via in vivo gene editing directly led to behavioral changes in adolescent primates, paving the way for the rapid generation of genetically engineered non-human primate models for neurobiological studies and therapeutic development.

Rett syndrome (RTT) is a rare neurologic disorder, characterized by severe behavioural and physiological symptoms. RTT is caused by mutations in the MECP2 gene in about 95% of cases and to date no cure is available. Recent evidence suggests that non-euphoric phytocannabinoids (pCBs) extracted from Cannabis sativa may represent innovative therapeutic molecules for RTT, with the cannabinoid cannabidivarin having beneficial effects on behavioural and brain molecular alterations in RTT mouse models. The present study evaluated the potential therapeutic efficacy for RTT of cannabidiolic acid (CBDA; 0.2, 2, 20 mg/kg through intraperitoneal injections for 14 days), a pCB that has proved to be effective for the treatment of nausea and anxiety in rodents. This study demonstrates that systemic treatment with the low dose of CBDA has anti-nociceptive effects and reduces the thermal hyperalgesia in 8 month-old MeCP2-308 male mice, a validated RTT mouse model. CBDA did not affect other behavioural or molecular parameters. These results provide support to the antinociceptive effects of CBDA and stress the need for further studies aimed at clarifying the mechanisms underlying the abnormal pain perception in RTT.

Epigenetics plays a role in many disorders that are present in the pediatric populations (e.g., in early human development, including embryologic and fetal life, infancy, childhood, and adolescence). In this chapter, we will first discuss specific diseases related to epigenetic disturbance. We will then discuss environmental and developmental disorders with a likely epigenetic mechanism. We will also discuss the role of epigenetics in informing Developmental Origins of Health and Disease (DOHaD) and public health strategies. Many syndromes were described because of their characteristic clinical features before epigenetic mechanisms were understood. Because time in development (age), as well as tissue-specific and gender-specific gene expression, is so important in early development, it is not surprising that disturbances in control of gene expression will lead to pathologic conditions. These groups of disorders are important in pediatrics and, thus, to the pediatrician.

Mammalian flexibility in response to environmental change is now recognized to play a role in transgenerational programming for many chronic diseases of adults that appear to have their genesis in very early life. Working out the pathways and mechanisms for these disorders holds the promise for therapy in the future. Small-for-dates (IUGR) newborns should avoid excess weight gain during infancy and early childhood as this may help prevent the development of some chronic diseases. Recording data on the placenta at birth as well as complications during the pregnancy is likely to be important for future research and possibly therapy. A new type of family history including multigenerational information on birth weights, pregnancy histories, and various types of parental stress and environmental exposures will be important for future medical care.

Rett syndrome (RTT) is a stern dominant progressive neurological development disorder linked with X chromosome ranking second for mental slowdown, exclusively in females after few months of birth with normal development and growth period. Genetically any defects in universally expressed methyl-CpG binding protein 2 (MeCP2) transcription regulator gene are considered as radix for RTT in almost all the previous studies. Our study mainly focuses in unraveling the genetic alterations like identifying MeCP2 gene polymorphisms, chromosomal abnormalities, or X-chromosome inactivation (XCI) as underlying cause of RTT in prototypes sorted through Diagnostic and Statistical Manual of Mental Disorders-Text Revised (DSM IV). In addition, we have examined the probable surrogates of brain function disabilities like serotonin, homocysteine (Hcy), calcium, potassium, and lead from blood in both RTT porotypes and their mothers. In our investigation, we have observed varied amino acid substitution of MeCP2 and varied frequency of skewed XCI in RTT prototype. Our study validates that the demonstration of chromosomal analysis, biochemical analysis, and genomic observations helps in concluding RTT condition and can be helpful in providing appropriate treatment and counseling as well as improve the currently available protocol of diagnosis.