Rett Syndrome: From Recognition to Diagnosis to Intervention: An update by Dr. Alan percy

Introduction Physicians and related healthcare professionals, basic scientists and educators are, generally, uninformed about Rett syndrome (RS). Indeed, RS is a relative newcomer to the field of neurodevelopmental disorders. Despite being one of the 'new kids on the block', significant advances have emerged in recent years regarding our understanding of the clinical features, molecular genetics and neurobiology of RS, particularly following the recognition that mutations in the gene, MECP2, are responsible. In the absence of a fundamental therapy, serious energies have been directed to developing rational intervention strategies. Excitement was generated within the past year by the potential for reversing the devastating consequences of RS. Through molecular manipulation, a mouse model for RS was engineered to allow activation of Mecp2 once the mice had developed the clinical phenotype associated with absence of this gene. At the same time, studies in another mouse model provided important new information on the molecular neurobiology of RS. These studies allowed the authors to suggest a neuroendocrine component involving elevated corticotrophinreleasing hormone (Crh) and elevated corticosterone levels (cortisol in humans) in the pathobiology of this disorder. Taken together, the findings support the notion that the clinical features of RS could be modified by appropriate interventions and suggest at least one potential avenue for doing so. This review provides background information on RS, including diagnosis, medical issues, genetics and molecular neurobiology, and explores potential therapeutic strategies. History Rett syndrome is a relatively rare neurodevelopmental disorder found almost exclusively in females. RS was recognized independently in the early 1960s by two European physicians, Andreas Rett in Vienna and Bengt Hagberg in Gothenborg, Sweden. However, widespread prominence was not achieved until 1983 following a chance meeting between them, which generated the first widely read description. Within a short time, RS was diagnosed throughout the USA and the International Rett Syndrome Association (IRSA) was formed, becoming the information clearing house in this country and, to some extent, worldwide. RS has been identified across the world in all racial and ethnic groups with an incidence approximating one in 10,000 females. In the 1980s, RS was already presumed to be X linked, based on almost total occurrence in females, twin studies and vertical transmission from an affected woman to her female offspring.[5] Detailed molecular studies taking advantage of an X chromosome–autosome translocation in one girl with RS, and several instances of familial recurrence[8,9] restricted the X-chromosome target area to Xq28, a gene-rich region where MECP2 resides. In 1999, the identification of mutations in MECP2 confirmed its genetic basis.[10] Nonetheless, RS is generally not transmitted from generation to generation but is sporadic: the recurrence risk is well below 0.5%. At present, more than 95% of females who fulfill consensus criteria for RS have a MECP2 mutation.[11] In the North American databases, 91% of females who met consensus criteria for RS and who had MECP2 testing had a mutation.[12] This number is lower because testing for large deletions was not conducted routinely in this cohort Diagnosis The diagnosis of RS is based on recently refined consensus diagnostic criteria.[13] These criteria provide the basis for standardized clinical diagnosis of RS throughout the world ( Box 1 ). Approximately 85% of females with RS will fulfill these criteria. The remaining 15% have one of several variant forms for which diagnostic criteria have also been elaborated ( Box 2 ).[13] These variants include an early-onset seizure type, a congenital form lacking normal early development, a preserved speech variant with some purposeful language, and a delayed form or forme fruste. Children who fulfill diagnostic criteria, whether typical or variant, should be tested for mutations in MECP2. This would include young females (6–24 months) who display only some features associated with RS, such as low muscle tone, deceleration in the rate of head growth or unexplained developmental delay or frank regression. With the advent of effective treatment strategies, early diagnosis prior to full expression of typical features will be crucial. Individuals meeting consensus criteria, of whom 95% or more will have a mutation,[12] should have molecular genetic testing for mutations in MECP2. Such testing should include sequencing of the four exons associated with this gene, as well as testing for large-scale deletions in those individuals who have negative results by standard sequencing analyses. For those mutations identified by sequencing, the vast majority will occur in exons 3 and 4. Of the individuals meeting variant criteria, 55–60% will have a mutation.[12] It is critical that healthcare providers be aware that sequencing and large deletion testing involve different methodologies. Analyses for large deletions should be requested whenever sequencing fails to identify a mutation. MECP2 testing should also be requested for females who demonstrate the characteristics of Angelman syndrome but have normal methylation or mutation studies at the Angelman locus. Males with features of RS or with X-linked mental retardation and normal Fragile X testing, and infants with unexplained neonatal or infantile encephalopathy, should also be tested.[14] Recently, males with cognitive delay, little or no speech and an unusual gait, with or without recurrent upper respiratory infections, have been described in association with duplications of MECP2.[15-20] These individuals should have deletion/duplication or comparative genomic hybridization (CGH) array testing.[19] MECP2 testing is available in a number of different clinical laboratories throughout the world. In the USA, these include the Baylor College of Medicine in Houston (TX) and the Greenwood Genetic Center in Greenwood (SC). Genetics More than 95% of females fulfilling criteria for RS have a MECP2 mutation.[11] More than 200 distinct mutations have been identified, but eight common mutations (four nonsense and four missense mutations) account for most individuals with RS. In the recently published North American database[12] and in the International Rett Syndrome Association-funded Australian RettBase, the four most common mutations are T158M, R168X, R255X and R270X. Missense mutations represent 39.0%, nonsense mutations 35.2%, large deletions 6.4% and C-terminal truncations 8.8% of the total mutations in the North American cohort. As stated previously, the number of large deletions is likely to be under-represented in this cohort because of incomplete testing in some females, especially those diagnosed prior to 2000. Phenotypic variability may be striking.[12] Females with such mutations may be completely normal or have clinical features ranging from mild learning disabilities, to Angelman syndrome, to autism and to RS. Females who appear normal or have learning disabilities generally share the same mutation as a sibling or child with RS, but lack features of RS owing to favorable skewing of X-chromosome inactivation. Females with features of Angelman syndrome represent the close clinical overlap during early childhood between the two disorders, and females who have autism but not RS reflect either favorable skewing in X-chromosome inactivation or less severe mutations. Rett syndrome has been identified in males who have an extra X chromosome (Klinefelter syndrome)[21] and in males with somatic mosaicism, some cells expressing normal X chromosomes and others expressing MECP2 mutation-bearing X chromosomes.[22] These males resemble females with balanced X-chromosome inactivation. Most males with MECP2 mutations display a much more severe progressive encephalopathy with motor and respiratory problems present from birth orearly infancy and premature death often by 1–2 years. Approximately 50% have the same mutation as an affected sibling(s), whereas others represent sporadic occurrences.[14,15] Still, other males demonstrate only X-linked mental retardation. A majority of these males will have a duplication of the Xq28 locus including MECP2. Based on the numbers presently reported, this MECP2 duplication disorder is likely to be the most common finding in males associated with MECP2 mutations or extra copy number.[15-20] Phenotype–genotype Correlations Clinical severity does depend on mutation type and position. A number of reports have appeared in recent years dealing with the relationships between the clinical picture and the specific MECP2 mutation. Over time, these analyses have been derived from increasingly larger datasets and, hence, provided greater reliability. In general, mutations more distal in the gene, such as R294X, R306C and C-terminal truncations, produce milder involvement.[23] However, strikingly milder features have also been associated with the R133C mutation that is much more proximal in the gene. Two papers in Neurology (one currently in press) support these findings and provide the most extensive published information to date.[11,24] The largest longitudinal dataset is currently held with the NIH-sponsored Rare Disease Clinical Research Network data repository. Preliminary analyses of data from more than 400 participants substantiate the general conclusions noted previously.[25] Any conclusions derived solely from mutation type and position will most probably be affected by other factors, the most prominent being imbalances in X-chromosome inactivation or differences in the specific neuronal po-pulations impacted by an abnormal MECP2. Clinical Picture Rett syndrome typically becomes evident between 6 and 18 months of age. Before then, most parents report normal pregnancy, birth and early motor and social development, but the infants are, perhaps, 'too good'. Early development may not be normal, but this is often difficult for parents to discern. Single words or phrases often appear. However, hypotonia or reduced muscle tone is commonly noted during this early period, and growth patterns are often problematic, including deceleration in the rate of head growth (as early as 3 months of age) and poor weight gain. It should be emphasized that deceleration in the rate of head growth does not necessarily mean microcephaly. Many of these children have head circumferences in the normal range, while in approximately 50% head circumferences decline into the microcephalic range. Indeed, a small number may demonstrate no decline in head circumference growth rate at all, despite having the other features of RS. Deceleration in the rate of linear growth typically follows the first birthday.[26] The first abnormal clinical signs may include increasing irritability with inconsolable crying and stagnation or plateauing in motor development, ultimately leading to frank regression and virtual loss of fine motor skills. It is during this period that autistic-like behavior appears, as contact with others diminishes or is lost. Other communication elements may also be lost, with some appearing as if they do not hear despite normal hearing assessments. Persistent stereotypic hand movements, noted only while awake, emerge during or shortly after this regression period. These may consist of hand-wringing, -patting, -clasping, -squeezing, or mouthing, picking at the clothes or skin, or twirling their hair. Generally, but not always, the hands are held in the midline. Each girl will have her own repertoire of stereotypies that continue to evolve over time. The hand stereotypies often dominate waking moments and tend to be exacerbated by stress or excitement. Approximately 80% develop independent ambulation (Percy AK, personal observations from the UAB database). However, the gait is dyspraxic and nonpurposeful, generally on a wide base. Toe-walking, repetitive shifting of weight from one foot to the other, and retropulsion (first step is backwards) are common. Other features may occur, but are not seen uniformly. These include seizures, teeth grinding (bruxism), disturbed sleep, and abnormal breathing patterns consisting of breath-holding or hyperventilation, or both. Abnormal breathing patterns, just like stereotypic hand movements, occur only during wakefulness and increase with excitement or stress. Scoliosis develops in most young females, with approximately 10% requiring surgery.[27,28] Additional features may include gastroesophageal reflux and other evidence of gastrointestinal dysmotility, including chewing and swallowing dysfunction, poor stomach emptying and constipation, cold, often purplish extremities (e.g., in the feet), osteopenia with increased incidence of fractures[29] and ECG changes, including prolonged QT syndrome.[30] Rett syndrome has a very consistent temporal profile ( Box 3 ). First comes the period of regression. Then, development stabilizes with disappearance of the autistic-like features and emergence of markedly improved interaction and socialization. Eye contact resumes to a variable degree and the ability to make choices can be developed. However, during this period, seizures and irregular breathing may be much more prominent. These tend to diminish during adolescence or somewhat later, along with reduced frequency and intensity of hand stereotypies. The absence of further cognitive loss provides strong support for employing therapies to maximize communication and socialization capabilities and to preserve motor function. This is also in line with the notion that RS is not a progressive disorder and could respond to fundamental therapies aimed at a cure. Although much work remains, we now have a better understanding of the natural history of RS, including survival and the many medical issues associated with it.[31] Medical Issues A wide variety of medical issues will be considered in the following paragraphs. Not all girls or women will manifest every one, but the care provider should be aware of them should problems arise that are otherwise unexplainable.[31,32] Growth Pervasive failure of growth is typical. As stated previously, the first evidence is often a deceleration in the rate of head growth beginning at 3 months of age.[26] Deceleration in the rate of weight gain also appears in the 1st year of life, and deceleration in the rate of linear growth becomes noticeable after the first birthday. The median value for head circumference approaches the second percentile value for the normal female population by 2 years of age. Hand and foot growth also diminish, more so for feet than hands.[33] Nonetheless, both tend to be smaller than in the general population. The decline in the rate of foot growth follows the pattern of decline in linear growth rate. Epilepsy Reported rates for occurrence of epilepsy are quite variable, ranging from a low of 20–25% to a high of more than 80%. Our experience, based on video-EEG monitoring, suggests that as few as 25% of clinical behaviors are epileptic seizures that require medical management.[34] Many of the events that parents, teachers and other caregivers report as seizures are not associated with abnormal cortical discharges when captured by video EEG. However, subtle clinical events identified by video EEG as representing seizures may not be appreciated by parents as such. Seizures may be generalized, focal, or atypical absence in character and, in some small numbers, may represent infantile spasms. Should any question arise regarding the possible occurrence of seizures, video-EEG monitoring is recommended or, at the very least, prolonged ambulatory monitoring with automatic spike-detection capabilities. The management of seizures in general involves available medications designed specifically to control epilepsy. A number of these medications may be effective (e.g., carbamazepine or oxcarbazepine, valproic acid or sodium valproate and newer medications including lamotrigine, zonisamide and levetiracetam). The importance of being certain that the diagnosis of seizures is correct should be emphasized in order to minimize the number of girls that are placed on these medications. Sleep Sleep is often disrupted with frequent night-time awakenings, in many instances without evidence of distress but, rather simply, playing in bed, occasionally punctuated by laughter for no apparent reason. On other occasions, sleep is interrupted by evidence of upset or fussiness in which case it is important to be certain that general care issues are addressed, such as the need for diaper change or hunger, or other medical issues, such as constipation, gastroesophageal reflux or even an intercurrent infection (e.g., an upper respiratory infection, otitis media or urinary tract infection). The importance of ruling out hunger and other gastrointestinal issues cannot be stressed too strongly. Evidence of excessive daytime sleepiness, that is, frequent napping, should alert the clinician to monitor night-time sleep for disruptions that might be caused by airway obstruction during sleep. When studied in detail, it is quite clear that stage rapid eye movement sleep is substantially reduced in RS. In addition, other sleep stages are also abnormal.[35] Good sleep hygiene is essential. Going to bed and awakening from sleep should occur at consistent times. In the morning, the use of a bright light will encourage arousal; in the evening, a dim light will promote going to sleep. If going to and staying asleep are problematic, such that the family life is disrupted or the quality of life is adversely impacted, medications should be considered. Clearly, if the parents are not obtaining adequate rest, their ability to care for their daughter will be adversely affected. Several medications may be effective for achieving sleep. These have included an antihistamine, such as hydroxyzine, as well as other classes, such as the benzodiazepines or melatonin. The antihistamines may have initial effectiveness, but for only a short time owing to the development of tolerance. Melatonin may be helpful in inducing sleep, although it may not decrease or prevent arousals during the night. Trazodone and the newer nonbenzodiazepine agents, such as zolpidem, may be helpful and safe in promoting a full night of sleep. Chloral hydrate is also an effective medication that can be used safely in quantities up to 50mg/kg/dose. However, it is most commonly formulated as a liquid that will often be refused because of its taste unless administered by gastrostomy. It may be found as a gel cap and some private pharmacies are able to formulate it in a capsule or as a suppository, very acceptable options. Breathing Irregularities Periodic breathing in the form of breath holding and hyperventilation, or both, is quite common.[35] These activities occur only during wakefulness and are modified by factors such as hunger, agitation and other stressful situations. Irregular breathing usually begins well after the other features of RS are clearly apparent but is typically present by early childhood with a peak of occurrence in the school-age period through to adolescence. Breath holding in particular may be quite prolonged and associated with color changes around the mouth or in the nailbed. These events are not known to cause additional medical problems. Breath holding can be quite subtle and often first apparent by the sudden expulsion of air (air puffing). Breath holding may also be accompanied by copious air swallowing, producing significant abdominal distension. This distension will dissipate on its own during periods of quiet breathing or sleep, but for some girls can be extremely distressful. Various approaches have been attempted to mitigate the irregular breathing with variable success. Naltrexone or magnesium citrate has been found to be effective in some individuals. Naltrexone has been utilized in doses ranging from 1 to 3mg/kg/day. In our own study, we were limited to 1mg/kg/day. In this study, it appeared that naltrexone was exerting its effect simply by producing sedation.[36] The utilization of any particular agent has not been promoted in the absence of systematic studies showing efficacy. Gastrointestinal Issues Gastrointestinal issues in RS may represent the single most vexing medical issue, covering the gamut from top to bottom.[37] Chewing and swallowing are often performed poorly with evidence on imaging studies of poor coordination in the movement of food from oropharynx to the hypopharynx and dysmotility in transiting the esophagus. Choking is a common complaint, particularly on thin liquids, often requiring the use of thickening agents to obviate possible aspiration. If laryngeal penetration is observed on swallowing or upper gastrointestinal imaging studies, caution should be given to continuing oral feeding (see later). Conversely, dry foods may require moistening with liquids, such as sauces or gravies. Gastroesophageal reflux is also common and can be confirmed by swallowing and upper gastrointestinal or pH probe studies. In some instances, gastroesophageal reflux rises to the larynx. As such, it is important to investigate both primary and secondary aspirations. Antireflux medications should be utilized as needed, to inhibit acid production and promote gastric emptying. In the absence of treatment, one runs the risk of developing a chronic or persistent esophagitis, Barrett's esophagus and secondary esophageal changes, including cancer, ulceration or stricture. Gastroesophageal reflux may be enhanced by delayed gastric emptying, for which the clinician should be alert and may wish to evaluate with radionuclide study. Constipation is also a significant problem, further reflecting the poor motility of the intestinal tract. In some instances, stool retention will produce marked enlargement of the colon. In order to prevent the adverse consequences of constipation, a variety of strategies can be employed including GlycoLax (also marketed as MiraLAX® or Milk of Magnesia®) increased fiber in the diet and adequate fluid intake. This should ensure that a bowel movement occurs every day or two. Some parents have complained that the generic form produces excessive gas. Finally, gallbladder disease, gallstones or reduced function, appears to be another consideration in RS, related to reduced motility and delayed emptying of the gallbladder. Gallbladder disease has been recognized in the pediatric age range, as well as in adulthood. Of those with gallbladder disease known to IRSA, nearly 40% are under the age of 20 years. In some instances, abdominal ultrasound fails to identify gallbladder disease, suggesting that a hepatobiliary iminodiacetic acid (HIDA) scan should be considered when other explanations for abdominal discomfort are lacking. In summary, any period of inconsolable crying, apparent distress, night-time awakenings or undue irritability should provoke a thorough evaluation of gastrointestinal function to c-onsider the problems noted previously. Nutrition Assuring adequate nutrition in RS is critical. It does appear that girls with RS have a well-above average daily protein and calorie intake requirement.[38] As such, particular attention must be given to meeting nutritional requirements and, if necessary, to providing high-calorie nutritional supplements or frequent snacks, including lower cost supplements, such as Carnation Instant Breakfast™, and milkshakes with additional nutritional ingredients. Appropriate vitamin supplementation should be provided. However, one should be aware that nutritional supplements are so enriched in vitamins that excessive vitamin intake must be avoided. As girls with RS tend to be small in stature, it is appropriate to monitor BMI as an indicator of nutritional well-being (BMI is calculated by multiplying the weight in kg by 10,000 and dividing by the square of the patient's height in cm). When adequate calories cannot be taken by mouth or primary aspiration prevents oral feeding, gastrostomy or gastrojejunostomy feedings are required. Depending on the presence of gastroesophageal reflux, a fundoplication may be required at the same time. Any girl who has failed to gain weight for 6 months to 1 year, or is losing weight, should be considered for this alternative feeding method. Osteopenia Osteopenia occurs quite commonly in RS, being more significant in those girls or women who have inadequate calorie and protein intake.[29] Even in those who have adequate protein calorie intake, osteopenia is present, but to a lesser degree. Owing to the frequency and extent of osteopenia, fractures are much more common.[29] As such, the possibility of a fracture should be considered if use of an extremity ceases suddenly despite the absence of complaints. Fractures often go undetected because of the girls' inability to express and localize pain. Regardless of age, consideration should be given to oral calcium supplementation, beginning in childhood based on recommended daily amounts (preadolescents: 800mg; adolescents: 1300mg; adults: 1000mg and postmenopausal women: 1500mg). Systematic studies on the occurrence of, and possible therapies for, osteopenia are ongoing at the Baylor College of Medicine (TX, USA) under the direction of Kathleen Motil. Scoliosis Scoliosis occurs with increasing frequency in RS, beginning as early as age 5 years and occurring in at least 80% of patients by the age of maturity.[28] Scoliosis tends to be more significant in those girls who are not ambulatory. Scoliosis often progresses dramatically and at curvatures above 40° corrective surgery should be considered. At lesser degrees of curvature, body jackets have been employed. No systematic studies have been performed to judge their efficacy. However, they remain an option in retarding the progression of scoliosis. Regardless of ambulation status, proper upright positioning is critical, including lateral supports for those in wheelchairs. Once girls with RS have matured and are through puberty, further progression of scoliosis is generally considered to be unlikely. Ambulation Up to 80% of girls with RS acquire the ability to walk (Percy AK, personal observations from the UAB database). During the course of their developmental regression, approximately a quarter of this group will lose this ability. Overall, approximately 60% remain ambulatory.[25] Toe walking is common, as well as abnormal foot postures in which case ankle/foot orthoses or other orthotic devices should be implemented. Significant effort should be allocated to maintaining ambulation as long as possible. In those girls who do not walk, standing frames should be employed both at home and at school along with parallel bars to promote supported ambulation. Sexual Maturation & Gynecologic Issues As adolescence approaches, puberty should be anticipated. Ordinarily, girls with RS will enter puberty at similar ages to their peers (Percy AK, personal observations from the UAB database). As these young women are quite vulnerable, appropriate consideration should be given to protecting them from unwarranted contact. Menstrual cycles generally occur with predictable regularity after they have become established (Percy AK, personal observations from the UAB database). Parents or other responsible caregivers may choose to deal with these in the usual fashion or may choose one of a variety of strategies to eliminate or minimize menstrual flow. These include Depo-Provera, birth-control pills, or endometrial oblation. Depo-Provera is not recommended, primarily because it adversely affects bone mineralization, but also because of its long period of action should other side effects arise. The use of birth-control pills is common and completely acceptable. Formulations of levonorgestrel/ethinyl estradiol provide menstrual management for 1 year (marketed as Lybrel®) or for 90 days (marketed as Seasonale® or Seasonique®), such that menstrual flow can be effectively eliminated or minimized. When unexplained irritability or inconsolable crying occurs, consideration should be given to a possible gynecologic issue. An abdominal ultrasound, as well as consultation with a gynecologist, should be considered strongly. Cardiac & Autonomic Systems A prolonged QT interval and ST-segment changes may be observed with increasing frequency through the age range.[30,32] As such, at the time of diagnosis, an ECG is recommended to assess QTc or other significant abnormality. If normal, repeating the cardiogram in 2–3 years is appropriate. If abnormal, referral to a cardiologist for consideration of an appropriate intervention strategy is indicated. Prolonged QTc is a quite treatable problem with medication and should not be overlooked. Should a QT-segment abnormality be identified, it would be appropriate to have other members of the patient's family screened in the event this finding is unrelated to the RS. The hands and feet tend to be cool to cold in RS. This is more typically noted in the lower extremities and may consist not only of cold but discolored feet, ranging from red and mottled to purple, and extending well up on the lower extremity. This finding is believed to be related to excess sympathetic nervous system tone. No specific intervention is available, but it is important to keep the hands and feet appropriately warm. Bruxism Bruxism (teeth grinding) occurs in virtually all girls at some point in time, varying in frequency and intensity, and exacerbated by anxiety, excitement or stressful situations. The sound created is really quite characteristic, having been described by Bengt Hagberg as that of slowly uncorking a bottle of wine. Efforts to reduce the teeth grinding are generally unrewarding. Over time, they diminish, in many actually disappearing after school age. Other Motor System Functions Hypotonia is the rule early in RS. Over time, muscle tone may be increased, representing rigidity rather than spasticity (upper motor neuron signs are generally absent). During this period, motor activities, including hand stereotypies and ambulation persist when present, but their speed slows down. Other disorders of movement occur, including tremor, myoclonus and choreiform activities.[39] Tremor is particularly apparent upon awakening from sleep or a nap, or when placed in an unstable position, such as upright on their feet or on the edge of a chair or lap. Myoclonus may be focal or multifocal and can also be exacerbated by excitement or anxiety. Focal or multifocal dystonia, especially at the ankles and feet, and at the wrists and hands, is common, particularly with advancing age. Without proper alignment, contractures can develop at the involved joints, particularly when sitting in a wheelchair for long periods of time without effective mobilization. In those girls who tend to keep their hands together in the midline at the chest level, contractures at the elbows and, to some extent, limitation of motion at the shoulders will develop. Ankle–foot orthoses or elbow splinting may reduce the risk for development of contractures. Botulinum toxin may be effective in all-eviating limitations of joint movement. Longevity Unlike the original suggestion that RS is a progressive neurodegenerative disorder, it is now understood to be a neurodevelopmental disorder, with prolonged survival likely. Current intervention strategies are much more aggressive than 20–30 years ago. This alone would be expected to lead to greater longevity. We are aware of many women that have survived into their 40s and 50s, and expect that with proper nutrition and medical care, prolonged survival is indeed likely to be the rule. Few systematic studies of survival have been conducted. A prior unpublished study conducted while the author was at Baylor noted that survival through age 10 years was normal, whereas survival through to age 35 years was approximately 70% of the normal female population. More recently, preliminary information from a cohort of more than 1900 US and Canadian females indicates at least 80% survival to the age of 20 years and 50% survival for those over 50 years of age (Percy AK, Glaze DG, Kozinetz C, del Junco D, unpublished observations). These data are similar to those recently reported from Australia.[6] The consequence of prolonged survival is that, in many cases, parents or other caregivers will themselves be elderly and due consideration must be given to long-term care of these women when their parents are no longer able to provide it. Sudden death has also been described in RS. In most cases the actual cause is unclear, but may well involve autonomic dysfunction or a cardiac conduction system abnormality, such as prolonged QT. Clinical Research Current clinical research is focused on refining the clinical aspects of RS, including its natural history and quality of life, both for the participants and their principal care providers, and on developing effective intervention strategies. The current US Clinical Research Consortium has three sites: Baylor College of Medicine, Greenwood Genetic Center in Greenwood (SC), the University of Alabama at Birmingham (AL) and a data coordinating site at the University of South Florida (FL) that ma-intains a public website.[101] Molecular Neurobiology Dramatic advances in our understanding of the neurobiologic aspects have occurred over the past few years. These have emanated in large part from molecular neurobiologic studies of MECP2 in mouse models for RS. Studies in a knockin mouse model demonstrated abnormal social behaviors and poor spatial and contextual fear memory.[3] Similar findings were reported from a knockout mouse model.[40] Knockout mice were shown to have reduced levels of brain-derived neurotrophic factor (BDNF), and mice lacking BDNF had features similar to Mecp2 knockout mice.[41,42] When BDNF was overexpressed in these mice, onset of Rett-like behaviors was delayed, but brain weight did not increase. Regulation of breathing is a key abnormality in RS. Reduced levels of norepinephrine and serotonin were observed in the brainstem (medulla) respiratory control pathways in knockout mice and supplementation of norepinephrine stabilized the respiratory control network.[43] Subsequent studies demonstrated that desipramine, an inhibitor of norepinephrine reuptake, improved breathing abnormalities substantially, prolonged survival by several weeks and increased the number of tyrosine hydroxylase-containing neurons in the brainstem.[44] Studies on the longitudinal expression pattern of MECP2 in cortical neurons from female knockout mice suggest a change in X-chromosome inactivation status with increasing age, that is, cells that express the wild-type protein increased from 50 to 70%. One might speculate that this change can be related to the pattern of stabilization and improved interaction in older girls.[45] Very recent results suggest a neuroendocrine component to RS. Studies in a knockin mouse model revealed increased corticosterone release and Crh expression, not only in the hypothalamus, but also in the amygdala and stria terminalis.[4] Furthermore, the authors showed that MECP2 binds to the Crh promoter and may regulate its expression. Mutant MECP2 does not bind to this promoter. The amygdala has important downstream connections to autonomic system nuclei subserving gastrointestinal, respiratory and cardiovascular functions. As anxiety and fear are common clinical features in RS, these results provide a potential therapeutic avenue. Studies in human tissues revealed abnormal developmental progression of serotonin-transporter binding in the brainstem from individuals with RS with increasing age, compared with controls, providing further evidence of dysregulation of gastrointestinal and cardiac responses.[46] Along the same lines, MECP2 also regulates IGFBP3 [47] and AVP in brain tissue.[48] Therapeutic Intervention Individuals with RS require a variety of therapies throughout their lives.[49] These include physical and occupational therapies to promote sitting, standing, ambulation and fine motor functions. Wherever necessary, standing frames and devices that assist with walking should be employed. In some cases, adaptive equipment, such as tricycles, may be considered and some girls may use small trampolines or treadmills. Hippotherapy and swimming are extremely popular and well tolerated by most individuals with RS. In terms of fine motor skills, goals in therapy must be realistic and, when improvements or advancements do occur, it often requires daily repetition to preserve them. As they mature, individuals with RS give intense eye contact, which may be employed to develop communication skills, such as choice making, using simple picture boards or sophisticated computer-based technologies. Some girls are able to use their hands to activate switches; others can use ocular devices to ac-tivate switches. These opportunities to make choices in all aspects of their lives, ranging from eating, to socializing, to types of entertainment, should be maximized. Music seems to be particularly appealing, with each girl or woman developing her own preference panel probably based on what music is played within the home. Emerging Therapeutic Strategies The complexities of gene therapy coupled with the specific requirement to correct only cells with abnormalities in MECP2 render gene-based therapies for RS problematic at this time. One promising strategy is to identify pharmacologic agents capable of correcting so-called 'stop' (nonsense) mutations by reading through the premature stop codon to produce a full-length protein. Efforts are currently underway to develop such agents. This strategy is particularly attractive in that approximately a third of MECP2 mutations in RS are the nonsense type. Alternatively, therapies directed at known MECP2 gene targets, as noted previously for the gene encoding Crh, suggest another avenue for therapy. Agents that would directly downregulate Crh might offer the possibility to intervene fundamentally in Crh-related systems and, in particular, to modify anxiety and fear that are quite prominent in individuals with RS. Efforts to identify or develop these agents should be considered. Alternatively, an indirect approach would be to employ available pharmacologic agents, such as the serotonin-reuptake inhibitor class of drugs, to address the core problems of anxiety and fear or the norepinephrine reuptake inhibitors to address p-eriodic breathing patterns. The previously noted abnormal development of serotonin-transporter binding in postmortem samples of brainstem nuclei from girls or women with RS[46] and the mouse model studies showing beneficial effects of desipramine, provide additional support for establishing clinical trials of serotonin and/or norepinephrine reuptake inhibitors in order to address the significant dysregulation of gastrointestinal and respiratory function. Expert Commentary & Five-year View The pace of advancement in understanding the clinical and basic science aspects of RS since the identification of mutations in the MECP2 gene in 1999 has been extraordinary. To date, studies have expanded the severity range of clinical involvement associated with such mutations to encompass not only RS, but also learning disabilities and autism on the one hand, and severe encephalopathy on the other, and have advanced clinical interventions dramatically such that the long-term outlook is very different today. Together with recent studies aimed at identifying the fundamental role of MECP2 in developmental neurobiology and the proof-of-principle studies in the mouse model regarding reversibility, clinical and basic science investigators seem poised for even more dramatic advances in the coming 5 years. During this period, the molecular targets of MECP2 will be more fully explained, enhancing possibilities for developing specific therapies. Published: 07/11/2008 Source: http://www.medscape.com/viewarticle/576129_13

The Story of Rett Syndrome: From Clinic to Neurobiology: An Interesting review article by Dr. Maria Chahrour and Dr. Huda Y. Zoghbi

Introduction Two severely disabled young girls were relentlessly wringing their hands as they sat in the laps of their respective mothers in the waiting room of a Vienna pediatric clinic. This coincidental occurrence prompted Dr. Andreas Rett to search for more patients with the same unusual behavior. A year later, in 1966, Dr. Rett described similar findings in 22 patients, reporting for the first time a unique clinical entity that now bears his name (Rett, 1966). But it would not be until 17 years later that Rett syndrome (RTT) became recognized in the medical community when Dr. Bengt Hagberg, a Swedish neurologist, and his colleagues reported 35 cases of RTT in the English language (Hagberg et al., 1983), and recognized this clinical entity as a condition “similar to a virtually overlooked syndrome described by Rett in the German literature.” In addition to the worldwide recognition of RTT, the 1980s witnessed major strides in another field, namely DNA methylation. For the first time, a connection was established between DNA methylation and heritable changes in gene expression. Scientists identified the CpG dinucleotide to be the site of almost all DNA methylation in mammalian genomes and began to explore the effects of this modification on gene activity. CpG islands were found to be unmethylated in the native state (with the exception of sequences on the inactive X chromosome), and methylation of CpGs was associated with gene silencing and alterations in chromatin structure. Two mechanisms have been demonstrated for DNA methylation-mediated gene repression. The first suggests that methylation of CpG sites within gene promoters will inhibit sequence-specific binding of transcription factors. In the second, more prevalent mechanism, the repression is mediated by proteins that specifically bind to methylated CpGs (methyl-CpG binding proteins) and alter chromatin structure, rendering it inaccessible to members of the transcription machinery. In 1992, Dr. Adrian Bird and his colleagues identified a novel mammalian protein that binds methylated CpGs, methyl-CpG binding protein 2 (MeCP2) (Lewis et al., 1992). The gene encoding MeCP2 was localized to the X chromosome in the mouse (Quaderi et al., 1994), and the protein repressed transcription in vitro (Nan et al., 1997). Soon afterwards, additional members of the methyl-CpG binding protein family were identified and included methyl-CpG binding domain proteins (MBD) 1–4 (Hendrich and Bird, 1998). Meanwhile, as the DNA methylation field was deciphering repression mechanisms mediated by methyl-CpG binding proteins, the RTT community was trying to understand the pathophysiology of this puzzling condition. The turning point for RTT research came with the discovery of its genetic basis in 1999. The surprising finding that mutations in the widely expressed MECP2 are at the root of RTT (Amir et al., 1999) brought together the fields of epigenetics and neurobiology. Clinical Features of RTT RTT (MIM 312750) is a postnatal progressive neurodevelopmental disorder that manifests in girls during early childhood; however, not all the symptoms are prominent initially, but rather appear over stages (Figure 1). Patients with RTT appear to develop normally up to 6–18 months of age. The child with RTT seemingly achieves appropriate milestones, including the ability to walk, and some patients even say a few words. One early indicator of neurological involvement is deceleration of head growth, leading to microcephaly by the second year of life. With the onset of developmental stagnation, the acquired microcephaly is accompanied by general growth retardation, weight loss, and a weak posture brought on by muscle hypotonia. As the syndrome progresses, patients lose purposeful use of their hands and instead develop stereotypic hand wringing or washing movements, and in some cases clapping, flapping, and mouthing of the hands. Social withdrawal and loss of language become apparent in addition to irritability and self-abusive behavior. Other autistic features also manifest, including expressionless face, hypersensitivity to sound, lack of eye-to-eye contact, indifference to the surrounding environment, and unresponsiveness to social cues (Nomura, 2005). The onset of mental deterioration is accompanied by loss of motor coordination and the development of ataxia and gait apraxia. The earliest autonomic perturbation is hyperventilation during wakefulness. Most girls with RTT suffer additional breathing anomalies, including breath-holding, aerophagia, forced expulsion of air and saliva, and apnea. One of the most arduous features of RTT is the occurrence of seizures, which range from easily controlled to intractable epilepsy, with the most common types being partial complex and tonic-clonic seizures (Jian et al., 2006). The seizures tend to decrease in severity after the teenage years and into adulthood, presenting minor problems after the age of forty. Amelioration of the social component of the autistic-like behavior occurs sometime between 5 to 10 years of age. Despite having a normal appetite, patients continue to lose weight and many suffer from osteopenia, scoliosis, and rigidity as they age. Behavioral abnormalities during this postregression phase include teeth grinding, night laughing or crying, screaming fits, low mood, and anxiety episodes elicited by distressful external events (Mount et al., 2001). Patients suffer devastating motor deterioration, generalized rigidity, dystonia, and worsening of scoliosis. Most girls with RTT lose mobility, and are often wheelchair-bound during the teenage years. Additional autonomic abnormalities include hypotrophic, cold blue feet, severe constipation, oropharyngeal dysfunction, and cardiac abnormalities, including tachycardia, prolonged corrected QT intervals, and sinus bradycardia. As patients get older they often develop Parkinsonian features ([Hagberg, 2005] and [Roze et al., 2007]). The condition reaches a plateau and some patients survive up to the sixth or seventh decade of life in a severely debilitated physical condition. After a period of normal development, a healthy-looking baby girl falls into developmental stagnation, followed by rapid deterioration, loss of acquired speech, and the replacement of purposeful use of the hands with incessant stereotypies, a characteristic of the syndrome. Patients also develop social behavior abnormalities and are often misdiagnosed as having autism. The condition worsens with loss of motor skills and profound cognitive impairment. In addition, patients suffer from anxiety, seizures, and a host of autonomic abnormalities. Genetic Basis of RTT Given that the vast majority of patients with RTT are females, early reports postulated an X-linked dominant mode of inheritance with fateful consequences in hemizygous males. RTT has an incidence of 1/10,000 female live births. However, since more than 99% of RTT cases are sporadic, it was very hard to map the disease locus by traditional linkage analysis. Using information from rare familial cases, exclusion mapping identified the Xq28 candidate region, and subsequent screening of candidate genes in RTT patients revealed mutations in MECP2 (Amir et al., 1999). Mutations in MECP2 are found in more than 95% of classic RTT cases; most arise de novo in the paternal germline and often involve a C to T transition at CpG dinucleotides ([Trappe et al., 2001] and [Wan et al., 1999]). The spectrum of mutation types includes missense, nonsense, and frameshift mutations, with over 300 unique pathogenic nucleotide changes described (Christodoulou et al., 2003), as well as deletions encompassing whole exons ([Archer et al., 2006b], [Pan et al., 2006] and [Ravn et al., 2005]). Eight missense and nonsense mutations account for 70% of all mutations, while small C-terminal deletions account for another 10%, and complex rearrangements constitute 6%. Several phenotype-genotype correlation studies have been reported and some general conclusions can be made. Mutations affecting the NLS of MeCP2 or early truncating mutations tend to cause more severe phenotypes than missense mutations, whereas C-terminal deletions are associated with milder phenotypes (Smeets et al., 2005). In addition, the R133C mutation causes an overall milder phenotype ([Kerr et al., 2006], [Leonard et al., 2003] and [Neul et al., 2007]), while the R270X mutation is associated with increased mortality (Bienvenu and Chelly, 2006). Because several mutations introduce premature stop codons throughout the gene and are predicted to result in a null allele, RTT results from loss of MeCP2 function. However, hypomorphic alleles may also occur and result, for instance, in truncated forms of MeCP2 that retain partial function. Phenotypic Variability in RTT Atypical forms of RTT that deviate from the classical clinical presentation occur. These variants range from milder forms with a later age of onset to more severe manifestations. Of the milder variants, the “forme fruste” (or “worn-down form”) has a later age of onset compared with the classical form, with regression occurring between 1 to 3 years of age; hand use is sometimes preserved with minimal stereotypic movements. The preserved speech variant is another benign form of RTT characterized by the ability of patients to speak a few words, although not necessarily in context. Patients with this variant have a normal head size and are usually overweight and kyphotic (Zappella et al., 2001). The more severe variants include the congenital form that lacks the early period of normal development, and a form of classical RTT with onset of seizures before the age of 6 months. Classical and atypical RTT phenotypes vary in severity and onset between different patients and in the same patient over time. A major source of the phenotypic variability associated with different MECP2 mutations in females is the pattern of X chromosome inactivation (XCI). In females, only one of the two X chromosomes is active in each cell and the choice of which X chromosome is active is usually random, such that half of the cells have the maternal X chromosome active and the other half have the paternal X chromosome active. Therefore, a female with a MECP2 mutation is typically mosaic, whereby half of her cells express the wild-type MECP2 allele and the other half express the mutant MECP2 allele. Occasionally, cells expressing the wild-type MECP2 allele divide faster or survive better than cells expressing the mutant allele, which therefore results in a nonrandom pattern of XCI and amelioration of the RTT neurological phenotypes. Depending on the extent of such favorable skewing, some patients can be mildly affected or are even asymptomatic carriers of MECP2 mutations. The latter cases are usually identified because of the occurrence of RTT in their offspring. The best examples for illustrating the dramatic effects of XCI patterns in RTT are monozygotic twins who manifest very different phenotypes (Dragich et al., 2000). In addition, skewed XCI patterns occur in brain regions of female mice heterozygous for a mutant MECP2 allele, where phenotypic severity correlates with the degree of skewing (Young and Zoghbi, 2004). Finally, somatic mosaicism in females with MECP2 mutations is another rare but important source of phenotypic variability (Bourdon et al., 2001). MECP2 Mutations in Males Although RTT was initially thought to occur exclusively in females, this dogma was broken with the identification of males presenting with classic RTT (Jan et al., 1999). The discovery of the disease-causing gene led to mutation screening in males with related neurological phenotypes and revealed that MECP2 mutations in males cause a host of neurological disorders, ranging from mental retardation to severe encephalopathy. MECP2 mutations that cause classic RTT in females typically lead to neonatal encephalopathy and death in the first year of life in males with a normal karyotype. The same mutations, however, result in an RTT phenotype in males with Klinefelter syndrome (47,XXY) or somatic mosaicism. In addition, some of these mutations have been reported in males with classic RTT and a normal karyotype, suggesting that genetic modifiers suppress the infantile lethality typically seen with loss-of-function MECP2 mutations ([Budden et al., 2005], [Dayer et al., 2007], [Maiwald et al., 2002] and [Masuyama et al., 2005]). Lastly, some MECP2 mutations that do not cause RTT in females can cause moderate, nonspecific to profound mental retardation or psychiatric disorders in males. Deficits in language and motor skills, obesity, autistic features, and epilepsy are also common in this category of mutations. CDKL5 Mutations Explain a Congenital RTT-like Disorder Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) were identified in patients with a rare congenital disorder with intractable early-onset seizures often accompanied by RTT-like features. Alterations in this gene were originally found to cause early-onset epilepsy and infantile spasms with severe mental retardation (Grosso et al., 2007). Although the majority of patients with CDKL5 mutations are female, there are rare occurrences in males suffering from severe mental retardation, infantile spasms, or early-onset epilepsy. The protein product of CDKL5, previously known as serine/threonine kinase 9 (STK9), autophosphorylates itself and phosphorylates MeCP2 in vitro, and this latter activity is eliminated in pathogenic CDKL5 mutants (Bertani et al., 2006). Disruption of NTNG1, which encodes the axon guiding molecule Netrin G1, was described in one female patient with atypical RTT and early-onset seizures (Borg et al., 2005). However, this may prove to be an isolated case because NTNG1 screening in a cohort of MECP2 and CDKL5 mutation-negative patients with RTT failed to identify any pathogenic mutations in this gene (Archer et al., 2006a). The MECP2 Duplication Disorder Perhaps one of the more intriguing findings regarding RTT and MeCP2 is the fact that loss of function and gain in MECP2 dosage result in clinically similar neurological disorders. In fact, duplications of Xq28 that span the MECP2 locus have been reported in males with progressive neurodevelopmental phenotypes. The patients suffer from mental retardation with facial and axial hypotonia, progressive spasticity, seizures, recurrent respiratory infections, and often premature death. In addition, autistic features and RTT phenotypes, including head growth deceleration, motor delay, ataxia, hand stereotypies, teeth grinding, and absence of speech, have been reported in these boys ([Friez et al., 2006], Lugtenberg et al., 2006 D. Lugtenberg, A.P. de Brouwer, T. Kleefstra, A.R. Oudakker, S.G. Frints, C.T. Schrander-Stumpel, J.P. Fryns, L.R. Jensen, J. Chelly and C. Moraine et al., Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH, J. Med. Genet. 43 (2006), pp. 362–370. View Record in Scopus | Cited By in Scopus (37)[Lugtenberg et al., 2006], [Meins et al., 2005] and [Van Esch et al., 2005]). A male patient with a triplication of the locus was also described with a worse early-onset neurological phenotype (del Gaudio et al., 2006). Only one duplication case has been identified in females, in a patient with the preserved speech variant of RTT (Ariani et al., 2004). Studies of transgenic mice that express wild-type MECP2 at twice the normal level reveal that doubling the dosage of this protein leads to a progressive neurological phenotype similar to that observed in human patients (Collins et al., 2004). These observations, together with the finding that MECP2 is the only common gene shared among all patients with the duplication syndrome, give credence to the notion that MECP2 is the gene within the 400 kb duplicated region that is responsible for these phenotypes. MECP2 Mutations in Other Neuropsychiatric Disorders Disruptions in MECP2 result in a host of neuropsychiatric disorders, of which classic and variant forms of RTT represent only one chapter (Table 1). On the milder side of the scale are females with mild mental retardation, learning disabilities, and autism spectrum disorders ([Carney et al., 2003] and [Lam et al., 2000]). MECP2 mutations can also cause severe mental retardation with epilepsy and Angelman-like syndrome in females ([Milani et al., 2005] and [Watson et al., 2001]). The spectrum of MECP2 disorders worsens with complex forms of severe mental retardation in males that are associated with epilepsy, ataxia, tremor, hyperactivity, autism, and bipolar disease (Klauck et al., 2002) or juvenile-onset schizophrenia (Cohen et al., 2002). In addition, MECP2 mutations were identified in males with mental retardation, psychosis, pyramidal signs, Parkinsonian features, and macroorchidism (PPM-X) (Klauck et al., 2002). The full spectrum of phenotypes in MECP2 disorders is far from being understood. Mutations that are predicted to result in a null allele suggest that the neurodevelopmental abnormalities are a result of MeCP2 loss of function. However, the MECP2 duplication disorder demonstrates that an increase in protein levels can be equally detrimental to the nervous system. Studies in loss-of-function and duplication models are beginning to provide insight on how variations in the level of this protein lead to neuronal dysfunction (see below). MeCP2 Expression and Functions MECP2 consists of four exons that code for two different isoforms of the protein, due to alternative splicing of exon 2 (Figure 2). The MeCP2 splice variants differ only in their N-termini; the more abundant MeCP2-e1 isoform (encoded by MECP2α) contains 24 amino acids encoded by exon 1 and lacks the 9 amino acids encoded by exon 2, whereas the start site for the MeCP2-e2 isoform (encoded by MECP2β) is in exon 2 ([Dragich et al., 2007], [Kriaucionis and Bird, 2004] and [Mnatzakanian et al., 2004]). In addition, MECP2 has a large, highly conserved 3′-untranslated region that contains multiple polyadenylation sites, which can be alternatively used to generate four different transcripts. Expression studies in mice showed that the longest transcript is the most abundant in brain, with higher expression during embryonic development, followed by postnatal decline, and subsequent increase in expression levels later in adult life ([Pelka et al., 2005] and [Shahbazian et al., 2002b]). These different MECP2 transcripts could be subject to tissue-specific and developmental stage-specific regulation. Although the MeCP2 protein is widely expressed, it is relatively more abundant in the brain, primarily in mature postmigratory neurons (Jung et al., 2003). MeCP2 protein levels are low during embryogenesis and increase progressively during the postnatal period of neuronal maturation ([Balmer et al., 2003], [Cohen et al., 2003], [Kishi and Macklis, 2004], [Mullaney et al., 2004] and [Shahbazian et al., 2002b]). The pattern of increasing expression in the cortex follows an inner-to-outer sequence akin to that of cortical development. In the olfactory epithelium, MeCP2 expression coincides with maturation of the olfactory receptor neurons (ORNs) and precedes the onset of synaptogenesis (Cohen et al., 2003). Both MeCP2 isoforms are nuclear and colocalize with methylated heterochromatic foci in mouse cells. A recent report suggests that MeCP2 translocates to the nucleus upon neuronal differentiation (Miyake and Nagai, 2007). Since MeCP2 is expressed in mature neurons and its levels increase during postnatal development, MeCP2 may play a role in modulating the activity or plasticity of mature neurons. Consistent with this, MECP2 mutations do not seem to affect the proliferation or differentiation of neuronal precursors. Although the mechanisms that regulate the complex MECP2 expression patterns are unknown, a recent study identified the core promoter and several cis-regulatory elements that drive MECP2 expression (Liu and Francke, 2006). These regulatory sequences may dictate the spatial and temporal patterns of MECP2 expression. MeCP2 is a member of the methyl-CpG binding protein family (Hendrich and Bird, 1998), and is composed of three domains: the MBD, the transcriptional repression domain (TRD), and a C-terminal domain, in addition to two nuclear localization signals (NLS). The MBD specifically binds to methylated CpG dinucleotides, with preference for CpG sequences with adjacent A/T-rich motifs (Klose et al., 2005). MBD also binds to unmethylated four-way DNA junctions with a similar affinity (Galvao and Thomas, 2005), implicating a role for the MeCP2 MBD in higher-order chromatin interactions. The more downstream TRD is involved in transcriptional repression through recruitment of corepressors and chromatin remodeling complexes. The C terminus facilitates MeCP2 binding to naked DNA and to the nucleosomal core, and it also contains evolutionarily conserved poly-proline runs that can bind to group II WW domain splicing factors (Buschdorf and Stratling, 2004). Although the C-terminal region of MeCP2 is not yet well characterized, it is clearly essential for protein function as evidenced by the numerous RTT-causing mutations that involve deletion of this domain, and the fact that a mouse model lacking the MeCP2 C terminus reproduces many RTT phenotypes (Shahbazian et al., 2002a). The function of MeCP2 as a transcriptional repressor was first suggested based on in vitro experiments in which MeCP2 specifically inhibited transcription from methylated promoters (Nan et al., 1997). When MeCP2 binds to methylated CpG dinucleotides of target genes via its MBD, its TRD recruits the corepressor Sin3A and histone deacetylases (HDACs) 1 and 2 ([Jones et al., 1998] and [Nan et al., 1998]). The transcriptional repressor activity of MeCP2 involves compaction of chromatin by promoting nucleosome clustering, either through recruitment of HDAC and histone deacetylation (Figure 3A) or through direct interaction between its C-terminal domain and chromatin (Nikitina et al., 2007). In addition, the interaction with Sin3A is not stable and appears to be dependent on MeCP2 being DNA-bound (Klose and Bird, 2004). This suggests that Sin3A is not an exclusive partner of MeCP2 and that other factors may interact with MeCP2 to modulate gene expression or other unknown functions. Additional MeCP2-interacting proteins include the catalytic component of the SWI/SNF chromatin-remodeling complex Brahma (at least in NIH 3T3 cells), the DNA methyltransferase DNMT1, the histone methyltransferase Suv39H1, the transcription factors TFIIB and PU.1, the corepressors c-Ski and N-CoR, LANA, and the SWI2/SNF2 DNA helicase/ATPase responsible for α-thalassemia/mental retardation syndrome X-linked (ATRX) (Harikrishnan et al., 2005 K.N. Harikrishnan, M.Z. Chow, E.K. Baker, S. Pal, S. Bassal, D. Brasacchio, L. Wang, J.M. Craig, P.L. Jones, S. Sif and A. El-Osta, Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing, Nat. Genet. 37 (2005), pp. 254–264. View Record in Scopus | Cited By in Scopus (92)[Harikrishnan et al., 2005], [Kaludov and Wolffe, 2000], [Kimura and Shiota, 2003], [Kokura et al., 2001] and [Nan et al., 2007]). However, the precise functional consequences of these protein-protein interactions remain unknown. To add to the complexity, MeCP2 also interacts with the RNA-binding protein Y box-binding protein 1 (YB1) to regulate splicing of reporter constructs (Figure 3B). The finding of aberrant RNA splicing patterns in a Mecp2 mutant mouse model of RTT (Young et al., 2005) together with the fact that MeCP2 can form complexes with RNA in vitro independently of its MBD (Jeffery and Nakielny, 2004) suggest that MeCP2 might modulate RNA splicing in vivo. Given that MeCP2 interacts with other proteins, chromatin, DNA, and RNA, it is clearly a multifunctional protein, with roles in chromatin remodeling and RNA splicing. Modeling RTT and Related Disorders in Mice To uncover the molecular changes that underlie RTT, three mouse models with different MeCP2 mutations were generated (Table 2). Mecp2 conditional knockout mice, lacking either exon 3 or both exons 3 and 4 ([Chen et al., 2001] and [Guy et al., 2001]), undergo a period of normal development, followed by severe progressive neurological dysfunction, leading to death at 8–10 weeks of age. Female Mecp2+/− mice have behavioral abnormalities as well, but with a later age of onset. Moreover, an embryonic Mecp2 deletion only in neurons, using a nestin-Cre transgene, results in a phenotype resembling the ubiquitous knockout, demonstrating that MeCP2 dysfunction in the brain is sufficient to cause the disease (Guy et al., 2001). Mecp2 null mice display no initial phenotype until 3–6 weeks of age, when male mutant mice develop a stiff, uncoordinated gait, hypoactivity, tremor, hindlimb clasping, and irregular breathing. Symptoms worsen and ultimately lead to weight loss and death by 10 weeks. The respiratory rhythm disturbances observed in the Mecp2 null mice have been attributed to alterations in bulbar postinspiratory discharges and to malfunction of the Kolliker-Fuse region of the pons that enhances postinspiratory activity and leads to the development of respiratory dysrhythmia and apnea (Stettner et al., 2007). The brains of Mecp2 null mice are smaller in size and weight than brains of wild-type littermates, but have no detectable structural abnormalities, except for smaller, more densely packed neurons. In addition, the olfactory neurons of Mecp2 null mice demonstrate abnormalities of axonal targeting in the olfactory bulb, suggesting a function for MeCP2 in terminal neuronal differentiation (Matarazzo et al., 2004). When Mecp2 is deleted in postmitotic neurons using a calcium-calmodulin-dependent protein kinase II (CaMKII)-Cre transgene, similar but less severe neurological phenotypes are observed with a later age of onset, confirming a critical role for MeCP2 in mature neurons. These mice display gait ataxia, increased anxiety, and impaired social behavior ([Chen et al., 2001] and [Gemelli et al., 2006]). In further support of this idea, when Mecp2 is expressed in postmitotic neurons of Mecp2 null mice under the control of the endogenous tau promoter, the neurological phenotypes of the knockout mice are rescued (Luikenhuis et al., 2004). These studies not only confirmed that neuronal MeCP2 dysfunction is responsible for the neurological phenotype, but also that MeCP2 is not very essential to early brain development. Recently, another Mecp2 null mouse model was reported in which exon 3 and part of exon 4 were deleted. The mice are hypoactive and display learning deficits and reduced anxiety. The same mutation was also generated on an XO background and resulted in similar phenotypes in Mecp2−/Y and Mecp2−/O mice, indicating that the Y-chromosome has no effect on the phenotypic manifestation in Mecp2 null mice (Pelka et al., 2006). Another RTT mouse model, generated by truncating MeCP2 at amino acid 308, results in a hypomorphic allele that retains the MBD, TRD, and NLS, and only eliminates the C-terminal region of the protein, similar to C-terminal deletions found in RTT patients (Shahbazian et al., 2002a). Mecp2308/Y mice appear normal until 6 weeks of age, when they develop progressive neurological phenotypes, including motor dysfunction, forepaw stereotypies, hypoactivity, tremor, seizures, kyphosis, social behavior abnormalities, decreased diurnal activity, increased anxiety-related behavior, and learning and memory deficits, reminiscent of the clinical picture in human girls with RTT. Female mice heterozygous for the truncation display milder and more variable features. In vivo, the truncated protein maintains normal chromatin localization, but histone H3 is hyperacetylated in the brain, indicating abnormal chromatin architecture ([McGill et al., 2006], [Moretti et al., 2005] and [Shahbazian et al., 2002a]). Two-fold overexpression of human MECP2 under the control of its endogenous promoter in mice (MECP2Tg) results in progressive neurological abnormalities with the onset of phenotypes around ten weeks of age. Initially MECP2Tg mice display increased synaptic plasticity, with enhancement in motor and contextual learning abilities. However, at 20 weeks of age, transgenic mice become hypoactive and develop forepaw clasping, aggressiveness, kyphosis, seizures, and motor abnormalities, and die by 1 year of age. In addition, higher levels of MeCP2 expression in other transgenic lines are associated with more severe phenotypes (Collins et al., 2004). Introducing a copy of the MECP2 transgene into Mecp2 null mice restores protein levels to normal and rescues the neurological abnormalities of both mouse models, indicating that phenotypes of the transgenic mice are indeed modulated by MeCP2 levels. Overexpression of MECP2 in adult neurons under control of the tau promoter also results in a progressive neurological syndrome. Studies of these mouse models suggest that MeCP2 levels must be tightly regulated even postnatally and that the slightest perturbation results in deleterious neurological consequences ([Collins et al., 2004] and [Luikenhuis et al., 2004]). The best illustration of the clinical relevance of such observations is the fact that duplication of the MECP2 locus in human male patients causes profound mental retardation and progressive neurodevelopmental disorders. Effects of MeCP2 Dysfunction on the Brain Neuropathological Changes Despite the profound neurological phenotype in RTT, the major morphological abnormalities detected in the central nervous system (CNS) are an overall decrease in the size of the brain and of individual neurons. Autopsy studies show a 12%–34% reduction in brain weight and volume in patients with RTT, the effect most pronounced in the prefrontal, posterior frontal, and anterior temporal regions (Armstrong, 2005). The RTT brain shows no obvious degeneration, atrophy, or inflammation, and there are no signs of gliosis or neuronal migration defects ([Jellinger et al., 1988] and [Reiss et al., 1993]). These observations indicate that RTT is a disorder of postnatal neurodevelopment rather than a neurodegenerative process. There is a decrease in the size of cortical minicolumns (Casanova et al., 2003), which could correlate with observations of reduced dendritic branching of layers III and V pyramidal neurons in the frontal, temporal and motor regions, and of layers II and IV of the subiculum (Armstrong, 2005). Neurons of hippocampal CA1 and occipital regions are relatively preserved (Armstrong et al., 1995). In addition, dendritic spines of the RTT frontal cortex are sparse and short, with no other apparent abnormalities (Belichenko et al., 1994). Although neuronal size is reduced in the cortex, thalamus, basal ganglia, amygdala, and hippocampus, there is an increase in neuronal cell packing in the hippocampus (Kaufmann and Moser, 2000). Hypopigmentation of the zona compacta of the substantia nigra and reduction in cell number in the nucleus basalis of Meynert have been reported (Kitt and Wilcox, 1995). Olfactory biopsies from RTT patients reveal that the mature ORNs are dysmorphic, and that there is a marked increase in the ratio of immature to mature ORNs (Ronnett et al., 2003). Delayed neuronal maturation and synaptogenesis were also detected in the Mecp2−/Y cerebral cortex (Fukuda et al., 2005). Neuropathological studies in both humans and mice were all performed well after the onset of symptoms. It would be interesting to determine if alterations exist in hippocampal or cortical neurons prior to the onset of symptoms, at least in the existing mouse models. Nonetheless, the results of neuropathological studies suggest a role for MeCP2 in neuronal maturation and maintenance. Additionally, a variety of neurochemical changes are documented in the cerebrospinal fluid and brain tissue of patients with RTT ([Blue et al., 1999], [Calamandrei et al., 2001], [Guideri et al., 2004], [Lipani et al., 2000], [Paterson et al., 2005], [Riikonen, 2003], [Saito et al., 2001], [Viola et al., 2007], [Wenk, 1997] and [Zoghbi et al., 1989]). However, it should be noted that the majority of these data result from small samples of postmortem tissue and none of these changes are diagnostic or occur in every patient. Neurophysiological Abnormalities Neurophysiological studies of patients with RTT suggest that both the CNS and the autonomic nervous system contribute to the pathophysiology of the disease. In RTT, the peripheral auditory and visual systems are normal, as demonstrated from evoked potential studies. Altered somatosensory evoked potentials and abnormal electroencephalogram (EEG) findings of focal, multifocal, and generalized epileptiform discharges, and the occurrence of rhythmic slow theta activity, all suggest altered cortical excitability in the RTT brain. However, the EEG patterns are not diagnostic of RTT, and they vary between patients and at different stages of the disorder (Moser et al., 2007). Electrocardiographic recordings demonstrate long corrected QT intervals and suggest perturbation of the autonomic nervous system (Glaze, 2005). Studies of Mecp2 mouse models reveal abnormalities in long-term potentiation (LTP) and impaired synaptic plasticity; LTP is reduced in Mecp2−/Y cortical slices (Asaka et al., 2006 Y. Asaka, D.G. Jugloff, L. Zhang, J.H. Eubanks and R.M. Fitzsimonds, Hippocampal synaptic plasticity is impaired in the Mecp2-null mouse model of Rett syndrome, Neurobiol. Dis. 21 (2006), pp. 217–227. Abstract | Article | PDF (360 K) | View Record in Scopus | Cited By in Scopus (38)Asaka et al., 2006) and in Mecp2308/Y cortical and hippocampal slices (Moretti et al., 2006). In contrast, LTP is enhanced with doubling of MeCP2 in Mecp2Tg1 hippocampal slices (Collins et al., 2004). In addition, loss of MeCP2 in mice results in reduced spontaneous activity in cortical slices due to a decrease in the total excitatory synaptic drive and an increase in the total inhibitory drive (Dani et al., 2005). Cultured hippocampal neurons from Mecp2 null mice have decreased frequency of spontaneous excitatory synaptic transmission, and an increase in short-term synaptic depression rate (Nelson et al., 2006). Chao et al. (2007) investigated the physiological consequences of either loss or doubling of MeCP2 on an individual neuron using autaptic cultured hippocampal neurons from Mecp2−/Y and MECP2Tg1 mice, and they found an approximately 50% reduction and 100% enhancement of synaptic outputs in glutamatergic Mecp2−/Y and MECP2Tg1 neurons, respectively. They went on to demonstrate that these physiological changes predominantly result from an altered number of glutamatergic synapses rather than dysfunction of the presynapse or postsynapse. Neurons from Mecp2−/Y mice have an approximately 40% reduction in VGLUT1-PSD95-positive puncta, whereas those from MECP2Tg1 mice have a 60% increase in such puncta in comparison with wild-type mice (Chao et al., 2007). The alterations in glutamatergic synapse number were observed both at the single-neuron level and in vivo during the early or “presymptomatic” disease stage (Chao et al., 2007). This finding is noteworthy in that it suggests that homeostatic compensatory changes must occur in response to altered synapse number. Altogether, these findings indicate that MeCP2 is essential in modulating synaptic function and plasticity, and that MeCP2 function is critical in regulating the number of excitatory synapses during early postnatal development. MeCP2 Target Genes and Relevance to RTT Pathogenesis Since RTT results from dysfunction of a putative transcriptional repressor, it is logical to pursue the transcriptional targets of MeCP2 to gain insight into disease pathogenesis. Transcriptional profiling studies using brain tissue from Mecp2 null mice did not reveal major gene expression changes, suggesting that MeCP2 may not be a global transcriptional repressor as previously thought, and that loss of MeCP2 must lead to subtle gene expression changes (Tudor et al., 2002). Alternatively, if MeCP2 regulates the expression of a select number of genes in different subsets of neurons, such transcriptional changes would be missed in samples of brain tissue containing a host of vastly different neuronal types. Several studies have used candidate gene approaches or samples from both human and mouse tissues and identified putative MeCP2 targets that might be relevant to the pathogenesis of RTT. Some of these targets have been confirmed while others yielded different results in different laboratories. A list of the targets that have been studied more extensively is shown in Table 3. In this section we will limit the discussion to a handful of targets that might provide insight about the function of MeCP2, RTT pathogenesis, or both. MeCP2 Regulates Gene Expression in an Activity-Dependent Manner Brain-derived neurotrophic factor (BDNF) was identified as a target of MeCP2 through a candidate gene approach ([Chen et al., 2003] and [Martinowich et al., 2003]). Rat Bdnf is under the control of four promoters, one of which, promoter III, is activated by calcium influx through L-type voltage-sensitive calcium channels (Tao et al., 1998). Chen et al. (2003) found that in the absence of neuronal activity in wild-type cultured neurons, MeCP2 is bound to the rat Bdnf promoter III and mediates its transcriptional repression. Membrane depolarization triggers the phosphorylation of MeCP2 at serine 421 (S421) through a CaMKII-dependent mechanism, and releases it from the promoter, thus allowing transcription ([Chen et al., 2003] and [Zhou et al., 2006]) (Figure 3A). In neurons expressing an S421A mutant form of MeCP2, the neuronal activity-dependent transcription of the mouse Bdnf promoter IV (which corresponds to rat promoter III) was impaired, suggesting that the unphosphorylatable form of MeCP2 cannot be released from the Bdnf promoter in response to membrane depolarization (Zhou et al., 2006). Another study found that the depolarization-induced increase in BDNF expression in mouse cortical cultures correlates with a decrease in methylation of CpGs in the Bdnf promoter IV region. Moreover, the increase in Bdnf transcription involved dissociation of MeCP2 and its associated corepressor complex from Bdnf promoter IV (Martinowich et al., 2003). So while these cortical culture studies provide evidence that MeCP2 binds the Bdnf promoter and regulates transcription of Bdnf in an activity-dependent manner, in vivo studies provide intriguing findings that question the role of MeCP2 as a repressor of Bdnf expression. Chang and colleagues reported that BDNF protein levels are decreased rather than increased in brains of symptomatic Mecp2−/Y mice. Moreover, conditional deletion of Bdnf in postmitotic neurons of mice mimicked some of the phenotypes observed in Mecp2 null mice, including hindlimb clasping, reduced brain weight, and reduced olfactory and hippocampal neuronal sizes. Furthermore, forebrain-specific deletion of Bdnf in Mecp2−/Y mice resulted in earlier onset of locomotor dysfunction and reduced lifespan, while forebrain-specific overexpression of Bdnf in Mecp2−/Y mice improved locomotor function and extended their lifespan (Chang et al., 2006). These results demonstrate in vivo interaction between Mecp2 and Bdnf, and a correlation between altered BDNF levels and neurologic impairment in Mecp2 null mice. One possible explanation for the discrepancy in observed BDNF levels between the in vivo and cell culture data is that neuronal activity was experimentally controlled in isolated cortical cultures (Chen et al., 2003), whereas in brains the neurons are inherently at different activity states (Chang et al., 2006). Since Bdnf is known to be upregulated in response to neuronal activity, the reduced cortical activity in Mecp2 null mice is expected to negatively affect Bdnf expression, hence masking the expected upregulation that would normally result from loss of repression in resting cortical neurons that lack MeCP2 (Chang et al., 2006). A recent study found that although Mecp2−/Y nodose ganglia and cortical neurons express less Bdnf mRNA and contain lower levels of the protein compared with wild-type neurons, they secrete a greater percentage of their total BDNF content. This enhanced secretion is due to an increase in BDNF stores available for release. Catecholamine release from Mecp2−/Y adrenal chromaffin cells is similarly affected, indicating general neurosecretory alterations in Mecp2 null mice (Wang et al., 2006). The fact that Bdnf null mice display a subset of phenotypes observed in Mecp2 null mice raises the possibility that the two proteins have certain overlapping effects. It is possible that MeCP2 dysfunction reduces overall neuronal activity and indirectly results in decreased BDNF. Alternatively, the alleviation of some of the Mecp2−/Y phenotypes upon BDNF overexpression may be a consequence of the general benefits of this neurotrophic factor. More studies are needed to determine the effects of MeCP2 on Bdnf expression in vivo and the role of BDNF in the initial stages of RTT pathogenesis. Irrespective of the effects of MeCP2 on Bdnf expression, there is compelling evidence that the phosphorylation of MeCP2 at S421 occurs in response to activity and that this phosphorylation leads to displacement of MeCP2 from the Bdnf promoter ([Chen et al., 2003] and [Zhou et al., 2006]). The finding that MeCP2 is phosphorylated at S421 in the suprachiasmatic nucleus in response to light exposure in mice after they are placed in 48 hr of darkness is an elegant demonstration of a role for this protein in activity-dependent modulation of gene expression. MeCP2 as a Modulator of Gene Expression The role of MeCP2 in regulating the expression of some genes that are involved in the stress response is consistent with its critical role in the context of altered neuronal activity. Stress is clearly an altered physiologic state that calls for dynamic changes in the expression of a variety of genes that control the activity of the hypothalamic-pituitary-adrenal axis, and the effector genes that are needed to modulate the activity of steroids and their receptors. Gene expression studies using RNA from Mecp2 null mouse brains reveal upregulation of the glucocorticoid-regulated genes, serum glucocorticoid-inducible kinase 1 (Sgk1) and FK506-binding protein 5 (Fkbp5). The reported expression changes were observed in presymptomatic, early symptomatic, and late symptomatic Mecp2 null mice. It is interesting that although these genes are induced in response to stress, the basal plasma glucocorticoid levels in these animals were not elevated, suggesting that the induced differences are not a result of high hormone levels. In addition, ChIP analysis showed that MeCP2 binds to the Fkbp5 and Sgk1 genes in wild-type brain. These findings raised the possibility that at least some RTT symptoms arise from the disruption of MeCP2 regulation of stress-responsive genes (Nuber et al., 2005). Following a phenotype-driven approach, McGill et al. identified a MeCP2 target gene that is potentially relevant to a specific RTT feature (McGill et al., 2006). The RTT mouse model, Mecp2308/Y, carrying a truncated Mecp2 allele, displays increased anxiety-like behavior ([McGill et al., 2006] and [Shahbazian et al., 2002a]). This is reminiscent of clinical reports of frequent high-anxiety episodes in RTT patients (Mount et al., 2001). To determine if stress underlies the anxiety in Mecp2308/Y mice, McGill and colleagues evaluated the physiological stress response in these animals and found that Mecp2308/Y mice have an enhanced physiologic response to stress manifested as increased corticosterone levels. This finding led to the evaluation of Crh as a target gene given that it encodes the neuropeptide corticotropin-releasing hormone (CRH), which modulates behavioral and physiologic responses to stress ([Bale and Vale, 2004] and [Bruhn et al., 1984]). RNA in situ hybridization and quantitative real-time RT-PCR on the paraventricular nucleus of the hypothalamus, the central amygdala, and the bed nucleus of the stria terminalis, three brain regions critical to the stress response, revealed increased Crh levels in Mecp2308/Y mice. ChIP analysis revealed that MeCP2 binds the Crh promoter, which is enriched for methylated CpGs. The enhanced Crh expression detected in Mecp2308/Y mice is due to a functional defect in the truncated MeCP2308 protein itself, since there were no differences in Crh promoter methylation patterns between the wild-type and mutant mice (McGill et al., 2006). This study suggests that overexpression of a specific MeCP2 target might explain a specific RTT phenotype. Crh is an attractive candidate in the investigation of therapeutic intervention strategies in RTT given that Crh receptor 1 (Crhr1) antagonists have been shown to reduce anxiety (Bale and Vale, 2004). It is noteworthy that the increase in Crh RNA levels results from upregulation of expression specifically where it is normally expressed, rather than from ectopic expression in neurons where it is normally repressed. These data argue that MeCP2 is not a silencer that turns off its targets where they are not normally expressed, but rather functions as a modulator of gene expression depending on the physiologic state of the organism. Metaphorically speaking, MeCP2 may be best thought of as the dimmer that regulates the amount of light rather than the switch that turns the lamp on and off. The facts that MeCP2 undergoes phosphorylation upon light exposure (a new physiologic state) and that it regulates RNA levels of a neuropeptide whose levels fluctuate according to the level of stress are consistent with this model. Rescuing RTT in Mouse Models The absence of neuronal degeneration in MeCP2 disorders begs the question of whether restoring MECP2 expression would restore normal neuronal function and reverse the resulting disease phenotypes. Recent work by Guy et al. (2007) and Giacometti et al. (2007) provide evidence supporting the feasibility of disease reversibility in mouse models of RTT. Guy et al. created a mouse in which endogenous Mecp2 is silenced by insertion of a Lox-Stop cassette and can be conditionally activated through Cre-mediated deletion of the cassette. The Mecp2lox-Stop allele behaved as a null mutation, and its activation was controlled by a tamoxifen-inducible (TM-inducible) Cre transgene. Acute TM injections caused sudden activation of Mecp2 and led to either rapid death or complete phenotypic rescue of the null mice. The associated toxicity resembled that caused by MeCP2 overexpression and was not due to the TM injections. A more gradual approach for induction using smaller and repeated doses of TM eliminated the toxicity and reversed the late-onset neurological phenotype of adult Mecp2lox-Stop/+; cre heterozygotes. The results indicate that MeCP2-deficient neurons are not permanently damaged, since Mecp2 activation leads to robust abrogation of advanced neurological defects in both young and adult animals. The authors propose a model in which neuronal MeCP2 target sites are defined by DNA methylation patterns that are preserved in its absence and that guide newly synthesized MeCP2 to its correct chromosomal positions. MeCP2 then resumes its role as interpreter of the DNA methylation signal required for normal neuronal function (Guy et al., 2007). Although the results do not provide immediate therapeutic strategies for RTT, they do establish that consequences of MeCP2 loss of function are reversible, and suggest that the neurological defects in RTT, and other MECP2 disorders, are not impervious to therapeutic possibilities. In an independent study, Giacometti et al., 2007 E. Giacometti, S. Luikenhuis, C. Beard and R. Jaenisch, Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2, Proc. Natl. Acad. Sci. USA 104 (2007), pp. 1931–1936. View Record in Scopus | Cited By in Scopus (26)Giacometti et al. (2007) demonstrated partial disease rescue by postnatal reactivation of MeCP2 in mutant animals. The investigators targeted a transgene carrying the mouse Mecp2-e2 cDNA downstream of a LoxP-Stop-LoxP (LSL) cassette to the Col1a1 locus, which provides a strong ubiquitous promoter. The LSL-Mecp2 transgene was then placed on a Mecp2 null background to determine if it could rescue upon excision of the Stop signal. Four different Cre transgenes, as follows, were tested to activate Mecp2: Nestin-Cre in neuronal and glial precursors; Tau-Cre in postmitotic neurons during embryogenesis; CaMKII-Cre 93 (C93) in the forebrain, hippocampus, midbrain, and brainstem at postnatal day (P) 0–P15; or CaMKII-Cre 159 (C159) in the forebrain at P15–P30. Although activation of the LSL-Mecp2 transgene prolonged the lifespan and delayed motor deterioration of Mecp2−/Y mice, the extent of rescue directly correlated with the time, level, and site of Cre expression. The most efficient symptomatic rescue was obtained in lines that provided early and wide Cre expression in most neurons (Nestin-Cre and Tau-Cre), extending the lifespan of mutant mice to 8 months. Postnatal MeCP2 activation in C93 and C159 lines extended the life span by 4 weeks, with the earlier C93 expression giving a more efficient rescue than the later C159 expression (Giacometti et al., 2007). Inappropriate Mecp2 expression levels resulting from use of nonendogenous promoters could account for the partial disease rescue obtained in this study, since this was not seen by Guy et al. when Mecp2 was activated under the control of its endogenous promoter. In fact, MeCP2 levels were much lower than the expected wild-type levels when induced by the C159 line, and neither line expressed the protein in the cerebellum. The findings from the C93 and C159 lines argue that it is critical that MeCP2 levels are restored to the expected wild-type levels and done so in all neurons, or at least in neurons outside of CaMKII domains, to achieve rescue. This conclusion is bolstered by another study in which induction of MeCP2 expression using a CaMKII promoter failed to rescue the lethality in MeCP2 null mice (Alvarez-Saavedra et al., 2007). Altogether, these data underscore the importance of proper expression of MeCP2, probably throughout the CNS or at least beyond the CaMKII expression domain, to achieve rescue. What is exciting, however, is that once proper expression of MeCP2 is achieved, symptoms can be reversed even in adult animals, implying that neurons might not be permanently damaged. New Questions and Challenges The past 8 years have witnessed many exciting discoveries that are relevant to MeCP2 function and RTT, yet many of these discoveries have inspired new questions and unveiled unexpected challenges. In the following section we will pose some of the questions that are worthy of consideration and potential new areas of investigation. What Is the Full Spectrum of MECP2 Disorders? The findings that MECP2 mutations cause RTT as well as a spectrum of neuropsychiatric phenotypes and that either loss or doubling of MeCP2 causes such phenotypes raise the possibility that regulatory mutations or posttranscriptional alterations that slightly alter MeCP2 levels might cause more restricted phenotypes such as autism, seizures, Parkinsonian features, or cognitive impairment (Coutinho et al., 2007). Given the importance of MeCP2 for postnatal neuronal function, it is conceivable that hypomorphic mutations might compromise neuronal plasticity such that overt phenotypes will become apparent only upon aging. It will take coordinated and large-scale efforts to establish if variations in MECP2 contribute to late-onset neurological disorders involving cognition and motor control. What Are the Functions of MeCP2 and How Are They Affected by the Various Mutations? Based on various in vivo studies, a few conclusions can be safely made about the function of MeCP2. It binds DNA at specific sites and this binding is affected by its phosphorylation state at S421, which in turn depends on neuronal activity. The finding that the expression changes in some of its targets are small argues that the protein modulates expression in response to activity and that such modulation is critical for normal neuronal function. Such modulatory effects would be easily missed when profiling RNA from heterogeneous neuronal populations. This is particularly true for targets that are expressed in specific neurons. The finding that either loss or doubling of MeCP2 results in progressive postnatal neurological disorders that have overlapping features is quite intriguing. The recent discovery that such loss and doubling result in decreases and increases (respectively) in excitatory synapse numbers argues that MeCP2 plays an important role in determining excitatory synapse number in early development. Whether this is achieved by modulating expression or splicing of factors critical for synapse formation (or both) remains to be determined. The finding, however, that loss and doubling of MeCP2 have opposing effects on synapses argues that certain expression or splicing changes might go in opposite directions in Mecp2 null versus duplication mice. Thus, these two models might provide an excellent resource to uncover some of the primary targets of MeCP2, which in turn would be an excellent framework to evaluate the functional consequences of some of the other RTT-causing MECP2 mutations. There are several alleles that alter one amino acid outside of the MBD or cause late truncations, raising the possibility that some of these might lead to a gain-of-function or hypermorphic effect as observed for the duplication. What Is the Anatomical Origin of Various RTT Phenotypes and What Are the Associated Molecular Changes? Given the diversity of phenotypes associated with MeCP2 dysfunction, a key question is whether individual phenotypes result from dysfunction of the protein in specific neurons, whether the phenotypes result from dysfunction of the entire neuronal network (the more compromised the network, the more phenotypes become apparent), or a combination of both (Figure 4). The identification of isolated partial phenotypes that are not always the same among patients with favorable XCI supports a model whereby dysfunction in specific neurons could explain one or more clinical features. The starting point of the disease mechanism could be cell-autonomous changes occurring during postnatal development that subsequently cause specific symptoms. Such select neuronal changes together with altered experiences might eventually lead to malfunction of an entire network, which is then manifested as additional neurobehavioral abnormalities. The molecular corollary of such a model is that the neuropsychiatric features of RTT and related disorders result from misregulation of some genes expressed in a specific set of neurons. Studies of large numbers of patients with essentially any one of the RTT phenotypes might identify females with partial symptoms and favorable XCI. Eventually, the contributions of specific neurons versus the networks of neurons to the RTT phenotype could be addressed in the mouse using neuron-specific deletions of Mecp2. The finding that anatomical changes, such as the decrease in glutamatergic synapses, are only detected in the presymptomatic stage (Chao et al., 2007) argues that compensatory changes might mask the primary consequences of MeCP2 dysfunction. Therefore, to get at some of the primary pathogenic events, one needs to study presymptomatic as well as early symptomatic animals. The Challenge of Developing Therapies for MECP2 Disorders The recent studies demonstrating that neurological deficits resulting from loss of MeCP2 can be reversed upon restoration of gene function are quite exciting. The next phase of research needs to assess how complete the recovery is. Clearly, lethality, level of activity, and hippocampal plasticity are rescued, but are the animals free of any other RTT symptoms such as social behavior deficits, anxiety, and cognitive impairments? Since postnatal rescue results in viability, it will be important to evaluate if even the subtler phenotypes of RTT and MECP2 disorders are rescued when protein function is restored postnatally. This is particularly important given emerging data about early neonatal experiences and their long-term effects on behavior in adults ([Plotsky et al., 2005] and [Sullivan et al., 2006]). The genetic rescue data are promising because they show that neurons that have suffered the consequences of loss of MeCP2 function are poised to regain functionality once MeCP2 is provided gradually and in the correct spatial distribution. This provides hope for restoring neuronal function in patients with RTT. However, the strategy in humans will require providing the critical factors that function downstream of MeCP2 because of the challenges in delivering the correct MeCP2 dosage only to neurons that lack it, given that the slightest perturbation in MeCP2 level is deleterious. Thus, therapeutic strategies necessitate the identification of the molecular mechanisms underlying individual RTT phenotypes and picking out the candidates that can be therapeutically targeted. Although the data are minimal, it is quite likely that there will be many genes whose expression is sufficiently altered to cause neuronal dysfunction, probably due to loss of normal homeostatic responses. While it is conceivable that some of these molecules could be investigated for potential therapeutics, it might prove challenging to restore levels of tens of targets. An alternative approach will be to identify proteins or pathways that suppress MeCP2 dysfunction phenotypes, which might prove easier to target therapeutically. The fact that there are human patients with milder phenotypes in spite of severe mutations (Dayer et al., 2007) argues that some variant in another protein or proteins might subdue the disease. Identifying such modifiers using various mouse models might prove very helpful. Lastly, given that loss and gain of MeCP2 have opposing effects on neurons, it will be important to establish whether certain mutant MECP2 alleles are functionally null or hyperactive when contemplating therapeutic trials. In closing, the RTT story started in the clinic, but today has inspired many exciting basic science studies in neurobiology and epigenetics. It is anticipated that the next chapter in this story will involve translating some of the discoveries back to the clinic to benefit patients with RTT and patients with related neurological disorders. Published in: Neuron, Volume 56, Issue 3, 8 November 2007, Pages 422-437 Source: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSS-4R2XTMN-4&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5996707d7ac7963fc1540579bde844e2

Another gene found for Rett syndrome

New research has revealed another gene for Rett syndrome which could lead to new treatments for the disorder. A research project by scientists from Australia and Israel has identified a genetic variation that influences the severity of symptoms in Rett syndrome. The scientists have established a correlation between the severity of clinical symptoms and a common brain-derived neurotrophic factor (BDNF) polymorphism and they say patients with the normal BDNF genetic variant had less severe symptoms, with later onset and frequency of seizures. Dr Helen Leonard from the Australian Rett Syndrome Study at the Telethon Institute for Child Health Research, says the finding was exciting in that it identifies a potential new target for treatment of the debilitating neurological disorder. Dr Leonard says previously it was known that there is a wide range in the onset and severity of symptoms in patients with Rett syndrome but it was difficult to give families a firm idea of how the disorder would progress and the new information is potentially helpful in predicting the clinical progression, but importantly, offers another area to explore for potential therapies. In the study, clinical information and DNA samples were gathered from 125 patients from the Australian Rett Syndrome Database and an Israeli cohort coordinated by Dr Bruria Ben Zeev at the Safra Pediatric Hospital, Sheba Medical Centre, Sackler School of Medicine, Tel Aviv. Professor John Christodoulou, from the NSW Centre for Rett Syndrome Research at the Children's Hospital at Westmead in Sydney and Dr Eva Gak from the Sagol Neuroscience Center at the Sheba Medical Centre carried out the genetic testing and say the new findings have established a correlation between the severity of clinical symptoms and BDNF polymorphism. Dr Christodoulou says those patients with the normal BDNF genetic variant had less severe symptoms, with later onset and frequency of seizures. He says they must now establish the nature of the interaction between MECP2 and BDNF and if it is possible to stimulate BDNF within patients with Rett syndrome, there is a chance that the onset of seizures can be delayed and some of the more debilitating aspects of the disorder reduced. Rett syndrome is a childhood neurodevelopmental disorder which almost always only affects females. Although Rett syndrome is a genetic disorder less than 1% of recorded cases are inherited or passed from one generation to the next - most cases are sporadic, which means the mutation occurs randomly and is not inherited. Initially Children with Rett's develop and grow normally but gradually symptoms appear which include a loss of purposeful use of the hands, distinctive hand movements, slowed brain and head growth, gait abnormalities, seizures, and mental retardation. The way Rett syndrome progresses, including the age it starts and the severity of symptoms, varies from child to child; mental and physical symptoms gradually appear such as a loss of muscle tone and as the syndrome progresses, the child loses purposeful use of their hands and the ability to speak - other early symptoms may include problems crawling or walking and diminished eye contact. The loss of functional use of the hands is followed by compulsive hand movements such as wringing and washing and this period of regression is sometimes sudden. Another symptom, apraxia - the inability to perform motor functions - is perhaps the most severely disabling feature of Rett syndrome, interfering with every body movement, including eye gaze and speech and children with Rett syndrome often exhibit autistic-like behaviours in the early stages. Other symptoms may include toe walking, sleep problems, wide-based gait, teeth grinding and difficulty chewing, slowed growth, seizures, cognitive disabilities, and breathing difficulties while awake such as hyperventilation, apnoea (breath holding), and air swallowing. Rett syndrome is caused by mutations in the MECP2 gene, which is found on the X chromosome and up to 80% of girls with Rett syndrome have the MECP2 genetic mutation. The remaining 20 to 30% of cases are suspected to be caused by mutations in other parts of the gene or by genes yet to be identified. Some children may require special equipment and aids such as braces to arrest scoliosis, splints to modify hand movements, and nutritional programs to help them maintain adequate weight; special academic, social, vocational, and support services may also be required in some cases. Despite the difficulties with symptoms, most individuals with Rett syndrome continue to live well into middle age and beyond. The finding is published in the international journal Neurology. 6a03bc68-77de-40cd-a513-b9c9db839e79|0|.0 Source: Medical Condition News http://www.news-medical.net/news/2009/04/22/48627.aspx