Rett Syndrome: Exploring the Autism Link

Abstract

The presence of autism in individuals with neurodevelopmental disorders, whether transient as in Rett syndrome (RTT) or enduring as in fragile X syndrome (FXS) or Down syndrome (DS), suggests the possibility of common neurobiological mechanisms whose elucidation could advance fundamentally their understanding. This review explores the commonalities and differences of autism and RTT at clinical and molecular levels with respect to current status and challenges for each, highlights recent findings from the Rare Disease Network Natural History study on RTT, and summarizes the broad range of phenotypes resulting from mutations in MECP2, the gene responsible for 95% of individuals with RTT. For RTT, animal models have been critical resources for advancing pathobiologic discovery and promise to be important testbeds for evaluating new therapies. Fundamental understanding of autism based on unique genetic mechanism(s) must await similar advances.

Introduction

Rett syndrome (RTT) is a neurodevelopmental disorder affecting females predominantly due to mutations in the methyl-CpG-binding protein 2 gene (MECP2) located at Xq28 in at least 95% of individuals meeting clinical criteria [1–5]. RTT is characterized by profound cognitive impairment, poor communication skills, stereotypic hand movements, and pervasive growth failure beginning between 6 and 18 months of age, following a period of apparently normal development including acquisition of fine motor skills and spoken communication. During the regression period, fine motor skills, effective eye contact, and communication are lost. Features of autism including limited eye contact and poor socialization or interaction are recognized along with inconsolable crying or irritability at this time. Typically, autistic features are transient lasting weeks to many months. By school age, intense eye gaze and interaction with others return, yet spoken communication does not. As such, individuals with RTT demonstrate a rather predictable temporal profile: apparently normal early development, arrest of developmental progress at 6–18 months followed by frank regression of social contact, language, and finger skills, and subsequent improvement in social contact and eye gaze by age 5. Effective interaction persists throughout life, yet motor functions gradually slow in adulthood.

Autism is a complex neurodevelopmental disorder involving poor communication and socialization and a limited repertoire of behaviors and interests [6]. Unlike RTT and a growing number of gene-based neurodevelopmental disorders noted below, mutations in a gene or specific set of genes have not been identified in non-syndromic autism. The diagnosis of autism has been confirmed in several neurodevelopmental disorders including fragile X syndrome (FXS), Down syndrome (DS), Angelman syndrome (AS), Prader-Willi syndrome, Smith-Magenis syndrome, Williams syndrome, neurofibromatosis type 1, tuberous sclerosis, San Filippo syndrome, phenylketonuria, adenylsuccinate lyase deficiency, and Smith-Lemli-Opitz syndrome. Specifically, up to 60% of individuals with FXS may have autism and up to 7% with DS. The diagnosis of autism in some, but not all individuals with these disorders, whether transient as in RTT or enduring as in FXS or DS, suggests the possibility of common neurobiological mechanisms whose elucidation could advance their understanding fundamentally. However, concern has been raised as to whether individuals with neurodevelopmental disorders demonstrating severe or profound cognitive impairment can be assigned an autism diagnosis [6].

In this review, the phenotypic variability and molecular complexities associated with MECP2 gene abnormalities and the clinical and molecular convergence between RTT and autism are explored.

Rett syndrome and Autism: Clinical Considerations

RTT and autism are regarded as neurodevelopmental disorders characterized clinically by apparently normal early development, failure of normal developmental progress, and absence of progressive deterioration and neurobiologically by fundamental failure of normal neuronal maturation and absence of progressive neuronal or glial pathology. As such, these disorders are potentially reversible. Autism and RTT share many common features, but clear differences exist (Table 1). Autism occurs predominantly in males, is associated at least initially with accelerated rate of head growth, lacks a specific genetic basis, and has greater incidence than RTT (1:100 births vs. 1:10,000 female births), which occurs mainly in females and is associated with postnatal deceleration in the rate of head growth and MECP2 mutations.

Table 1.

Comparative Features of Autism and Rett syndrome

Characteristic	Autism	Rett syndrome
Regression	Some regress	Universal
Eye gaze	Poor	Good except during regression period
Socialization	Poor	Good except during regression period
Head Circumference	Infants: large Adults: normal	Postnatal deceleration
Hand Skills	Generally good	Poor to absent
Gait	Good	Dyspraxic/None
Periodic breathing	Uncommon	Common

A clear understanding of the relationship between autism and RTT is lacking. For example, does brain function differ in autism and RTT, are similar brain regions affected, and do these change over time? Innovative technologies such as fMRI should be able to provide important insights.

Autism and RETT Syndrome: Molecular Convergence

A potential molecular convergence exists for autism and RTT involving MECP2 and EGR2 (early growth response gene-2), an activity-dependent immediate-early gene (IEG) [7]. EGR2, the only member of the EGR family restricted to CNS neurons, encodes a DNA-binding zinc finger protein important for cerebral development and synaptic plasticity. EGR2 and MECP2 expression increase coordinately in mouse and human cortex; regulation of EGR2 and MECP2 is disrupted in autism and RTT; MECP2 expression is decreased in autism cortex; and EGR2 expression is decreased in autism and RTT cortex and in cortex of a Mecp2 knock-out mouse. EGR2 has a predicted binding site in the MECP2 promoter region and the MECP2 family of methyl-binding proteins binds the EGR2 enhancer region.

AS, due principally to 15q11-q13 deletions, represents another potentially important link with autism and RTT. Mutations in UBE3A/E6AP (ubiquitin protein ligase E3A), a gene contained within this deletion, result in AS. Both UBE3A/E6AP and GABRB3 (β3 subunit of GABA_A receptors located within the deleted region) expression are reduced in Mecp2 deficient mice and in human brains from individuals with RTT, AS, and autism [8].

Functional Role of MECP2

Methyl-CpG-binding protein 2, a member of the methyl-binding protein family is capable of transcriptional regulation. However, specific gene targets are not defined fully [9]. MECP2 is ubiquitous in mammalian tissues and is highly expressed in brain, functioning in development and maintenance of neurons. During brain development, MECP2 appears in a caudal-rostral gradient, cortical neurons being the last to express. In the early prenatal period, forebrain expression is limited to Cajal-Retzius neurons. Initially proposed as a transcriptional repressor, recent evidence suggests that MECP2 participates in up- or down-regulation of a large number of genes [9].

Brain Morphology in Rett syndrome

In the human, brain morphology in RTT is characterized by reduced volume, particularly fronto-temporal lobes and caudate, reduced brain weight, and reduced or absent melanin pigmentation notably in substantia nigra [10]. At the microscopic level, cortical neurons are small and demonstrate simplified dendrites with reduced and immature dendritic spines [10]. Importantly, no evidence of recognizable disease progression, especially neuronal loss, is evident. This pattern appears to be a common theme among other neurodevelopmental disorders. Autism is associated with increased packing density, decreased cell size, and increased spine density; DS with reduced dendritic branches and spines after early infancy; and AS with reduced dendritic branches and AS and FXS with immature spines. These commonalities suggest a convergence of molecular mechanisms underlying their pathobiology.

Cellular Role of MECP2

Tightly regulated MECP2 dosing is critical to neuronal morphology, dendritic spine density, and morphology in RTT and animal models. As noted above, human brain revealed dendritic spine abnormalities in CA1 hippocampal neurons. In cultured rat embryonic hippocampal neurons, knock-down of normal MECP2 produces shorter dendrites with normal axon length whereas mutant MECP2 results in shorter axons and dendrites [11]. Conversely, over-expression (~2-fold) of MECP2 yields longer axons and dendrites. Both knockdown and over-expression (~2-fold) of MECP2 produce higher BDNF levels while over-expression of Bdnf partially corrects the adverse effect of mutant MECP2. In postnatal rat hippocampal slice cultures, knockdown and mutant MECP2 reduce spine density whereas spine density in wild-type MECP2 (~2-fold) overexpression is similar to controls (Figure) [12].

Figure. Pyramidal Neuron Dendritic Spines from Rat Hippocampal Slice Cultures and Human Hippocampus.

Upper panels: Rat CA1 neurons (96 hours post-transfection with enhanced yellow fluorescent protein, eYFP) from slice cultures showing similar spine density to controls with wildtype MECP2 over-expression (~2-fold) and decrease in spine density with mutant R106W (p< 0.05) and T158M (p< 0.01) MECP2 expression when compared with control slice cultures. Data in each bar graph represent the mean and standard deviation. The number of observations represented are control = 30 cells from 14 slices; T158M = 31 cells from 23 slices; R106W 16 cells from 11 slices; and wild-type MECP2 = 18 cells from 16 slices.

Lower panels: CA1 pyramidal neurons from human hippocampus stained with DiI showing marked reduction and aberrant morphology of dendritic spines (p< 0.01) from child (age 5) and adult (age 21) with RTT, compared to non-MR controls, child (age 3) and adult (age 35), respectively. Data in each bar graph represent the mean and standard deviation of ten RTT and nine control individuals, respectively.

[Unpublished data graciously provided courtesy of Christopher Chapleau and Lucas Pozzo-Miller.]

RETT SYNDROME: Role of MECP2

Prior to 1999, diagnosis of RTT was strictly clinical. In 1999, MECP2 mutation testing became available and identification of a MECP2 mutation implied the diagnosis was RTT: Maybe Yes … Maybe No.

Diagnosis of RTT is based on specific clinical criteria Table 2 [5]. Approximately 95% of girls with classic RTT have a MECP2 mutation [4]. More than 250 specific mutations are associated with RTT, 4 missense mutations, 4 nonsense mutations, several 3’ deletions, and entire exon deletions accounting for 70–80%. MECP2 mutations are generally sporadic (de novo), the majority of paternal origin. Familial RTT represents <<1%. RTT is NOT synonymous with MECP2 mutations. MECP2 mutations also occur without Rett syndrome. Thus, MECP2-related phenotypes involve both males and females and extend well beyond classic RTT (Table 3).

Table 2.

RTT Clinical Criteria [5]

Classic or typical RTT
1. Regression followed by recovery or stabilization
2. Main and exclusion criteria required*
	Main criteria:
	Partial or complete loss of acquired purposeful hand skills
	Partial or complete loss of acquired spoken language
	Gait abnormalities: Impaired or absent
	Stereotypic hand movements
	Exclusion criteria:
	Brain injury: trauma, metabolic disease, or infection
	Abnormal psychomotor development in first 6 months
Variant or atypical RTT
1. Regression followed by recovery or stabilization
2. 2 of 4 Main criteria (as above)
3. 5 of 11 supportive criteria
	Supportive criteria
	Breathing disturbances when awake
	Bruxism when awake
	Impaired sleep pattern
	Abnormal muscle tone
	Peripheral vasomotor disturbances
	Scoliosis/kyphosis
	Growth retardation
	Small cold hands and feet
	Inappropriate laughing/screaming spells
	Diminished response to pain
	Intense eye communication - “eye pointing”

^*Supportive criteria are not uniformly present and are not required for diagnosis.

Table 3.

MECP2-related Phenotypic Variants

Females

Rett syndrome

Preserved speech variant

Delayed onset variant

Congenital or early onset seizure variant

Autistic-like variant

Angelman syndrome

Mild learning disability

Normal carriers

Males

Severe encephalopathy

Classic RTT: Klinefelter syndrome or somatic mosaicism

X-Linked MR ± progressive spasticity

MECP2 duplications ± frequent respiratory infections

Continuum of MECP2 Associated Phenotypes

In females, mutation-related phenotypes range from normal to learning disabilities to RTT to congenital variants (Table 3). RTT comprises the largest group, the other presentations being greatly under-represented due to their phenotypic differences from RTT. Individuals with normal function, learning disabilities, or mild cognitive impairments are transmitting females with both female and male offspring affected adversely by the MECP2 mutation. The favorable status of transmitting females generally results from skewed or unbalanced X chromosome inactivation (XCI). Other non-RTT presentations represent a combination of factors: milder or more severe mutations, skewed XCI, or perhaps variable distribution of normal MECP2 within critical brain regions. In males, the picture is quite different (Table 3). Severe encephalopathy with markedly shortened survival was first noted in a boy from a multiplex family including two females with RTT and one with mild learning disability [13]. Subsequently, more than fifteen males with severe encephalopathy have been described [14]. Typical RTT has been diagnosed in males with Klinefelter syndrome (47XXY) and somatic mosaicism, both yielding populations of cells with distinct X chromosomes. More recently, the much more common (>100 reported) MECP2 duplication disorder in males was described involving duplication of variable portions of the X chromosome including Xq28 [15]. This disorder presents a phenotype quite distinct from RTT, including recurrent respiratory infections in the majority.

Medical Issues in Rett syndrome

Despite emphasis on neurodevelopmental aspects of RTT, attention should also be given to associated medical issues. These are multisystemic, but particularly involve gastrointestinal functions including ineffective chewing and swallowing, GE reflux, delayed gastric emptying, constipation, and an unusually high frequency of gallbladder disease. Growth and nutrition are problematic. In addition to abnormal deceleration in rate of head growth in infancy, weight and height also demonstrate a fall-off, typically by the second year of life. Longevity is less than normal but average survival is >50 years [16]. Other issues requiring attention are epilepsy [17], scoliosis [18], sleep, and anxiety. Importantly, differentiation of seizures from anxiety or other behaviors is often difficult, requiring video-EEG assessment. EEGs are uniformly abnormal by age 3 with background slowing and multifocal epileptiform patterns, especially during sleep. Cardiac conduction may be abnormal with prolonged QTc and non-specific ST segment changes for which annual evaluations are recommended. Onset of menstruation mirrors general female patterns. As such, appropriate measures are required to protect this vulnerable group.

Animal Models

Several knock-out and knock-in mouse models for RTT are now available for study. Knock-in models representing two common human mutations, R168X and R255X, are now emerging and the less common A140V mutation associated with cognitive impairment in males, but not RTT in females.

Notable findings have emerged from these models. One addressed the question: Is Mecp2 reversible? Using an estrogen receptor controlled Mecp2 promoter, the Mecp2 knock-out phenotype could be reversed in both male and in female mice, indicating that identification of effective therapies could be beneficial in reversing some if not all of the clinical abnormalities [19]. This study, while not applicable to humans mechanistically, provided proof of principle that RTT, and perhaps other neurodevelopmental disorders, could be reversible regardless of stage of involvement.

Two, studies in a knock-in model demonstrated impaired hippocampus-dependent social, spatial, and contextual fear memory, impaired long-term potentiation and depression, and reduced post-synaptic densities [20]. Extending these studies to hypothalamus, enhanced anxiety and fear were related to elevated levels of blood corticosterone and of corticotropin-releasing hormone (Crh) in hypothalamus, central nucleus of amygdala, and bed nucleus of stria terminalis. Importantly, Mecp2 binds to the Crh promoter methylated region, producing enhanced Crh expression and underscoring the central role of anxiety in RTT [21]. Further, amygdala have direct input into hypothalamus and brainstem autonomic nuclei correlating with clinical problems of respiration and GI function. Trials directed specifically at Crh expression or at downstream effects associated with anxiety with approved SSRIs are ongoing.

Three, available read-through compounds show promise in providing a full length protein in individuals with nonsense or stop mutations. These agents are being evaluated in the knock-in models, R168X and R255X.

Comment

Rett Syndrome and Autism: Current Status and Challenges

At present, our understanding of clinical aspects of RTT has been accelerated by an NIH-funded RTT Natural History Study (NCT00299312) and an international investigator consortium. Medical and behavioral management has improved and advances in molecular genetics have refined diagnostic methodologies and broadened recognition of clinical and molecular heterogeneity. Development of animal models has expanded neurobiologic understanding and provided testbeds for evaluating promising treatment strategies. Current challenges include adequate information dissemination to broader medical communities, implementation of consensus criteria, refinement of medical management, and development of fundamental therapies, both targeted and gene-based. Clinical trials of new and repurposed agents are hampered by a lack of reliable behavioral, cognitive, and neurophysiologic outcome measures.

Autism has penetrated the public domain widely and differs dramatically from RTT with respect to national focus and commitment, strong collaborative networks and interdisciplinary approaches, and standardized, broad-based evaluations. Identifying specific molecular markers is a national priority. Innovative in vivo investigations including fMRI and interactive computer techniques are increasingly utilized and strong emphasis has been directed at behavioral management. Nonetheless, significant challenges exist. These include lack of unifying genetic defect(s), paucity of human neuroanatomic and functional data, lack of a gene-based animal model as in RTT, and similar clinical trials challenges. While an inbred animal model with autistic features exists and recent pharmacologic interventions demonstrate reduction of repetitive behaviors but not improved socialization in this model, a specific gene defect has not been elucidated in this model [22]. For clinical trials, what are strategic targets, how should they be stratified, and what outcome measures should be employed? While autism and RTT may share common neurobiologic mechanisms, fundamental understanding of both remains a critical topic for study.