All nine boys with the MECP2 duplication syndrome exhibited features consistent with those observed in children with idiopathic autism including difficulties with eye gaze, limited shared enjoyment in interactions, limited range of facial expression, the use of another person's hand as a tool to communicate, several unusual sensory interests and aversions, and repetitive behaviors and interests. Anxiety was also prevalent. The language of the boys who speak consisted largely of repetitive word use. These behaviors cannot be fully accounted for by significant cognitive and language delays since typically developing infants acquire social communication milestones (e.g. a responsive social smile, gaze monitoring) that were not present in these boys. It is also important to note that these behaviors were quite prevalent, regardless of the functional level of the patient. For example, the higher functioning boys (subjects 1, 5, 7, and 8) exhibited significant symptoms of autism and their symptoms were even more prominent (related to anxiety, gaze avoidance, restricted interests, and use of repetitive language). In addition to autism and mental retardation, the majority of boys in our sample also manifest hypotonia with lower extremity spasticity, recurrent respiratory infections, hand sterotypies, choreiform movements, aerophagia, drooling, bruxism, and epilepsy.
All of the female MECP2 duplication carriers exhibited phenotypic changes despite favorable skewing of XCI. They exhibited psychiatric symptoms (anxiety, depression, compulsions) that preceded the birth of their children. They all exhibited less flexible personality traits, and the majority met formal criteria for the broad autism phenotype. Although cognitive abilities varied, most of the women exhibited higher nonverbal reasoning as compared to verbal reasoning skills.
In aggregate, phenotypic findings from the boys and the female carriers suggest that gain of MeCP2 function contributes to certain behaviors associated with autism and the BAP. This is evident with regard to repetitive behaviors in the boys and compulsions in the female carriers, anxiety, rigidity, and difficulties with the functional use of language for social purposes. The BAP is the milder expression of underlying genetic liability for autism and associated neuropsychiatric symptoms that are manifest in non-autistic relatives29
. It is an intermediate phenotype that refers to personality and language characteristics that reflect the phenotypic expression of this genetic liability29
. Utilizing family history and direct observational methods, researchers found that parents of children with autism are more likely to have inflexible personalities, to exhibit difficulty with transitions, to be aloof, blunt, anxious, and hypersensitive to criticism, and to have fewer high quality friendships compared to control parents of children with Down Syndrome30, 31
. These characteristics parallel the social communication deficits and the repetitive/stereotypic behaviors observed in children with autism.
Our data also suggest that menstrual irregularities, premature menopause, and endocrine and/or autoimmune disease may be more prevalent in female duplication carriers. It is possible that MeCP2 contributes to these phenotypes via methylation sensitive estrogen or progesterone responsive elements. Alternatively, the IRAK1 protein has been postulated to have a role in the regulation of autoimmunity32
and may contribute to these phenotypes. Others have suggested that increased IRAK1
dosage may contribute to the recurrent infection phenotype in affected boys15
, a hypothesis that warrants further investigation.
Clinically, the high prevalence of sterotyped and repetitive behaviors (commonly manifested by midline hand movements), breathing abnormalities, epilepsy, hypotonia, autism, mental retardation, and anxiety in our series suggests that Rett syndrome and the MECP2
duplication syndrome are more similar than previously suspected. Of note, the EEGs of the boys with MECP2
duplication were less abnormal than girls with RTT and MECP2
mutations. For example, by age three most girls with RTT have EEGs characterized by epileptiform abnormalities and background activity that is moderately to severely slow in contrast to these boys33
. Longitudinal studies of the EEG in these boys may reveal a characteristic EEG pattern. Longitudinal studies have shown that girls with RTT undergo typical clinical and developmental changes with time; similar longitudinal studies of boys with MECP2
duplication will provide important prognostic information.
The fact that loss and gain of MeCP2 function lead to very similar and predominantly neurological syndromes indicates that tight regulation of MECP2
is critical for the appropriate development and maintenance of neuronal function21
. Furthermore, the fact that female MECP2
duplication carriers manifest the BAP supports the hypothesis that MeCP2 participates in dosage-sensitive neuronal pathways which directly influence the phenotypic expression of autism spectrum behaviors. Interestingly, hemizygous male mice that express twice the endogenous level of Mecp2
have significant anxiety and manifest abnormal social interaction, suggesting that this transgenic mouse model reproduces the autism phenotype observed in the boys (Samaco and Zoghbi, unpublished data). The neurobiology of MeCP2 function suggests that the protein has an essential role in fine-tuning gene expression changes in response to neuronal activity. For example, phosphorylation of MeCP2 at S421 occurs in response to neuronal activity and is necessary for MeCP2 to modulate dendritic growth and spine maturation34
, and MeCP2 regulates the expression of Bdnf
, an activity dependent gene34-36
. The Mecp2308/y
Rett syndrome mouse model demonstrates exaggerated physiological responses to stress manifested by heightened anxiety and increased corticosterone levels37
. The Crh
gene is a direct target of MeCP2, and altered Crh
expression occurs only in brain regions where it is normally expressed37
. These data reinforce the hypothesis that MeCP2 is a modulator of neuronal function and suggest that failure of MeCP2 to appropriately modulate internal and external stimuli at the molecular level may underlie the BAP and autism phenotypes observed in our subjects.
The observation that MECP2
duplication may lead to neuropsychiatric symptoms in women despite nearly 100% skewing of XCI and wild-type expression levels in peripheral blood is interesting. Perhaps duplication of the IRAK1
gene, an important kinase in the Toll-like receptor signaling pathway32
, results in selective survival of cells with wild-type XCI patterns in blood but not in brain. It is also possible that extreme skewing of X-inactivation itself contributes to the observed phenotypes. The women in our study may have some neurons expressing the duplicated allele throughout the brain, in specific brain regions or nuclei, or during specific developmental periods, and one can imagine that even small MECP2
dosage increases in certain brain regions may lead to mild changes in gene expression or synapse number and the mild neuropsychiatric and cognitive phenotypes observed. We cannot exclude the possibility that other additive environmental or genetic factors also contribute to the phenotypes observed in carrier females. Heterozygous, transgenic, female mice with MECP2
duplication express lower levels of MeCP2 protein than hemizygous males likely due to XCI, yet they still manifest neurological phenotypes8
. Conditional knock-out of MECP2
from Sim1-expressing neurons in the mouse hypothalamus led to mice with abnormal responses to stress, abnormal aggressive behavior, hyperphagia, and obesity providing a precedent for the idea that altered MECP2
dosage in discrete brain regions can be sufficient to cause significant biological phenotypes38
The data presented in this study reveal that the full extent of neuropsychiatric phenotypes resulting from MeCP2 dysfunction is not yet appreciated and awaits large-scale studies searching for non-conventional mutations in large cohorts of subjects with neuropsychiatric disorders and appropriate controls. Recent mouse data suggest that even a 50% reduction in MeCP2 dosage is sufficient to cause neurological phenotypes and predict that human patients with mild reductions in MeCP2 protein exist20
. Subtle alteration of MeCP2 levels (decrease or increase) is likely to result from non-coding mutations that might alter transcription, mRNA stability, or translation. Some hypomorphic coding region mutations might also affect protein stability or protein-protein interactions. Given the detrimental developmental effects of MECP2
duplication, we predict that mutations that slightly enhance MeCP2 function will also cause neuropsychiatric phenotypes. The fact that autism is penetrant in 100% of boys in this cohort suggests that MECP2
duplication should be added to the several dozen other rare causes of syndromic autism, and suggests that MECP2
gain of function may be another excellent autism model. Males with the appropriate phenotype should be tested for MECP2
duplication, and since the duplication appears to be inherited frequently from carrier mothers, genetic counseling should be offered to the family.