Overview Rett Syndrome (RTT) is a neurodevelopmental disorder that predominantly affects females, it arises from mutations in the X-linked gene methyl-CpG binding protein 2 (MeCP2).The diagnosis of RTT is based on clinical features (Neul et al. 2010)                                                                                                                                                                                                                                                                                                                                     . It is characterized by a broad clinical spectrum of signs and symptoms involving the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of seemingly normal development.  It was first described in 1983 when a cohort of females were observed with strikingly similar clinical features of “progressive autism, loss of purposeful hand movements, ataxia, and acquired microcephaly” Haberg 1983 It occurs with an average frequency of about 1:10,000 in girls Laurvick 2006. After the first identification of the gene in 1999 Amir 1999 , MECP2 mutations have been identified in 90–95 % of the Classical RTT cases. AK Percy 2008Classification of RTTRTT syndrome is broadly categorised into ‘Classical’ and ‘Atypical’. Recently revised criteria has recognized that some individuals present many of the clinical features of RTT, such as regression, but do not fit the criteria established for the diagnosis of Classical RTT Neul et al 2010. These have been termed “atypical” RTT. These RTT atypical forms  have been found to cluster in some distinct clinical groupings, such as preserved speech variant, early seizure variant, and congenital variant.Preserved Speech Variant or “Zappella variant” (Z-RTT) is characterized by milder clinical abnormalities and by the appearance of some degree of speech. Mutations in other genes such as cyclin-dependent kinase like 5 (CDKL5) and forkhead box G1 (FOXG1) can cause phenotypes overlapping with those seen in RTT (Archer et al., 2006; Ariani et al., 2008); however, several features, such as congenital onset and ealry onset of seizures in CDKL5-mutant patients, and congenital onset and hypoplasia of the corpus callosum in FOXG1-mutant patients, distinguish these disorders from typical RTT (Kortüm et al., 2011)   In these last two variants, the mutation and several clinical features are notably different, hence they are considered as distinct clinical and molecular entities.All these forms have different severity degrees in terms of comorbidities, behaviour, prognosis and involvement of the autonomic nervous system. Clinical pathological features of RTT A 4 stage system of clinical features was outlined to facilitate the characterization of disease patterns and profiles from infancy to adolescence. This was derived from a synthesis of clinical observations over the years. Hamberg 1986,2002Stage 1: early onset stagnation corresponds to an interruption of growth, and a decrease in the head circumference growth. Stage 2: developmental regression is characterised by a loss of acquired skills/ communication (language, motor abilities, and purposeful use of hands), appearance of seizures, hand stereotypes, alteration in the cardiorespiratory function, problems with the autonomic system, and autistic features such as social withdrawal, avoidance of eye contact and indifference to visual or auditory stimuli.Stage 3: pseudostationary period is characterized by a reduction of autistic symptoms, appearance of scoliosis and seizures.This differentiates RTT from progressive degenerative brain disorders.Stage 4: ‘Late motor deterioration’ is defined by wheelchair dependency with severe disability, wasting and distal distortion. Prominent features include reduced mobility, curvature of the spine, and muscle weakness, rigidity, spasticity, and increased muscle tone with abnormal posturing of an arm or leg. Transitions between stages can be indistinct and there are difficulties in  separating them accurately for research purposes. However ,the staging system was found to be a useful instrument for a more systematic registration when examining and monitoring RTT patients in routine clinical work.Movement abnormalities are a major issue in RTT (FitzGerald et al., 1990a; FitzGerald et al., 1990b), with the most obvious being the repetitive hand stereotypies, which seem to interfere with volitional hand use. Gait is almost always disrupted, with evidence of ataxia and apraxia. Dystonia is common, seen first in the ankles and eventually progressing to many joints. Axial hypotonia is present early in the disease course but, as children become young adults, increased tone with features of rigidity becomes more prominent. Additional movement abnormalities include tremor, myoclonus, chorea, facial grimacing and severe teeth grinding. Most individuals with RTT have scoliosis, and some require surgical intervention (Percy et al., 2010).Nutrition and gastrointestinal function are also major clinical issues in RTT, and there is marked growth failure in most affected individuals (Tarquinio et al., 2012). It has long been recognized that head growth is impaired, resulting in acquired microcephaly (Hagberg et al., 1983), and height and weight are usually markedly diminished (Schultz et al., 1993). However, a subset of individuals with RTT are overweight or obese (Renieri et al., 2009), a feature that is often associated with higher functioning and possibly improved oromotor skills (Motil et al., 1999). Many individuals with RTT have various gastrointestinal problems, including significant chewing and swallowing difficulties, gastroesophageal reflux, gastrointestinal dysmotility and severe constipation, which severely decrease the quality of life for patients and their families (Motil et al., 2012).Dysregulation of breathing and autonomic homeostasis are very common in RTT. Respiratory abnormalities, which include periods of forceful breathing (hyperventilation), severe pauses in breathing (including breath holds) that can cause cyanosis and even loss of consciousness, and abnormal cardiorespiratory coupling, are more severe during wakefulness than during sleep (Elian and Rudolf, 1991; Julu et al., 2001; Julu and Witt Engerström, 2005; Marcus et al., 1994; Weese-Mayer et al., 2008; Weese-Mayer et al., 2006) and can be exaggerated during periods of excitement or stress. Autonomic abnormalities include periods of vasomotor disturbance (usually associated with cold hands and feet), abnormal sweating, decreased heart rate variability, evidence of sympathetic-parasympathetic imbalance and prolongation of corrected QT interval (an indication of abnormal cardiac electrical activity) in a subset of individuals (Guideri et al., 2004; McCauley et al., 2011; Sekul et al., 1994). One quarter of deaths in RTT are sudden and unexpected (Kerr et al., 1997), and might result from complications of cardiorespiratory dysfunction.Despite the severity and phenotypic complexity of RTT, the brains of individuals with RTT do not show gross neuropathological changes, nor evidence of neuronal or glial atrophy, degeneration, gliosis, or demyelination, indicating that RTT is not a neurodegenerative disorder (Jellinger et al., 1988; Reiss et al., 1993).Genetic Basis of RTT 99% of RTT cases are sporadic therefore mapping the disease locus is difficult by traditional linkage analysis.Exclusion mapping of rare familial cases identified  Xq28 as the candidate region and subsequent sequencing of candidate genes revealed mutations in the gene encoding Methyl-CpG-binding protein 2 (MECP2) (Amir 1999) Mutations in MECP2 have been identified in more than 95% of typical RTT patients. Knowing the causative gene on the X chromosome clarified the female bias of the disorder. In the rare cases of familial transmission mothers with favourably skewed X chromosome inactivation (XCI) can transmit a mutant allele to viable hemizygous male offspring but these males typically die in infancy or are born stillborn if they inherit total loss of function alleles. Weaving 2005 Males with milder mutations survive and exhibit varying degrees of intellectual disability and autistic features. These male patients help to broaden the spectrum in phenotype of MECP2 mutation. Phenotypic VariabilityBecause the mutated gene that causes Rett syndrome is located on the X chromosome, females have twice the likelihood of developing a mutation. Females with Rett syndrome usually have one mutated X chromosome and one normal X chromosome. The pattern of X chromosome inactivation is a major source of phenotypic variability.  Only one chromosome will be active in each cell and this is usually random, where in one half the maternal X chromosome is active and in the other half the paternal X chromosome is active. A female MECP2 patient is therefore typically mosaic, where the wildtype MECP2 allele is expressed in half her cells, and the other half express the mutant MECP2 allele. A non random pattern of XCI occurs when cells expressing the wild type allele divide faster than than cells expressing the mutant allele. This allows for compensation of lost MeCP2 function and results in  amelioration of the RTT neurological phenotype.Zogbhi-Charour 2007 This allows most females with Rett syndrome to survive infancy. However because most boys have only one X chromosome, when a mutation occurs on the MeCP2 gene the detrimental effects are not compensated by the presence of a second, normal X chromosome. As a result, many males with Rett syndrome are stillborn or do not live past infancy. Weaving 2005 In order to understand RTT pathogenesis, both the function and location of the gene product, MeCP2, must be evaluated.MeCP2 Function MeCP2 is a multifunctional protein with roles in transcriptional regulation and modulation of chromatin structure.  Different domains of MeCP2 have been associated in facilitating multiple functions through direct DNA binding, interaction with protein partners or recruitment of other factors Guy 2011. MeCP2 is made up of three domains, the methyl- CpG binding domain (MBD), the transcriptional repression domain (TRD) and a C-terminal domain. Transcriptional regulation: The transcriptional regulatory role of MeCP2 appears to be dependent on interacting protein partners.  MeCP2 has been shown to repress transcription by recruitment of the nuclear receptor co-repressor (NCOR)-SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) co-repressor complex. MeCP2 has also been shown to specifically repress methylated reporter genes. Bird 2015. MeCP2 has also been shown to activate transcription by recruiting the co-activator cyclic AMP-responsive element binding protein 1 (CREB1). Recent studies DellaRagione 2012 have defined MeCP2 as a genome wide transcriptional modulator instead of a transcriptional regulator due to its diverse nature of MeCP2 target genes and the opposing effects (activation/repression) on the studied genes. RNA Splicing: Altered expression of a few specific genes could not explain the broad spectrum of MeCP2 phenotypes. Tudor 2002. The role of MeCP2 in RNA splicing was implied by alternative splicing of genes in a mouse model of RTT. Young 2005 MeCP2 has been shown to interact with RNA binding protein YB-1 Young 2005 and spliceosome-associated protein PRPF3 Long 2011 Aberrant alternative splicing events have been shown to occur in MeCP2 deficient human cell lines. Altered RNA splicing of synaptic genes has also been observed in autism. Smith-Sadee 2011. MicroRNA Regulation: a role of MeCP2 in the expression of microRNA has been implicated. MicroRNA-137 (MiR-137) has been shown to be epigenetically regulated by MeCP2, where MeCP2 deficiency in adult neural stem cells activates MiR-137s involvement in proliferation and differentiation. Szulwach 2010 MeCP2 deficiency was also found to activate and repress other MiRs. Regulation of miRNAs in RTT pathology was further compounded by observed transcriptional dysregulation of long noncoding RNAs in the brains of Mecp2-null RTT mouse model Petazzi 2013MeCP2 has also been suggested to have a role in the regulation of chromatin structure, loss of a chromatin loop was reported in Mecp2-knockout mice. Horike-Cheng2005 However, an essential role for MeCP2 outside the central nervous system (CNS) cannot be excluded, since the phenotype of the mice was analyzed primarily with regard to neuronal function, and more subtle deficiencies outside the CNS may have been overlooked.MECP2 Expression Previous studies have  looked at the expression of  MECP2 RNA transcripts in mouse and human tissues.  This revealed the presence of three alternatively spliced MECP2 transcripts (1.9, 7.5 and 10 kb) produced by differential polyadenylation poly(A) site usage (Reichwald 1999). At least one of these transcripts is present in all human tissues, but the abundance varies between tissues. Expression and half-life of the alternative MECP2 transcripts. To investigate whether the putative, unusually long 38 UTR is related to MECP2/Mecp2, we performed Northern blot analysis with probes from various regions of the human MECP2 gene. A cDNA probe (acc. no. Y12643) as well as probes specific for exon 2, exon 3, or the translated portion of exon 4 simultaneously detected two transcripts of 1.9 kb and ?10 kb (Fig. 3a and data not shown). Both transcripts are ubiquitously expressed but show different tissue expression patterns; for example, the shorter transcript is barely detectable in brain and lung, but very abundant in heart, skeletal muscle, and spleen. On the other hand, the ?10-kb transcript is barely detectable in lung and liver, and absent in ovary, but relatively abundant in skeletal muscle, kidney, pancreas, and brain. Furthermore, there is a third, weak transcript of >7.5 kb. It is mainly expressed in heart, brain, skeletal muscle, and pancreas, but also present in other tissues.MeCP2 is widely expressed in many organs, and its highest expression is detected in brain, lung and spleen, compared with which the expression levels are lower in liver, heart, kidney and small intestines (Shahbazian et al. 2002b). However, the brain-specific expression of MeCP2 is extensively studied, as the majority of RTT phenotypes are neurological. Additionally, abolishing Mecp2 expression in the embryonic brain results in similar RTT phenotypes caused by Mecp2 null mutations affecting all murine tissues (Chen et al. 2001; Guy et al. 2001). Nonetheless, non-neuronal RTT symptoms such as scoliosis, breathing/respiratory abnormalities, cardiac problems, difficulty in feeding and limb movements indicate the importance of MeCP2 expression outside the central nervous system (Guideri and Acampa 2005; Ogier and Katz 2008; Nomura and Segawa 1992; Isaacs et al. 2003; Ezeonwuka and Rastegar 2014).Within the brain, both the distribution and levels of MeCP2 have been shown to be different. For instance, we have recently demonstrated the distribution profile of MeCP2 in the adult murine brain regions, specifically, in the olfactory bulb, cortex, striatum, hippocampus, thalamus, cerebellum and brain stem (Zachariah et al. 2012; Olson et al. 2014). Analysis of whole cell extracts isolated from these brain regions indicated the highest MeCP2 expression in the cortex and cerebellum among the studied brain regions (Zachariah et al. 2012). In contrast, analysis of nuclear extracts from the same brain regions indicated relatively even levels of MeCP2E1 and differential levels of MeCP2E2 (Olson et al. 2014). Implicating the importance of brain region-specific MeCP2 expression in Rett syndrome pathogenesis, others have shown that the expression levels of MeCP2 in different mouse brain regions correlated with impaired behavioral phenotypes in a RTT mouse model (Wither et al. 2013). Several RTT mouse models have been generated by deleting the Mecp2 expression in specific brain regions and/or specific cell types within the brain regions that show varying degrees of RTT phenotypes. For example, loss of MeCP2 expression in the neurons in basolateral amygdala causes increased anxiety-like behavior and impaired cue-dependent fear learning (Adachi et al. 2009; Wu and Camarena 2009). Several studies have shown abnormal social behaviors, anxiety and also autistic features in RTT mouse models lacking MeCP2 expression in the neurons of forebrain (Gemelli et al. 2006; Chen et al. 2001; Chao et al. 2010). Moreover, MeCP2 deletion from hypothalamic neurons resulted in abnormal physiological stress response, hyper-aggressiveness and obesity (Fyffe et al. 2008).Within the brain, cellular expression of MeCP2 is predominantly detected in neurons. Other than neurons, MeCP2 expression has also been demonstrated in astrocytes, oligodendrocytes and microglia (Zachariah et al. 2012; Ballas et al. 2009; Rastegar et al. 2009; Derecki et al. 2012; Liyanage et al. 2013; Olson et al. 2014). Our studies also show that MeCP2 is localized to chromocenters in neurons, astrocytes and oligodendrocytes (Zachariah et al. 2012; Rastegar et al. 2009; Liyanage et al. 2013; Olson et al. 2014).tInaccessibility of brain Expression screenings of MeCP2 has shown MECP2 function in other neuropsychiatric disordersAlthough mutations in MECP2 can be found in 95–97% of individuals with typical RTT,  3–5% of individuals who strictly meet clinical criteria for RTT do not have an identified mutation in MECP2. Neul 2008 This indicates that a mutation in this gene is not required to make the diagnosis of typical RTT. The situation is more dramatic in atypical cases, with only 50–70% having identified mutations in MECP2.AKPercy 2007Similarly, known RTT causing mutations in MECP2 have been identified in individuals who do not have the clinical features of RTT. Neul 2010 Typical and atypical RTT only constitutes a section of MECP2 associated disorders. MECP2 mutations result in a multitude of neuropsychiatric disorders such as schizophrenia Cohen 2002, autism spectrum disorders Beyer 2002 and Angelmann syndrome.Watson 2001 This highlights the importance of understanding the function of MECP2 mutations in RTT as well as other MeCP2 related disorders. It also illustrates the implications of the identification of biomarkers of MECP2 mutations in RTT and in the broader range of neuropsychiatric disorders. The most important action of MECP2 is regulating epigenetic imprinting and chromatin condensation, but MECP2 influences many different biological pathways on multiple levels although the molecular pathways from gene to phenotype are currently not fully understood.Importance of animal modelsAnimal mutants in Mecp2 recapitulate the signs of the disorder and therefore represent valid models for studying the neurobiology of RTT and test candidate treatments.Gross motor dysfunction and shortened lifespan are common features of diverse RTT mouse models on different genetic backgrounds and broadly recapitulate the clinical presentation in individuals with RTT. The prototypical features of human RTT neuropathology – such as smaller neurons, higher neuronal packing density, reduced dendritic arbors and abnormal dendritic spines – have all been found in Mecp2-mutant mice models analyzed thus farImportance of finding a biomarker Rett syndrome is a clinical concept. In 2001, a diagnosis still has to rely on abattery of characteristic co-existing clinical criteria and a sequence of stages, combined with a procedure of differential diagnostic exclusions. CLINICAL MANIFESTATIONS AND STAGES OF RETT SYNDROME Hamberg 2002 Given the emergence of new therapeutic leads in the field (cf. Abdala et al., 2010; De Filippis et al., 2012; Deogracias et al., 2012; Kron et al., 2012; McCauley et al., 2011; Nag and Berger-Sweeney, 2007; Ogier et al., 2007; Roux et al., 2007; Schmid et al., 2012; Tropea et al., 2009; Zanella et al., 2008), RTT models hold great promise for translational research, particularly for prioritizing and validating potential treatment strategies prior to launching costly clinical trials. Conclusion Despite considerable progress in describing the pathophysiological consequences of Mecp2 mutations in mice, crucial gaps in knowledge remain that must be addressed in order to accurately assess potential therapeutics..Developing a non invasive biomarker of disease progression and amelioration would facilitate translation to clinical studies and