rtt ( omim i d : 312750 ) occurs with a frequency of up to 1  :  10,000 live female births . causative mutations in the x linked methyl - cpg binding protein 2 gene ( mecp2 ) are detectable in up to 95% of cases , although a wide genetical and phenotypical heterogeneity is well established . approximately 80% of rtt clinical cases show the so - called  typical  clinical picture ; after an apparently normal development for 618 months , rtt girls lose their acquired cognitive , social , and motor skills in a typical 4-stage neurological regression . it has become apparent that there is a spectrum of severity in rtt , as some patients may present with atypical features , sometimes overlapping with those suggestive of autism spectrum disorders ( asds ) [ 35 ] . autistic features are typically transient in rtt , although this condition has long been considered as a genetic / epigenetic model of asds [ 6 , 7 ] , rtt has been recently distinct from the asds group [ 8 , 9 ] . recently , the gene sequence analysis indicates that several hundreds of gene mutations appear to be associated with the mecp2 gene mutation and , therefore , are to be considered as potential disease modifiers , although the genetic mechanisms of rtt have been explored to an extraordinary extent , to date the details of the biological mechanisms linking the mecp2 gene mutation to protein expression as a function of clinical phenotype , and yet to be clarified . in particular , with the single exception of a proteomic study on a mouse model , very little information exists on possible rtt - related protein changes . although the neuropathology of rtt is well understood , the cellular and molecular mechanisms , leading to the disease initiation and progression , have yet to be elucidated . several lines of evidence indicate the presence of an early immune activation in asds with an associated peripheral and central chronic inflammation [ 1216 ] , with a particular focus on mast cells dysfunction and cytokines dysregulation [ 12 , 14 ] . to date , experimental and clinical evidence has generated the idea that several serum proteins , considered as biomarkers , are strictly correlated with the pathophysiology of the autistic disorder [ 1720 ] . in particular , significant changes in inflammation - related proteins suggested that at least some autistic children display a subclinical inflammatory state . during inflammation , particularly during the apr , there is a known reduction in several proteins potentially affecting cholesterol transport and inhibiting oxidation phenomena . it is thought that reduction in these proteins , associated with an increase in positive apr proteins , may change the high density lipoprotein from anti - inflammatory into proinflammatory particles . in the present study , we investigated the occurrence of a plasma apr in stage ii ( i.e. ,  pseudo - autistic  ) rtt patients by using routine haematology / clinical chemistry and proteomic 2-de / maldi - tof analyses . the study included 25 female patients with clinical diagnosis of typical rtt ( median age : 5.0 years inter - quartile range 36 , values range 310 years ) with demonstrated mecp2 gene mutation ( r306c ( n = 5 ) , t158 m ( n = 5 ) , r168x ( n = 8) , and large deletions ( deletions of exons 3 and 4 , n = 7 ) ) carrying different phenotype severity , and 40 age - matched healthy controls ( median age : 5.0 years inter - quartile range 35.5 , values range 310 years ) . rtt diagnosis and inclusion / exclusion criteria were based on the recently revised rtt nomenclature consensus . given the specific aims of the study , subjects with clinically evident inflammatory conditions either acute or chronic were excluded , as well as individuals on anti - inflammatory drugs , or undergoing supplementation with known antioxidants , such as -3 polyunsaturated fatty acids . all the patients were consecutively admitted to the rett syndrome national reference centre of the university hospital of the azienda ospedaliera universitaria senese ( aous ) . rtt clinical severity was assessed using either the total clinical severity score ( css ) , a validated clinical rating specifically designed for rtt , or the  compressed  css both based on 13 individual ordinal categories measuring clinical features common in rtt , and phenotypical severity was also measured by the use of the rett syndrome behaviour questionnaire ( rsbq ) . blood samplings in the patients ' group were performed during the routine follow - up study at hospital admission , while the samples from the control group were carried out during routine health checks , sports , or blood donations , obtained during the periodic clinical checks . the healthy control subjects were gender ( given that over 98% of rtt patients are females , we selected a female control group ) and age - matched . the study was conducted with the approval by the institutional review board and all informed consents were obtained from either the parents or the legal tutors of the enrolled patients . all samplings from rtt patients and healthy controls were carried out around 8 a.m. after overnight fasting . blood was collected in heparinized tubes and all manipulations were carried out within 2  h after sample collection . the blood samples were centrifuged at 2400  g for 15  min at 4c ; the platelet poor plasma was saved and the buffy coat was removed by aspiration . the test 1 analyzer , a closed automated analyzer , determines the length of sedimentation reaction in blood in a standard - size primary tube with a perforating stopper . the principle of measurement is the study of the aggregation capacity of red blood cells ( rbcs ) by telemetry . the tubes are placed in appropriate racks , and their contents are rotated slowly for about 2  min . the sample loader simultaneously accepts 4 racks containing 15 tubes each . by using a closed aspiration needle , the blood is directly drawn from the collection tube , distributed in a capillary , and centrifuged at about 20  g. the sensing area temperature is maintained at 37c . the system uses an infrared ray microphotometer with a light wavelength of 950  nm and performs 1,000 readings during 20 seconds . the electrical impulses , collected using a photodiode detector , are directly correlated to the aggregation of rbcs present at each capillary level . for each sample a mathematical algorithm converts the raw data obtained from evaluation of optical density signals into esr results , which are transformed to comparable westergren values . the system operates at a rate of 180 specimens per hour in continuous loading , providing a result every 20 seconds , and requires 150  l of blood for each sample [ 26 , 27 ] . a modular analytics p module ( roche , hitachi ) was used to determine serum crp by immunoturbidimetry using 3rd generation crp tina - quant reagent ( roche , hitachi ) . 2-de is a protein separation technique that combines two different electrophoretic methods : isoelectric focusing ( ief ) in the first dimension , in which proteins are separated according to their isoelectric points ( pi ) , and sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) in the second dimension , in which proteins are separated according to their molecular weights ( mw ) . samples containing 60  g of protein as determined by bradford   were denatured with 10  ml of a solution containing 10% of sds and 2.3% of dithiothreitol ( dtt ) . afterwards , samples were combined with 350  ml of solubilizing buffer containing 8  m urea , 2% of 3-[(3-cholamidopropyl)-dimethylammonium]-1-propane sulfonate ( chaps ) , 0.3% dtt , and 2% of immobilized ph gradient ( ipg ) buffer , and loaded into 18  cm ipg strips 310 nonlinear on an ettan ipgphor ( ge healthcare ) apparatus system , and rehydrated for 7  h. ief was carried out for a total of 32  kv  h. after focusing , the strips were equilibrated with the buffer containing 50  mm tris - hcl , ph 8.8 , 6  m urea , 2%  w / v sds , 30%  v / v glycerol , and 1%  w / v dtt for 15  min . subsequently , strips were equilibrated again with the same equilibration buffer described above , except that it contained 4%  w / v iodoacetamide instead of dtt and a trace of bromophenol blue . the second dimension was performed on an ettandalt six electrophoresis system ( ge healthcare ) . ipg strips and a mw standard were embedded at the top of a 1.5  mm thick vertical polyacrylamide gradient gel ( 816%  t ) using 0.5%  w / v agarose and run at a constant current of 40  ma / gel at 20c . images of gels were analyzed using imagemaster 2d platinum v7.0 software ( ge healthcare ) . the reference gel for each group ( i.e. , healthy controls , rtt , r306c , t158 m , r168x , and large deletions in exons 3 and 4 ) was defined and used for the comparative analyses . the background was subtracted from all gels using the average - on - boundary method . spot volume was expressed as a ratio of the total protein percentage volume ( % v ) detected from the entire gel to minimize differences between gels ( gel normalization ) , for pooling them . only spots appearing in all gels of the same group were matched with those of the reference gel . 2-de / maldi - tof is a complex and advanced technique to identify and characterize proteins from biological fluids and/or tissues whose results are considered to be reliable and reproducible when fitting a series of different parameters , such as peptide matches , sequence coverage ( % ) , mowse score , and pi / relative molecular mass ( mr , kda ) . a 2-de / maldi - tof approach is helpful in order to reveal global protein pattern changes in a given tissue / body fluid for a given condition . however , the technique has assay sensitivity limitations when applied to either less abundant or small proteins ( i.e. , cytokines ) [ 22 , 31 ] . after mass spectrometry compatible silver staining , the preparative gel was matched to the master gel in the analytical gel match set . a spot - picking list was generated and exported to ettan spot picker ( ge healthcare ) . the spots were excised and delivered into 96-well microplates where they were destained and dehydrated with acetonitrile ( acn ) for subsequent rehydration with trypsin solution . each protein spot digest ( 0.75  ml ) was spotted into the maldi instrument target and allowed to dry . then 0.75  ml of the instrument matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid in 50% acn and 0.5%  v / v trifluoroacetic acid ) was applied to dried samples and dried again . mass spectra were obtained , as described in , using an ultraflextreme maldi - tof / tof ( bruker corporation , billerica , ma , usa ) . after tryptic peptide mass acquisition , mass fingerprint searching was carried out in swiss - prot / trembl and ncbinr databases using mascot ( matrix science , london , uk , http://www.matrixscience.com ) . a mass tolerance of 100  ppm was allowed and only one missed cleavage site was accepted . alkylation of cysteine by carbamidomethylation was assumed as a fixed modification , whereas oxidation of methionine was considered a possible modification . the criteria used to accept identifications included the extent of sequence coverage , number of matched peptides , and probabilistic score . possible connections among identified plasma proteins with significant variations as compared to healthy controls expression levels were analyzed by a protein and gene network software . for each protein , uniprotkb entry numbers and related gene names were acquired in uniprotkb and used for network generation by the use of string 9.0 ( http://www.string-db.org/ ) . the uniprotkb entry numbers were inserted into the input form as  multiple proteins  and  homo sapiens  was selected as the reference organism . all variables were tested for normal distribution ( d'agostino - pearson test ) and data were presented as means  sd for normally distributed variables . statistical analysis for protein expressed differently in the groups was carried out using graphpad prism software using student 's t - test and one - way anova test . unmatched spots or spots with significantly different % v were considered  differently expressed  in the groups . comparisons between differently expressed proteins as a function of mecp2 mutation type were evaluated using either mann - whitney rank sum test or kruskal - wallis test . the effects of small population sizes on possible type i ( )/type ii (  ) errors in the data interpretation were examined using a sampling size algorithm . the medcalc version 12.1.4 statistical software ( medcalc software , mariakerke , belgium ) was used . median clinical severity score for the whole rett population was 17 ( inter - quartile range 921.2 , values range 731 ) . elevated esr values ( median 33.0  mm / h versus 8.0  mm / h , p < 0.0001 ) were detectable in rtt ( figure 1 ) , whereas crp levels were within the reference range ( rtt median 0.04  mg / dl inter - quartile range 0.020.08  mg / dl , values range 0.010.18 versus control median 0.04  mg / dl inter - quartile range 0.0010.11  mg / dl , values range 0.010.86 , p = 0.6343 ) . the existence and relevance of an unrecognized subclinical inflammatory status in rett syndrome are strongly suggested by the evidence of relationships between esr values and phenotypical severity of the disease ( figure 4 ) . changes for a total of 17 different proteins were identified and expressed as folds changes relative to controls ( figures 2(a)2(e ) and see supplementary table in supplementary material available online at http://dx.doi.org/10.1155/2014/480980 ) and shown in 2-de maps ( figures 3(a)3(e ) ) . protein identification as well as peptide matches , sequence coverage , and the probabilistic score were obtained using the mascot software . actually , the majority of the proteins were categorized as either positive apr proteins ( n = 6 spots , such as complement factor b ( cfab , spot 1 ) , alpha-1-antitrypsin ( a1at , spot 5 ) , fibrinogen gamma chain ( fibg , spot 6 ) , haptoglobin ( hpt , spots 8 and 15 ) , and serum amyloid a-1 protein ( saa1 , spot 17 ) ) or negative apr proteins ( n = 9 spots , such as serum transferrin ( trfe spot 2 ) , albumin ( albu , spot 3 as whole protein while spots 7 , 11 , and 14 as c terminal fragments ) , transthyretin ( tthy , spots 10 and 16 ) , apolipoprotein a1 ( apoa1 , spot 12 ) , and retinol - binding protein 4 ( ret4 , spot 13 ) ) and , to a lesser extent , as proteins involved in the immune system ( n = 2 spots , such as alpha-2-hs - glycoprotein ( fetua , spot 4 ) and ig gamma-2 chain c region ( ighg2 , spot 9 ) ) . some proteins have known overlapping functions on metabolism and signal transduction ( n = 10 spots , such as trfe ( spot 2 ) , albu ( spots 3 , 7 , 11 , and 14 ) , fibg ( spot 6 ) , tthy ( spots 10 and 16 ) , apoa1 ( spot 12 ) , and ret4 ( spot 13 ) ) . the number of protein changes was found to be proportional to the severity of the mecp2 gene mutation . saa1 , aiat , and cfab were all overexpressed in the examined mecp2 mutation types . the behaviour of hpt appears to be discrepant among different mecp2 mutations rtt , with the 40  kda spot being overexpressed in t158 m and large deletions , while underexpressed in the r306c and normally expressed in the r168x mutation type . the htp 18  kda spot appears to be normally expressed in r306c and t158 m while being overexpressed in the plasma samples from patients with r168x and large deletions . a1at ( spot 5 ) was significantly overexpressed ( p < 0.001 ) in all mecp2 mutation types and varied from + 2.96 in r306c to + 5.15 in large deletions when compared to controls ( figure 2 ) . furthermore , our data indicate that fetua is overexpressed in all the mecp2 mutations examined . on the other hand , ret4 and apoa1 were significantly underexpressed in three of the four examined mecp2 gene mutation classes , that is , t158 m , r168x , and large deletions . the tthy 15  kda spot ( very close to the theoretical mw of 15,900  da ) was found to be underexpressed in all the examined mutation types and the trfe spot appeared to be underexpressed in the r168x mutation type . a detailed analysis of protein expression patterns as a function of mecp2 gene mutation types is reported as supplementary material ( protein expression patterns in the different mecp2 mutation types : detailed analysis ) . in the rtt population identified proteins undergoing significant changes , potentially classifiable as positive apr proteins , negative apr proteins and immune response proteins , appear to be closely connected . string software created a prediction of protein - protein interaction networks ( ppi ) based on confidence ( figure 5(a ) ) , evidence ( figure 5(b ) ) and actions ( figure 5(c ) ) . in the confidence ppi , the thickness of the lines shows how strong the interactions are ( threshold : 0.4 , medium confidence ) . the strongest interactions were between tthy and ret4 ( combined association score : 0.997 ) , apoa1 and albu ( combined association score : 0.994 ) , and tthy and apoa1 ( combined association score : 0.994 ) ( figure 5(a ) ) . positive apr proteins , negative apr proteins , and immune response proteins are highlighted in three different ellipses . in the evidence ppi , the colours of the lines represent the types of evidence which characterized the protein - protein association . more evidence was between tthy and ret4 ( combined association score : 0.997 ) , albu and apoa1 ( combined association score : 0.994 ) , and tthy and apoa1 ( combined association score : 0.993 ) ( figure 5(b ) ) . in the actions ppi , the different colours of the lines represent the mode of action of proteins . code lines : black is reaction between tthy and apoa1 ( combined association score : 0.993 ) , a1at and albu ( combined association score : 0.951 ) , and apoa1 and trfe ( combined association score : 0.982 ) while blue is binding between tthy and ret4 ( combined association score : 0.997 ) , trfe and albu ( combined association score : 0.988 ) , hpt and apoa1 ( combined association score : 0.975 ) , and tthy and trfe ( combined association score : 0.970 ) ( figure 5(c ) ) . etiology of autism is still unknown although several explanations have been proposed , including environmental factors combined to particular genotype , vaccines , and phthalates . experimental and clinical evidence accumulated in the last decades indicates the presence of an early immune activation in asds with an associated peripheral and central chronic inflammation [ 1216 ] . in particular , a subclinical chronic inflammatory condition has been previously suggested to occur in autism . inflammation , as the first nonspecific defensive response in mammalians , is a component of the innate immunity , a self - protective process whose aim is to remove harmful stimuli in order to maintain the homeostasis . chronic inflammation differs from the acute inflammatory processes because of the duration , types of involved cells and primary mediators ( i.e. , cytokines , reactive oxygen species , hydrolytic enzymes , and growth factors versus eicosanoids and vasoactive amines ) , site ( tissue versus vascular ) , and outcome . besides a few isolated studies regarding complement factors , trfe and a1at [ 4143 ] , not consistent information regarding a correlation between our individual identified plasma protein and asds or rtt exist in literature . in particular , a recent report analyzing the response of astrocytes during activation by proinflammatory stimulation   proposes that the protective phenotype against iron - mediated oxidative stress in cell death involves a complex change in the expression and activity of several genes involved in the control of the cellular redox state . therefore , it is plausible that abnormal redox status and unrecognized pro - inflammatory stimuli would converge in rtt patients to functionally damage astrocytes . the present study indicates , for the first time , the occurrence of a subclinical persistent inflammatory status in rtt patients with stage ii ( i.e. ,  pseudo - autistic  ) . in addition , our findings show a direct relationship between the number of the plasma protein quantitatively modified and the clinical severity of the disease . a moderately increased esr value was the only standard laboratory clue for an underlying inflammatory process , whereas , interestingly , other standard routine tests ( i.e. , crp ) appear to be unchanged . at the molecular level , production of crp is induced by proinflammatory cytokines il-1 , il-6 , and il-17 . it is plausible that a cytokine dysregulation may exist in rett syndrome , although no clear demonstration has been so far brought in this sense . a cytokine dysregulation can be inferred by an interesting parallel between rtt and apr protein variations in perinatal human plasma . plasma proteome analysis by 2-de / maldi - tof is known to be able to identify even subtle changes with high specificity in protein identification and recognition of structural changes and posttranslational modifications . this approach has allowed in the present study to unveil the upregulation of several positive apr proteins , such as saa1 and a1at [ 52 , 53 ] , as well as the downregulation of known negative apr proteins , such as apoa1 and ret4 [ 54 , 55 ] . saa1 , a1at , and cfab , known as positive apr proteins , are all overexpressed in the examined mecp2 mutation types . saa1 is a major acute phase reactant and an apolipoprotein belonging to the hdl complex . inflammatory cells chemotaxis , positive regulation of cytokines secretion , and platelet activation are some of its functions . a1at is a multifunctional protein involved in anti - inflammatory and tissue protective properties by protecting tissues from enzymes of inflammatory cells . more broadly , a1at plays an important role in modulating immunity , inflammation , apoptosis , and possibly cellular senescence programs [ 53 , 56 ] . by comparing r168x to t158 m it is interesting to note that a1at ( spot 5 ) is underexpressed , whereas one c - terminal fragment of albu ( spot 11 ) appears to be overexpressed , thus supporting the hypothesis that in the most severe rtt phenotypes more proteolysis occurs along with a lower activity by protease inhibitors , including a1at . cfab , a component of the alternate pathway of the complement system which contributes to generate c3 or c5 convertase and plays a role in the hemolytic uremia complex , appears to be overexpressed in t158 m and large mecp2 gene deletions . hence , we can suppose that the  pseudo - autistic  phase of rtt is characterized by a tissue damage to which an adaptive / defensive response ensues . to this regard , the mounting evidence of a persistent redox imbalance in rtt [ 6 , 58 ] appears to be related to the wider context of a chronic inflammatory process whose fine mechanisms remain to be elucidated . as predicted by the string software ( figure 5 ) the most strong interaction was represented by tthy and ret4 ( combined association score : 0.997 ) and demonstrated by the binding of ret4 to tthy when circulating in plasma ( in a 1 to 1 stoichiometry ) . in vitro tthy has been suggested to be a transporter for thyroxin from the bloodstream to the brain . to this regard , subtle changes in the levels of the thyroid hormones have been reported in rtt , although no evidence of clinical hypothyroidism is present in the affected patients . tthy shows in the proteomic maps , a contrasting behavior , with the 15  kda spot being uniformly underexpressed in all the examined rtt patients , while the 30  kda spot appears to be normally expressed in the r306c and t158 m patients , but overexpressed in the r168x and mecp2 large deletion mutation types . in the light of our findings , the observed underexpression of the tthy 15  kda spot could be interpreted as mirroring the behavior of a negative apr protein . the tthy spot which appears to be corresponding to a higher mw ( exactly 34,400  da ) already reported in the literature a likely source for the mw discrepancy could reside in the reducing experimental condition of 2-de which could lead to splitting of the whole original protein into two subunits . another tthy interaction was with apoa1 ( combined association score : 0.994 ) , demonstrated as relevant not only in physiological condition but also in amyloidosis ( reactome pathways as of october 2012 , visit http://www.reactome.org/ for the latest updates ) . apoa1 and ret4 were found to be significantly underexpressed , as compared to controls , in three out of four examined mecp2 gene mutations ( i.e. , t158  m , r168x , and large deletions ) . both proteins are involved in the lipid metabolism and have been suggested to be involved in the dyslipidemia of children with metabolic syndrome [ 54 , 55 , 63 ] . on the other hand , their involvement in rtt appears to support the concept of an altered lipid metabolism in this condition featuring hyperleptinemia   and hypercholesterolemia   and for which the coexistence of a  fatty acids paradox  has been suggested , given that an excessive endogenous fatty acids oxidation is paradoxically ameliorated by administration of the same exogenous fatty acids [ 66 , 67 ] . interestingly , also the interaction of apoa1 with albu ( combined association score : 0.994 ) takes place in the lipid metabolism particularly in the pathway of hdl - mediated lipid transport . in contrast , the interaction of apoa1 with trfe ( combined association score : 0.993 ) is involved in the release of platelet secretory granule components ( reactome pathways as of october 2012 , visit http://www.reactome.org/ for the latest updates ) . in our findings , trfe was underexpressed in the r168x mutation type , a variation which is in accordance with the classical negative apr protein changes . in another biochemical pathway , hpt can bind apoa1 and impairs its stimulation of lecithin : cholesterol acyltransferase ( lcat ) . hpt is a protein which captures and combines with free plasma hemoglobin to allow hepatic recycling of heme iron in order to prevent kidney damage [ 69 , 70 ] . its behaviour appears to be discrepant among different mecp2 mutations rtt , with the 40  kda spot being overexpressed in t158 m and large deletions , while underexpressed in the r306c and normally expressed in the r168x mutation type . the htp 18  kda spot appears to be normally expressed in r306c and t158 m while being overexpressed in the most severe examined mutations ( i.e. , r168x and large deletions ) . significant changes in rtt plasma were evidenced in fetua and ighg2 both involved in the immune response . in particular , fetua is directly connected with apoa1 ( combined association score : 0.874 ) . this protein is known to promote endocytosis , possess opsonic properties , and influence the mineral phase of bone . , cumulating evidence indicates that osteoporosis is a phenotypic feature of rtt [ 72 , 73 ] . of course , several unsolved questions arise from the present study , including the causes of apr in the autistic phase of rtt . therefore , inflammation seems to be a previously unrecognized feature in autistic and cognitive disorders , which could play a role in the evolution of the pathology and modulations of phenotypical severity . cumulating evidence indicates that rtt is a multisystemic disorder , with the involvement of lung , bone [ 72 , 73 ] , heart , and gastrointestinal apparatus [ 75 , 76 ] , besides the central nervous system . for the first time , we evidenced a subclinical chronic inflammatory status related to the severity carried by mecp2 gene mutations in the  pseudo - autistic our detection of a persistent inflammatory status is compatible with a systemic disease and adds a new perspective in the pathogenesis and future therapeutic strategies for rtt   and asds .
inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment . 
 rett syndrome ( rtt ) is a devastating neurodevelopmental disorder , mainly caused by de novo loss - of - function mutations in the gene encoding mecp2 . here , we investigated plasma acute phase response ( apr ) in stage ii ( i.e. ,  pseudo - autistic  ) rtt patients by routine haematology / clinical chemistry and proteomic 2-de / maldi - tof analyses as a function of four major mecp2 gene mutation types ( r306c , t158 m , r168x , and large deletions ) . elevated erythrocyte sedimentation rate values ( median 33.0  mm / h versus 8.0  mm / h , p < 0.0001 ) were detectable in rtt , whereas c - reactive protein levels were unchanged ( p = 0.63 ) . 
 the 2-de analysis identified significant changes for a total of 17 proteins , the majority of which were categorized as apr proteins , either positive ( n = 6 spots ) or negative ( n = 9 spots ) , and to a lesser extent as proteins involved in the immune system ( n = 2 spots ) , with some proteins having overlapping functions on metabolism ( n = 7 spots ) . 
 the number of protein changes was proportional to the severity of the mutation . 
 our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the  pseudo - autistic  phase of rtt , which is related to the severity carried by the mecp2 gene mutation .