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A Plasma Proteomic Approach in Rett Syndrome: Classical versus Preserved Speech Variant

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A Plasma Proteomic Approach in Rett Syndrome: Classical versus Preserved Speech Variant
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  Hindawi Publishing CorporationMediators of InflammationVolume 󰀲󰀰󰀱󰀳, Article ID 󰀳󰀸󰀶󰀵󰀳, 󰀱󰀰 pageshttp://dx.doi.org/󰀱󰀰.󰀱󰀱󰀵󰀵/󰀲󰀰󰀱󰀳/󰀳󰀸󰀶󰀵󰀳 Research Article  A Plasma Proteomic Approach in Rett Syndrome:Classical versus Preserved Speech Variant  Alessio Cortelazzo, 1,2 Roberto Guerranti, 1 Claudio De Felice, 3 Cinzia Signorini, 4 Silvia Leoncini, 2,4  Alessandra Pecorelli, 2,4 Claudia Landi, 5 Luca Bini, 5 Barbara Montomoli, 2 Claudia Sticozzi, 6 Lucia Ciccoli, 4 Giuseppe Valacchi, 6,7 and Joussef Hayek  2 󰀱 Department of Medical Biotechnologies, University of Siena, Via A. Moro 󰀲, 󰀵󰀳󰀱󰀰󰀰 Siena, Italy  󰀲 Child Neuropsychiatry Unit, University Hospital Azienda Ospedaliera Universitaria Senese (AOUS), Viale M. Bracci 󰀱󰀶,󰀵󰀳󰀱󰀰󰀰 Siena, Italy  󰀳 Neonatal Intensive Care Unit, University Hospital AOUS, Viale M. Bracci 󰀱󰀶, 󰀵󰀳󰀱󰀰󰀰 Siena, Italy   Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro 󰀶, 󰀵󰀳󰀱󰀰󰀰 Siena, Italy  󰀵 Department of Life Science, University of Siena, Via A. Moro 󰀲, 󰀵󰀳󰀱󰀰󰀰 Siena, Italy  󰀶  Department of Life Sciences and Biotechnology, University of Ferrara, Via Borsari 󰀶, 󰀱󰀰󰀰 Ferrara, Italy  󰀷  Department of Food and Nutrition, Kyung Hee University, 󰀱 Hoegi-dong, Dongdaemun-gu, Seoul 󰀱󰀳󰀰-󰀷󰀰󰀱, Republic of Korea Correspondence should be addressed to Joussef Hayek; j.hayek@ao-siena.toscana.itReceived 󰀱󰀹 September 󰀲󰀰󰀱󰀳; Revised 󰀱󰀶 October 󰀲󰀰󰀱󰀳; Accepted 󰀱󰀷 October 󰀲󰀰󰀱󰀳Academic Editor: Paul AshwoodCopyright © 󰀲󰀰󰀱󰀳 Alessio Cortelazzo et al. is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 󰀲 (MeCP󰀲). Although over 󰀲󰀰󰀰 mutations types have been identified so far, nine of which the most frequentones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, includingthe classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasmaproteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same  MECP󰀲 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysedby 󰀲-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to thePSV siblings. A total of five out of six (i.e., 󰀸󰀳.󰀳%) of the overexpressed proteins were well-known acute phase response (APR)proteins,includingalpha-󰀱-microglobulin,haptoglobin,fibrinogenbetachain,alpha-󰀱-antitrypsin,andcomplementC󰀳.erefore,theexaminedRTTsiblingspairsprovedtobeanimportantbenchmarkmodeltotestthemolecularbasisofphenotypicalexpression variability and to identify potential therapeutic targets of the disease. 1. Introduction Rettsyndrome(RTT;OMIMno.󰀳󰀱󰀲󰀷󰀵󰀰),withafrequencyof  ∼ 󰀱:󰀱󰀰󰀰󰀰󰀰–󰀱:󰀱󰀵󰀰󰀰󰀰 females, is a severe and complex neuro-developmental disorder, as well as the second most commoncause of severe mental retardation in the female gender [󰀱].RTT presents in about 󰀷% of cases in a classical form(typical presentation); a󰀀er 󰀶–󰀱󰀸 months of an apparently normal development girls lose their acquired cognitive,social, and motor skills in a typical -stage neurologicalregression. A wide phenotypical heterogeneity is a well-known feature of the disease, which includes at least fourmajor different clinical presentations, that is, classical, pre-served speech (PSV), early seizure (ESV), and congenital variants [󰀲]. Studies have implicated  de novo  mutations of the X-linked methyl-CpG-binding protein 󰀲 (  MECP󰀲 ) gene(OMIM ∗ 󰀳󰀰󰀰󰀰󰀰󰀵) in the majority of the RTT cases, whilemutations in cyclin-dependent kinase-like 󰀵 ( CDKL󰀵 ) andforkheadboxG󰀱( FOXG󰀱 )havebeenmorerarelyreported[󰀳–󰀵]. Typical RTT has been described worldwide, whereas PSV  󰀲 Mediators of Inflammationismorerarelyreported.GirlsaffectedbyPSVhavebeeno󰀀enmisreported with various diagnoses ranging from autism tomental retardation [󰀶, 󰀷]. While the available RTT literature is mainly focusing onthe molecular genetics aspects, very little is known aboutpossible disease-related protein changes, with the singleexceptionofaproteomicstudyonamousemodel[󰀸].Amongthe several hundred RTT sporadic patients examined in theChild Neuropsychiatry Unit of the University Hospital of Siena, Italy, we have encountered two rare familial cases con-sistingoftwopairsofsisterswithRTTthatarephenotypically discordant as previously reported [󰀹]; that is, individuals ineach pair demonstrate extremes of the RTT spectrum, thatis, classical RTT and PSV-RTT. X chromosome inactivation(XCI) status is able to modulate X-linked disorders [󰀱󰀰].However, all four mentioned individuals show a balancedXCI,indicatingthatotherfactorsbeyondXCImaycontributetothephenotypicoutcome[󰀷,󰀱󰀱,󰀱󰀲].Aimofthestudy wasto unveil possible relationships between plasma proteome andphenotypic expression in two cases of familial RTT repre-sented by two pair of sisters, harbor the same  MECP󰀲  genemutation while being dramatically discrepant in phenotype,that is, classical RTT versus PSV. 2. Materials and Methods 󰀲.󰀱. Subjects.  Two pairs of sisters with discordant phenotypeand identical mutation for each pair (pair 󰀱: c.󰀱󰀱󰀵󰀷del󰀳󰀲; pair󰀲: de novo MECP󰀲  deletionincludingexon󰀳andpartofexon) were enrolled in the present study [󰀱󰀲]. Siblings no. 󰀱 (󰀲years old) and no. 󰀲 (󰀳󰀳 years old) exhibits classical RTTand PSV-RTT, respectively. Both sisters showed a balancedXCI and inherited the same mutation from their unaffectedmother, who had a completely skewed XCI [󰀷]. Siblings no.󰀳 (󰀳 years old) and no.  (󰀰 years old) exhibits classicalRTT and PSV-RTT, respectively. XCI status analysis in thiscouple of sisters revealed balanced XCI in both [󰀱󰀲]. eunrelated classical RTT individuals no. 󰀱 and no. 󰀳 couldnot speak and walk and had a profound intellectual deficit,while the PSV individuals no. 󰀲 and no.  could speak andwalk and had a moderate intellectual disability (PSV-RTT).Striking differences in somatic, neurodevelopmental, andneurovegetative features between the sisters were present.A full clinical description of the affected siblings has beenalready reported by Grillo et al. [󰀹]. e diagnostic criteriafor the PSV form of RTT have been previously reported[󰀱󰀳]. Mean classical RTT and PSV scores were, respectively, 27.5 ± 5.3  and  13.8 ± 5.9  (see the two pedigrees in Figure 󰀱).Gender and age-matched controls were also enrolled inthestudy.Bloodsamplingsinthecontrolgroup(  = 10 )werecarriedout,duringroutinehealthchecksorblooddonations,always followed by written informed consent. is study wasapproved by the institutional review board of AOUS, Siena,Italy. 󰀲.󰀲. Blood Sampling.  All samplings from RTT patientsand healthy controls were carried out around 󰀸a.m. a󰀀erovernight fasting. Blood was collected in heparinized tubesandallmanipulationswerecarriedoutwithin󰀲ha󰀀ersamplecollection. e blood samples were centrifuged at 󰀲󰀰󰀰g for󰀱󰀵min at  ∘ C; the platelet poor plasma was saved and thebuffy coat was removed by aspiration. Plasma samples werestored at  − 󰀷󰀰 ∘ C until use. 󰀲.󰀳.  2 -   Analysis.  󰀲-DE was performed according to G¨orget al. [󰀱] with slight modifications. Samples containing󰀶󰀰  g of protein as determined by Bradford [󰀱󰀵] were dena-tured with 󰀱󰀰mL of a solution containing 󰀱󰀰% of sodiumdodecyl sulfate (SDS), 󰀲.󰀳% of dithiothreitol (DTT) heatedto 󰀹󰀵 ∘ C for 󰀵min. e sample was then combined with󰀳󰀵󰀰mL of solubilizing buffer containing 󰀸M urea, 󰀲% of 󰀳-[(󰀳-cholamidopropyl)-dimethylammonio]-󰀱-propane sul-fonate (CHAPS), 󰀰.󰀳% DTT, 󰀲% of immobilized pH gradient(IPG)buffer,andatraceofbromophenolblueandloadedinto󰀱󰀸cm IPG strips 󰀳–󰀱󰀰NL on an Ettan IPGphor (GE Health-care) apparatus system and rehydrated for 󰀷h. Isoelectricfocusing (IEF) was carried out for a total of 󰀳󰀲kVh. A󰀀erfocusing, the strips were first equilibrated with equilibrationbuffer containing 󰀵󰀰mM Tris-HCl, pH 󰀸.󰀸, 󰀶M urea, 󰀲% w/v SDS, 󰀳󰀰% v/v glycerol, and 󰀱% w/v DTT for 󰀱󰀵min; then they were equilibrated again with the same equilibration bufferdescribed above, except that it contained % w/v iodoac-etamide instead of DTT and a trace of bromophenol blue.e strips were washed further for 󰀱󰀰min with Tris-glycinebuffer.eseconddimensionwasperformedonanEttanDaltSix Electrophoresis system (GE Healthcare). IPG strips anda molecular weight standard were embedded at the top of a󰀱.󰀵mm thick vertical polyacrylamide gradient gel (󰀸–󰀱󰀶%T)using 󰀰.󰀵% w/v agarose and run at a constant current of 󰀰mA/gel at 󰀲󰀰 ∘ C. Each sample was carried out in triplicateunder the same conditions. e exposure time for silverstaining was also optimized to avoid overexposure of somegels with respect to others. 󰀲.. Tryptic Digestion and MALDI-TOF MS.  A󰀀er massspectrometry compatible silver staining [󰀲󰀸], the preparativegel was matched to the master gel in the analytical gel matchset. A spot-picking list was generated and exported to EttanSpot Picker (GE Healthcare). e spots were excised anddeliveredinto󰀹󰀶-wellmicroplateswheretheyweredestainedanddehydratedwithacetonitrile(ACN)forsubsequentrehy-dration with trypsin solution. Tryptic digestion was carriedout overnight at 󰀳󰀷 ∘ C. Each protein spot digest (󰀰.󰀷󰀵mL)was spotted into the MALDI instrument target and allowedto dry. en 󰀰.󰀷󰀵mL of the instrument matrix solution(saturated solution of    -cyano--hydroxycinnamic acid in󰀵󰀰% ACN and 󰀰.󰀵% v/v trifluoroacetic acid) was applied todried samples and dried again. Mass spectra were obtained,as described [󰀲󰀹], using an ultrafleXtreme MALDI-ToF/ToF(Bruker Corporation, Billerica, MA, USA). 󰀲.󰀵. Protein Identification by MS.  A󰀀er tryptic peptide massacquisition, mass fingerprint searching was carried out inSwiss-Prot/TREMBLandNCBInrdatabasesusingMASCOT(Matrix Science, London, UK, http://www.matrixscience.com/). A mass tolerance of 󰀱󰀰󰀰ppm was allowed and only one missed cleavage site was accepted. Alkylation of cysteine  Mediators of Inflammation 󰀳 RTT sisters family 1No. 1No. 2No. 4No. 3Severity score: 30Severity score: 10RTT sisters family 2Severity score: 33Severity score: 7Healthy control(kDa)150100755037252015345678910pHNo. 1, classical RTT(kDa)150100755037252015345678910pHNo. 2, PSV-RTT(kDa)150100755037252015345678910pHNo. 3, classical RTT(kDa)150100755037252015345678910pHNo. 4, PSV-RTT(kDa)150100755037252015345678910pH F󰁩󰁧󰁵󰁲󰁥 󰀱: In the pedigrees the two RTT sisters families are represented by grey circles (milder variant = preserved speech variant, PSV-RTT)or black circles (more severe phenotype = classical RTT) with their respective clinical scores as derived by the approbation of phenotypicalseverity scale [󰀹]. In the 󰀲-DE maps typical control plasma proteome (healthy control), RTT sisters Family 󰀱 (no. 󰀱, no. 󰀲) and RTT sistersFamily󰀲(no.󰀳,no.)areshown.Arrowsindicatetheproteinspotswithsignificantvariationsintheirmajororminorrelativevolume;circlesare used to indicate the absence of the spots (i.e., qualitative variations). by carbamidomethylation was assumed as a fixed modifica-tion, whereas oxidation of methionine was considered a pos-sible modification. e criteria used to accept identificationsincludedtheextentofsequencecoverage,numberofmatchedpeptides, and probabilistic score. 󰀲.󰀶. Image and Statistical Analysis.  Images of gels wereanalyzed using ImageMaster 󰀲D Platinum v󰀷.󰀰 so󰀀ware (GEHealthcare). e reference gel for each group (i.e., RTT,controls, classical RTT, PSV-RTT, RTT sisters Family 󰀱, RTTsisters Family 󰀲, and cases no. 󰀱, no. 󰀲, no. 󰀳, and no. )wasdefinedandusedforthecomparativeanalyses.Statisticalanalysis for protein differently expressed in the groups wascarriedoutusingGraphPadPrismso󰀀wareandtheMedCalc version󰀱󰀲.󰀱.statisticalso󰀀warepackage(MedCalcSo󰀀ware,Mariakerke, Belgium) was used. All variables were tested   Mediators of Inflammation T󰁡󰁢󰁬󰁥 󰀱: Identification of plasma proteins in RTT patients and healthy controls by MS.ID AC a Protein name ShortnamepI/Mr(kDa)predictedpI/Mr (kDa)experimentalPeptidematchesSequencecoverage(%)MOWSEscoreBiological process involved;molecular function; references󰀱 P󰀰󰀲󰀷󰀶󰀰 Alpha-󰀱-microglobulin AMBP 󰀵.󰀰󰀷/󰀳󰀰.󰀹 󰀵/󰀳󰀱.󰀱 󰀹/󰀱󰀵 󰀲󰀵 󰀷󰀷 Host-virus interaction; trypsinand plasmin inhibitor; [󰀱󰀶]󰀲 P󰀱󰀰󰀹󰀰󰀹 Clusterin CLUS .󰀹/󰀳󰀶.󰀹 .󰀸/󰀳󰀶. 󰀱󰀲/󰀱󰀸 󰀲󰀵 󰀱󰀶Chaperone; preventsstress-induced aggregation of blood plasma proteins; [󰀱󰀷]󰀳 P󰀰󰀰󰀷󰀳󰀸 Haptoglobin HPT 󰀵./󰀱󰀶.󰀸 󰀵.󰀲/󰀱󰀷 󰀶/󰀱 󰀱󰀹 󰀷󰀵Immunity; captures hemoglobin,antimicrobial, and antioxidant;[󰀱󰀸] P󰀰󰀰󰀷󰀳󰀸 Haptoglobin HPT 󰀶.󰀰󰀷/󰀱󰀶.󰀸 󰀵.󰀹/󰀱󰀷 󰀶/󰀱󰀳 󰀱󰀹 󰀷󰀳Immunity; captures hemoglobin,antimicrobial, and antioxidant;[󰀱󰀸]󰀵 P󰀰󰀲󰀶󰀷󰀵 Fibrinogen beta chain FIBB 󰀶./󰀵󰀵.󰀲 󰀶.󰀶/󰀵󰀵.󰀲 󰀳󰀷/󰀸󰀸 󰀶󰀰 󰀲󰀳󰀱 Blood coagulation andhemostasis; [󰀱󰀹]󰀶 P󰀰󰀲󰀷󰀶󰀸 Serum albumin ALBU 󰀵.󰀶/󰀶󰀷.󰀷 󰀵.󰀸/󰀶󰀸 󰀵/󰀷 󰀸 󰀵󰀶Regulation of the osmotic bloodpressure; binds ions, hormones,and fatty acids; [󰀲󰀰]󰀷 P󰀰󰀱󰀵󰀹󰀱 Immunoglobulin J chain IGJ .󰀵/󰀲󰀳. .󰀵/󰀲 󰀵/󰀱󰀸 󰀳󰀲 󰀶󰀱 Immunity; links two monomerunits of either IgM or IgA; [󰀲󰀱]󰀸 P󰀶󰀸󰀸󰀷󰀱 Hemoglobin subunit beta HBB 󰀶.󰀸/󰀱󰀰.󰀵 󰀶./󰀱󰀲.󰀵 󰀶/󰀹 󰀵󰀳 󰀱󰀱󰀰 Oxygen transport; [󰀲󰀲]󰀹 P󰀰󰀱󰀰󰀰󰀹 Alpha-󰀱-antitrypsin A󰀱AT .󰀸/󰀵󰀰.󰀳 .󰀸/󰀵󰀰.󰀲 󰀱󰀰/󰀱 󰀳󰀲 󰀱󰀱 Serine proteases inhibitor; [󰀲󰀳]󰀱󰀰 P󰀶󰀸󰀸󰀷󰀱 Hemoglobin subunit beta HBB 󰀷.󰀰󰀵/󰀱󰀰.󰀵 󰀶.󰀸/󰀱󰀲.󰀵 󰀱󰀵/󰀳󰀱 󰀹󰀵 󰀲󰀲󰀰 Oxygen transport; [󰀲󰀲]󰀱󰀱 P󰀰󰀱󰀸󰀵󰀹 Ig gamma-󰀲 chain C region IGHG󰀲 󰀶.󰀱/󰀲. 󰀶/󰀲.󰀶 󰀷/󰀰 󰀱󰀷  Immunity; antigen binding; [󰀲]󰀱󰀲 P󰀰󰀲󰀷󰀸󰀷 Serum transferrin TRFE 󰀶.󰀳/󰀸󰀰.󰀷 󰀶.󰀳/󰀷󰀹.󰀳 󰀳󰀶/󰀷󰀱 󰀵 󰀳󰀱󰀱Iron binding transport proteinswhich can bind two Fe 󰀳+ ions;[󰀲󰀵]󰀱󰀳 P󰀰󰀱󰀰󰀲 Complement C󰀳 CO󰀳 󰀶.󰀶/󰀷󰀰.󰀶 󰀶.󰀸/󰀶󰀹.󰀷 󰀱󰀶/󰀲󰀱 󰀱 󰀱Immunity; central role in theactivation of the complementsystem; [󰀲󰀶]󰀱 P󰀰󰀲󰀷󰀶󰀶 Transthyretin TTHY 󰀵.󰀵/󰀳󰀵.󰀳 󰀵./󰀳. 󰀹/󰀲󰀷 󰀷󰀷 󰀱󰀳󰀶 yroid hormone-bindingprotein; [󰀲󰀷] Spot ID refers to that shown in 󰀲-DE maps (Figure 󰀱).  a Accession numbers of Swiss-Prot or GenBanK databases. fornormaldistribution(D’Agostino-Pearsontest).Datawereexpressed as median values and interquartile range, unlessotherwisestated.Unmatchedspotsorspotswithsignificantly differentpercentagevolume(%V)wereconsideredas“differ-ently expressed”. Differences between groups were tested by thenonparametricMann-WhitneyranksumtestorKruskal-Wallis analysis of variance, as appropriate. A two-sided   <0.05  was considered to indicate statistical significance. 3. Results To better characterize the RTT plasma protein pattern, wecarried out a proteomic analysis based on 󰀵 different analyti-cal groups: (󰀱) classical RTT versus PSV-RTT, (󰀲) RTT versuscontrols,(󰀳)RTTsistersFamily󰀱versusRTTsistersFamily󰀲,()no.󰀱classicalRTTversusno.󰀳classicalRTT,and(󰀵)no.󰀲PSV-RTT versus no.  PSV-RTT. Among these groups therewere significant quantitative and qualitative variations in 󰀱protein spots subsequently identified by mass spectrometry.Protein name as well as peptide matches, sequence coverage,and the probabilistic score obtained using the MASCOTso󰀀warearesummarized(Table 󰀱).Alltheidentifiedproteinsareknowntobeinvolvedinspecificbiologicalprocesses[󰀱󰀶–󰀲󰀷]. Proteomic plasma maps of healthy control, RTT sistersFamily 󰀱, and RTT sisters Family 󰀲 with the protein spotsare represented (Figure 󰀱). Black arrows indicate the spotswithquantitativevariationswhilealltheidentifiedqualitative variations are reported with black circles.As shown in Figure 󰀲, significant changes appeared inalpha-󰀱-microglobulin(AMBP),haptoglobin(HPT/Hp,spots󰀳 and ), fibrinogen beta chain (FIBB), complement C󰀳(CO󰀳), and transthyretin (TTHY) in classical RTT siblingsas compared to PSV-RTT sisters. In addition, quantitativeand qualitative protein variations values as derived from theexamined RTT sister pairs and healthy controls comparativeanalyses were reported (Tables 󰀲 and 󰀳). RTT patients, when compared to control group, showed󰀶 underexpressed protein spots including FIBB, hemoglobinsubunit beta (HBB), serum transferrin (TRFE), HPT, Iggamma-󰀲 chain C region (IGHG󰀲), and CO󰀳, while 󰀱 spot of clusterin (CLUS) is overexpressed (Table 󰀳).  Mediators of Inflammation 󰀵    C    h  a  n  g  e  s    (    f  o    l    d  s    ) Classical RTT versus PSV-RTTKruskal-wallis ANOVA,  P < 0.0001−4−2 02468    A   M   B   P   C   L   U   S   H   P   T  s   p  o   t   #   3   H   P   T  s   p  o   t   #   4   F   I   B   B   A   L   B   U   I   G   J   H   B   B  s   p  o   t   #   8   A   1   A   T   H   B   B  s   p  o   t   #   1   0   I   G   H   G   2   T   R   F   E   C   O   3   T   T   H   Y F󰁩󰁧󰁵󰁲󰁥 󰀲: Plasma proteins expression in sisters with classical Rettsyndrome as protein expression ratios of classical RTT versus PSV-RTT plasma proteome. Data are expressed as box-and-whiskersplots. Results of the Kruskal-Wallis ANOVA are shown. Family 󰀱 versus Family 󰀲 (third group in Table 󰀳) showeda significant underexpression of TTHY, a significant overex-pression of HBB, and the appearance of one protein spot of albumin (ALBU). e comparison between the two classicalforms showed underexpression of  protein spots (AMBP,CLUS, IGHG󰀲, and TTHY) and appearance of 󰀳 proteinspots (ALBU, HBB, and A󰀱AT) in no. 󰀱 as compared to no.󰀳. Significant qualitative variations are most evident in thecomparisonbetweenthetwoPSVvariantsinwhich󰀵proteinspots appeared (AMBP, CLUS, ALBU, immunoglobulin Jchain, IGJ and CO󰀳) while 󰀲 proteins (HBB and TTHY)disappeared in no. 󰀲 as compared to no. . Moreover, inthe same comparative group, an overexpression of HPT wasobserved(Table 󰀳).Inaddition,anothercomparativeanalysisof each RTT patient versus healthy controls has resulted inseveral significant quantitative and qualitative variations inplasma proteome (see Table  in Supplementary Materialavailableonlineathttp://dx.doi.org/󰀱󰀰.󰀱󰀱󰀵󰀵/󰀲󰀰󰀱󰀳/󰀳󰀸󰀶󰀵󰀳)andanumberofproteinspotschangeslikelyduetothesizeeffectsrcinating from the healthy control group were detected butconsidered to be not significant (data not shown) and notcomparable ( n.c. , Table 󰀲). 4. Discussion Proteomic analysis has proven effective in identifying vari-ations in proteins with biological and/or clinical signifi-cance [󰀳󰀰]. e examined RTT siblings pairs represented aninteresting benchmark model to test the molecular basis of phenotypical expression variability and to identify potentialtherapeutic targets of the disease.Rettsyndromeistheresultofamonogenicmutation,thatis, the X-linked  MECP󰀲  gene in the overwhelming majority of cases. As RTT is an X-linked trait and the  MECP󰀲  locus issubject to X inactivation, different patterns of X inactivationmay lead to different phenotypes within a group of patientswho carry the same mutation. Based on these data someauthorsspeculatethattheremightbeagroupofRTTpatientswith milder phenotypes owing to skewed X inactivation,who have not so far been identified because of their atypicalphenotypes. Nonetheless, variations in XCI are known toexplain only 󰀱/󰀵 of the variance in severity of the disease [󰀳󰀱],thus not fully accounting for the phenotype severity rangetypically seen in RTT [󰀳󰀲].We can safely state that in our patients, as well as in themajority of Rett syndrome patients reported in the literature,Xinactivationwasfoundtobebalanced.us,itisreasonableto assume that the clinical phenotype of our pairs of sistersappears to be determined mainly by the type and location of the  MECP󰀲  mutations.Statistical analysis, represented by the fold changes,revealed in the classical RTT versus PSV-RTT comparisona significant overexpression of proteins involved in APR including AMBP, HPT, FIBB, A󰀱AT, and CO󰀳 [󰀱󰀶, 󰀱󰀸, 󰀲󰀳, 󰀲󰀶]. Although little is known about the APR and the frequency of infections in RTT [󰀳󰀳], our findings evidenced a lack of some key APR components. Possible explanations for thesefindings may include a continuous stimulation of cytokine-mediated liver protein synthesis, an accelerated turnover of APRproteins,oracombinationofboth.eevidencethattheRTT patients present chronic terminal bronchiolitis and anincreaseinintestinalmicrobiomeduetoconstipationsuggestthe coexistence of recurrent infections [󰀳, 󰀳󰀵]. Evidence of  the involvement of inflammatory events in RTT, was mainly represented by the significant variations of AMBP and A󰀱AT(fi󰀀h and sixth comparative groups in Table 󰀳), two serineprotease inhibitors linked to the acute phase reaction, whichlimit the damage caused by activated neutrophils and theirenzymeelastase[󰀱󰀶,󰀲󰀳].Amajorrolefortheimmunesystem in RTT pathogenesishas been previouslydocumented by thefact that transplantation of wild-type bone marrow restoreswild-type microglia and arrests pathology in a mouse modelof RTT [󰀳󰀶].e other finding on a partial deficit in an oxygentransport HBB [󰀲󰀲] could be compatible with our priorfinding of a subclinical hypoxia with an altered redox statusin RTT patients with the classical phenotype [󰀳󰀷].Interestingly, TRFE was significantly underexpressed inRTT as compared to healthy controls, confirming the asso-ciation previously reported in autism [󰀳󰀸]. Alterations inthe TRFE levels may lead to abnormal iron metabolismin RTT; it has been suggested for autism [󰀳󰀸]. On theother hand, TRFE is also a negative APR protein whoseexpressionlevelsdecreaseduringinflammation[󰀲󰀵].ustheunderexpression of TRFE in RTT suggests once again thatinflammatoryprocessmayplayakeyroleinthepathogenesisof the disease.More intriguing is the finding of an overexpressed CLUS,which may reflect a counterbalanced response to exces-sive proteins accumulation, namely, the unfolded proteinresponse [󰀱󰀷]. Abundant evidences demonstrated that CLUSexpression is increased during cellular stress [󰀱󰀷]. e chap-erone action of CLUS could be cytoprotective in either or
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