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

A Plasma Proteomic Approach in Rett Syndrome: Classical versus Preserved Speech Variant
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  Hindawi Publishing CorporationMediators o In󿬂ammationVolume 󰀲󰀰󰀱󰀳, Article ID 󰀴󰀳󰀸󰀶󰀵󰀳, 󰀱󰀰 pages󰀱󰀰.󰀱󰀱󰀵󰀵/󰀲󰀰󰀱󰀳/󰀴󰀳󰀸󰀶󰀵󰀳 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 Jousse Hayek; j.hayek@ao-siena.toscana.itReceived 󰀱󰀹 September 󰀲󰀰󰀱󰀳; Revised 󰀱󰀶 October 󰀲󰀰󰀱󰀳; Accepted 󰀱󰀷 October 󰀲󰀰󰀱󰀳Academic Editor: Paul AshwoodCopyright © 󰀲󰀰󰀱󰀳 Alessio Cortelazzo et al. Tis 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 (R) 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 identi󿬁ed so ar, nine o which the most requentones. A wide phenotypical heterogeneity is a well-known eature o the disease, with different clinical presentations, includingthe classical orm and the preserved speech variant (PSV). Aim o the study was to unveil possible relationships between plasmaproteome and phenotypic expression in two cases o amilial R represented by two pairs o sisters, harbor the same  MECP󰀲 gene mutation while being dramatically discrepant in phenotype, that is, classical R versus PSV. Plasma proteome was analysedby 󰀲-DE/MALDI-OF MS. A signi󿬁cant overexpression o six proteins in the classical sisters was detected as compared to thePSV siblings. A total o 󿬁ve out o six (i.e., 󰀸󰀳.󰀳%) o the overexpressed proteins were well-known acute phase response (APR)proteins,includingalpha-󰀱-microglobulin,haptoglobin,󿬁brinogenbetachain,alpha-󰀱-antitrypsin,andcomplementC󰀳.Tereore,theexaminedRsiblingspairsprovedtobeanimportantbenchmarkmodeltotestthemolecularbasisophenotypicalexpression variability and to identiy potential therapeutic targets o the disease. 1. Introduction Rettsyndrome(R;OMIMno.󰀳󰀱󰀲󰀷󰀵󰀰),witharequencyo  ∼ 󰀱:󰀱󰀰󰀰󰀰󰀰–󰀱:󰀱󰀵󰀰󰀰󰀰 emales, is a severe and complex neuro-developmental disorder, as well as the second most commoncause o severe mental retardation in the emale gender [󰀱].R presents in about 󰀷󰀴% o cases in a classical orm(typical presentation); afer 󰀶–󰀱󰀸 months o 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 eature o the disease, which includes at least ourmajor different clinical presentations, that is, classical, pre-served speech (PSV), early seizure (ESV), and congenital variants [󰀲]. Studies have implicated  de novo  mutations o the X-linked methyl-CpG-binding protein 󰀲 (  MECP󰀲 ) gene(OMIM ∗ 󰀳󰀰󰀰󰀰󰀰󰀵) in the majority o the R cases, whilemutations in cyclin-dependent kinase-like 󰀵 ( CDKL󰀵 ) andorkheadboxG󰀱( FOXG󰀱 )havebeenmorerarelyreported[󰀳– 󰀵]. ypical R has been described worldwide, whereas PSV  󰀲 Mediators o In󿬂ammationismorerarelyreported.GirlsaffectedbyPSVhavebeenofenmisreported with various diagnoses ranging rom autism tomental retardation [󰀶, 󰀷]. While the available R literature is mainly ocusing onthe molecular genetics aspects, very little is known aboutpossible disease-related protein changes, with the singleexceptionoaproteomicstudyonamousemodel[󰀸].Amongthe several hundred R sporadic patients examined in theChild Neuropsychiatry Unit o the University Hospital o Siena, Italy, we have encountered two rare amilial cases con-sistingotwopairsosisterswithRthatarephenotypically discordant as previously reported [󰀹]; that is, individuals ineach pair demonstrate extremes o the R spectrum, thatis, classical R and PSV-R. X chromosome inactivation(XCI) status is able to modulate X-linked disorders [󰀱󰀰]. However, all our mentioned individuals show a balancedXCI,indicatingthatotheractorsbeyondXCImaycontributeto the phenotypic outcome [󰀷, 󰀱󰀱, 󰀱󰀲]. Aim o the study was to unveil possible relationships between plasma proteome andphenotypic expression in two cases o amilial R repre-sented by two pair o sisters, harbor the same  MECP󰀲  genemutation while being dramatically discrepant in phenotype,that is, classical R versus PSV. 2. Materials and Methods 󰀲.󰀱. Subjects.  wo pairs o sisters with discordant phenotypeand identical mutation or each pair (pair 󰀱: c.󰀱󰀱󰀵󰀷del󰀳󰀲; pair󰀲:  de novo MECP󰀲  deletion includingexon 󰀳 and part o exon󰀴) were enrolled in the present study [󰀱󰀲]. Siblings no. 󰀱 (󰀴󰀲years old) and no. 󰀲 (󰀳󰀳 years old) exhibits classical Rand PSV-R, respectively. Both sisters showed a balancedXCI and inherited the same mutation rom their unaffectedmother, who had a completely skewed XCI [󰀷]. Siblings no. 󰀳 (󰀳󰀴 years old) and no. 󰀴 (󰀴󰀰 years old) exhibits classicalR and PSV-R, respectively. XCI status analysis in thiscouple o sisters revealed balanced XCI in both [󰀱󰀲]. Te unrelated classical R individuals no. 󰀱 and no. 󰀳 couldnot speak and walk and had a proound intellectual de󿬁cit,while the PSV individuals no. 󰀲 and no. 󰀴 could speak andwalk and had a moderate intellectual disability (PSV-R).Striking differences in somatic, neurodevelopmental, andneurovegetative eatures between the sisters were present.A ull clinical description o the affected siblings has beenalready reported by Grillo et al. [󰀹]. Te diagnostic criteria or the PSV orm o R have been previously reported[󰀱󰀳]. Mean classical R 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 ollowed by written inormed consent. Tis study wasapproved by the institutional review board o AOUS, Siena,Italy. 󰀲.󰀲. Blood Sampling.  All samplings rom R patientsand healthy controls were carried out around 󰀸a.m. aferovernight asting. Blood was collected in heparinized tubesandallmanipulationswerecarriedoutwithin󰀲hafersamplecollection. Te blood samples were centriuged at 󰀲󰀴󰀰󰀰g or󰀱󰀵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 perormed according to G¨orget al. [󰀱󰀴] with slight modi󿬁cations. Samples containing󰀶󰀰 󝠵 g o protein as determined by Bradord [󰀱󰀵] were dena-tured with 󰀱󰀰mL o a solution containing 󰀱󰀰% o sodiumdodecyl sulate (SDS), 󰀲.󰀳% o dithiothreitol (D) heatedto 󰀹󰀵 ∘ C or 󰀵min. Te sample was then combined with󰀳󰀵󰀰mL o solubilizing buffer containing 󰀸M urea, 󰀲% o 󰀳-[(󰀳-cholamidopropyl)-dimethylammonio]-󰀱-propane sul-onate (CHAPS), 󰀰.󰀳% D, 󰀲% o immobilized pH gradient(IPG)buffer,andatraceobromophenolblueandloadedinto󰀱󰀸cm IPG strips 󰀳–󰀱󰀰NL on an Ettan IPGphor (GE Health-care) apparatus system and rehydrated or 󰀷h. Isoelectricocusing (IEF) was carried out or a total o 󰀳󰀲kVh. Aferocusing, the strips were 󿬁rst equilibrated with equilibrationbuffer containing 󰀵󰀰mM ris-HCl, pH 󰀸.󰀸, 󰀶M urea, 󰀲% w/v SDS, 󰀳󰀰% v/v glycerol, and 󰀱% w/v D or 󰀱󰀵min; then they were equilibrated again with the same equilibration bufferdescribed above, except that it contained 󰀴% w/v iodoac-etamide instead o D and a trace o bromophenol blue.Te strips were washed urther or 󰀱󰀰min with ris-glycinebuffer.TeseconddimensionwasperormedonanEttanDaltSix Electrophoresis system (GE Healthcare). IPG strips anda molecular weight standard were embedded at the top o a󰀱.󰀵mm thick vertical polyacrylamide gradient gel (󰀸–󰀱󰀶%)using 󰀰.󰀵% w/v agarose and run at a constant current o 󰀴󰀰mA/gel at 󰀲󰀰 ∘ C. Each sample was carried out in triplicateunder the same conditions. Te exposure time or silverstaining was also optimized to avoid overexposure o somegels with respect to others. 󰀲.󰀴. Tryptic Digestion and MALDI-TOF MS.  Afer 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). Te spots were excised anddeliveredinto󰀹󰀶-wellmicroplateswheretheyweredestainedanddehydratedwithacetonitrile(ACN)orsubsequentrehy-dration with trypsin solution. ryptic digestion was carriedout overnight at 󰀳󰀷 ∘ C. Each protein spot digest (󰀰.󰀷󰀵mL)was spotted into the MALDI instrument target and allowedto dry. Ten 󰀰.󰀷󰀵mL o the instrument matrix solution(saturated solution o    -cyano-󰀴-hydroxycinnamic acid in󰀵󰀰% ACN and 󰀰.󰀵% v/v tri󿬂uoroacetic acid) was applied todried samples and dried again. Mass spectra were obtained,as described [󰀲󰀹], using an ultra󿬂eXtreme MALDI-oF/oF(Bruker Corporation, Billerica, MA, USA). 󰀲.󰀵. Protein Identi󿬁cation by MS.  Afer tryptic peptide massacquisition, mass 󿬁ngerprint searching was carried out inSwiss-Prot/REMBLandNCBInrdatabasesusingMASCO(Matrix Science, London, UK, A mass tolerance o 󰀱󰀰󰀰ppm was allowed and only one missed cleavage site was accepted. Alkylation o cysteine  Mediators o In󿬂ammation 󰀳 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 R sisters amilies are represented by grey circles (milder variant = preserved speech variant, PSV-R)or black circles (more severe phenotype = classical R) with their respective clinical scores as derived by the approbation o phenotypicalseverity scale [󰀹]. In the 󰀲-DE maps typical control plasma proteome (healthy control), R sisters Family 󰀱 (no. 󰀱, no. 󰀲) and R sisters Family 󰀲 (no. 󰀳, no. 󰀴) are shown. Arrows indicate the protein spots with signi󿬁cant variations in their major or minor relative volume; circlesare used to indicate the absence o the spots (i.e., qualitative variations). by carbamidomethylation was assumed as a 󿬁xed modi󿬁ca-tion, whereas oxidation o methionine was considered a pos-sible modi󿬁cation. Te criteria used to accept identi󿬁cationsincludedtheextentosequencecoverage,numberomatchedpeptides, and probabilistic score. 󰀲.󰀶. Image and Statistical Analysis.  Images o gels wereanalyzed using ImageMaster 󰀲D Platinum v󰀷.󰀰 sofware (GEHealthcare). Te reerence gel or each group (i.e., R,controls, classical R, PSV-R, R sisters Family 󰀱, Rsisters Family 󰀲, and cases no. 󰀱, no. 󰀲, no. 󰀳, and no. 󰀴)wasde󿬁nedandusedorthecomparativeanalyses.Statisticalanalysis or protein differently expressed in the groups wascarried out using GraphPad Prism sofware and the MedCalc version󰀱󰀲.󰀱.󰀴statisticalsofwarepackage(MedCalcSofware,Mariakerke, Belgium) was used. All variables were tested  󰀴 Mediators o In󿬂ammation 󰁡󰁢󰁬󰁥 󰀱: Identi󿬁cation o plasma proteins in R patients and healthy controls by MS.ID AC a Protein name ShortnamepI/Mr(kDa)predictedpI/Mr (kDa)experimentalPeptidematchesSequencecoverage(%)MOWSEscoreBiological process involved;molecular unction; reerences󰀱 P󰀰󰀲󰀷󰀶󰀰 Alpha-󰀱-microglobulin AMBP 󰀵.󰀰󰀷/󰀳󰀰.󰀹 󰀵/󰀳󰀱.󰀱 󰀹/󰀱󰀵 󰀲󰀵 󰀷󰀷 Host-virus interaction; trypsinand plasmin inhibitor; [󰀱󰀶] 󰀲 P󰀱󰀰󰀹󰀰󰀹 Clusterin CLUS 󰀴.󰀹/󰀳󰀶.󰀹 󰀴.󰀸/󰀳󰀶.󰀴 󰀱󰀲/󰀱󰀸 󰀲󰀵 󰀱󰀴󰀶Chaperone; preventsstress-induced aggregation o blood plasma proteins; [󰀱󰀷] 󰀳 P󰀰󰀰󰀷󰀳󰀸 Haptoglobin HP 󰀵.󰀴/󰀱󰀶.󰀸 󰀵.󰀲/󰀱󰀷 󰀶/󰀱󰀴 󰀱󰀹 󰀷󰀵Immunity; captures hemoglobin,antimicrobial, and antioxidant;[󰀱󰀸]󰀴 P󰀰󰀰󰀷󰀳󰀸 Haptoglobin HP 󰀶.󰀰󰀷/󰀱󰀶.󰀸 󰀵.󰀹/󰀱󰀷 󰀶/󰀱󰀳 󰀱󰀹 󰀷󰀳Immunity; captures hemoglobin,antimicrobial, and antioxidant;[󰀱󰀸]󰀵 P󰀰󰀲󰀶󰀷󰀵 Fibrinogen beta chain FIBB 󰀶.󰀴/󰀵󰀵.󰀲 󰀶.󰀶/󰀵󰀵.󰀲 󰀳󰀷/󰀸󰀸 󰀶󰀰 󰀲󰀳󰀱 Blood coagulation andhemostasis; [󰀱󰀹] 󰀶 P󰀰󰀲󰀷󰀶󰀸 Serum albumin ALBU 󰀵.󰀶/󰀶󰀷.󰀷 󰀵.󰀸/󰀶󰀸 󰀵/󰀷 󰀸 󰀵󰀶Regulation o the osmotic bloodpressure; binds ions, hormones,and atty acids; [󰀲󰀰] 󰀷 P󰀰󰀱󰀵󰀹󰀱 Immunoglobulin J chain IGJ 󰀴.󰀵/󰀲󰀳.󰀴 󰀴.󰀵/󰀲󰀴 󰀵/󰀱󰀸 󰀳󰀲 󰀶󰀱 Immunity; links two monomerunits o either IgM or IgA; [󰀲󰀱]󰀸 P󰀶󰀸󰀸󰀷󰀱 Hemoglobin subunit beta HBB 󰀶.󰀸/󰀱󰀰.󰀵 󰀶.󰀴/󰀱󰀲.󰀵 󰀶/󰀹 󰀵󰀳 󰀱󰀱󰀰 Oxygen transport; [󰀲󰀲] 󰀹 P󰀰󰀱󰀰󰀰󰀹 Alpha-󰀱-antitrypsin A󰀱A 󰀴.󰀸/󰀵󰀰.󰀳 󰀴.󰀸/󰀵󰀰.󰀲 󰀱󰀰/󰀱󰀴 󰀳󰀲 󰀱󰀴󰀱 Serine proteases inhibitor; [󰀲󰀳]󰀱󰀰 P󰀶󰀸󰀸󰀷󰀱 Hemoglobin subunit beta HBB 󰀷.󰀰󰀵/󰀱󰀰.󰀵 󰀶.󰀸/󰀱󰀲.󰀵 󰀱󰀵/󰀳󰀱 󰀹󰀵 󰀲󰀲󰀰 Oxygen transport; [󰀲󰀲] 󰀱󰀱 P󰀰󰀱󰀸󰀵󰀹 Ig gamma-󰀲 chain C region IGHG󰀲 󰀶.󰀱/󰀲󰀴.󰀴 󰀶/󰀲󰀴.󰀶 󰀷/󰀴󰀰 󰀱󰀷 󰀴󰀴 Immunity; antigen binding; [󰀲󰀴]󰀱󰀲 P󰀰󰀲󰀷󰀸󰀷 Serum transerrin RFE 󰀶.󰀳/󰀸󰀰.󰀷 󰀶.󰀳/󰀷󰀹.󰀳 󰀳󰀶/󰀷󰀱 󰀴󰀵 󰀳󰀱󰀱Iron binding transport proteinswhich can bind two Fe 󰀳+ ions;[󰀲󰀵]󰀱󰀳 P󰀰󰀱󰀰󰀲󰀴 Complement C󰀳 CO󰀳 󰀶.󰀶/󰀷󰀰.󰀶 󰀶.󰀸/󰀶󰀹.󰀷 󰀱󰀶/󰀲󰀱 󰀱󰀴 󰀱󰀴󰀴Immunity; central role in theactivation o the complementsystem; [󰀲󰀶] 󰀱󰀴 P󰀰󰀲󰀷󰀶󰀶 ransthyretin HY 󰀵.󰀵/󰀳󰀵.󰀳 󰀵.󰀴/󰀳󰀴.󰀴 󰀹/󰀲󰀷 󰀷󰀷 󰀱󰀳󰀶 Tyroid hormone-bindingprotein; [󰀲󰀷] Spot ID reers to that shown in 󰀲-DE maps (Figure 󰀱).  a Accession numbers o Swiss-Prot or GenBanK databases. or normal distribution (D’Agostino-Pearson test). Data wereexpressed as median values and interquartile range, unlessotherwisestated.Unmatchedspotsorspotswithsigni󿬁cantly different percentage volume (%V) were considered as “differ-ently expressed”. Differences between groups were tested by the nonparametricMann-Whitneyrank sum test or Kruskal-Wallis analysis o variance, as appropriate. A two-sided   <0.05  was considered to indicate statistical signi󿬁cance. 3. Results o better characterize the R plasma protein pattern, wecarried out a proteomic analysis based on 󰀵 different analyti-cal groups: (󰀱) classical R versus PSV-R, (󰀲) R versuscontrols,(󰀳)RsistersFamily󰀱versusRsistersFamily󰀲,(󰀴)no.󰀱classicalRversusno.󰀳classicalR,and(󰀵)no.󰀲PSV-R versus no. 󰀴 PSV-R. Among these groups therewere signi󿬁cant quantitative and qualitative variations in 󰀱󰀴protein spots subsequently identi󿬁ed by mass spectrometry.Protein name as well as peptide matches, sequence coverage,and the probabilistic score obtained using the MASCOsofwarearesummarized(able 󰀱).Alltheidenti󿬁edproteinsare known to be involved in speci󿬁c biological processes [󰀱󰀶–󰀲󰀷]. Proteomic plasma maps o healthy control, R sistersFamily 󰀱, and R sisters Family 󰀲 with the protein spotsare represented (Figure 󰀱). Black arrows indicate the spotswithquantitativevariationswhilealltheidenti󿬁edqualitative variations are reported with black circles.As shown in Figure 󰀲, signi󿬁cant changes appeared inalpha-󰀱-microglobulin(AMBP),haptoglobin(HP/Hp,spots󰀳 and 󰀴), 󿬁brinogen beta chain (FIBB), complement C󰀳(CO󰀳), and transthyretin (HY) in classical R siblingsas compared to PSV-R sisters. In addition, quantitativeand qualitative protein variations values as derived rom theexamined R sister pairs and healthy controls comparativeanalyses were reported (ables 󰀲 and 󰀳). R patients, when compared to control group, showed󰀶 underexpressed protein spots including FIBB, hemoglobinsubunit beta (HBB), serum transerrin (RFE), HP, Iggamma-󰀲 chain C region (IGHG󰀲), and CO󰀳, while 󰀱 spot o clusterin (CLUS) is overexpressed (able 󰀳).  Mediators o In󿬂ammation 󰀵    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 o classical R versus PSV-R plasma proteome. Data are expressed as box-and-whiskersplots. Results o the Kruskal-Wallis ANOVA are shown. Family 󰀱 versus Family 󰀲 (third group in able 󰀳) showeda signi󿬁cant underexpression o HY, a signi󿬁cant overex-pression o HBB, and the appearance o one protein spot o albumin (ALBU). Te comparison between the two classicalorms showed underexpression o 󰀴 protein spots (AMBP,CLUS, IGHG󰀲, and HY) and appearance o 󰀳 proteinspots (ALBU, HBB, and A󰀱A) in no. 󰀱 as compared to no.󰀳. Signi󿬁cant qualitative variations are most evident in thecomparisonbetweenthetwoPSVvariantsinwhich󰀵proteinspots appeared (AMBP, CLUS, ALBU, immunoglobulin Jchain, IGJ and CO󰀳) while 󰀲 proteins (HBB and HY)disappeared in no. 󰀲 as compared to no. 󰀴. Moreover, inthe same comparative group, an overexpression o HP wasobserved(able 󰀳).Inaddition,anothercomparativeanalysis o each R patient versus healthy controls has resulted inseveral signi󿬁cant quantitative and qualitative variations inplasma proteome (see able 󰀴 in Supplementary Materialavailable onlineat󰀱󰀰.󰀱󰀱󰀵󰀵/󰀲󰀰󰀱󰀳/󰀴󰀳󰀸󰀶󰀵󰀳)anda number o protein spots changes likely due to the size effectsrcinating rom the healthy control group were detected butconsidered to be not signi󿬁cant (data not shown) and notcomparable ( n.c. , able 󰀲). 4. Discussion Proteomic analysis has proven effective in identiying vari-ations in proteins with biological and/or clinical signi󿬁-cance [󰀳󰀰]. Te examined R siblings pairs represented aninteresting benchmark model to test the molecular basis o phenotypical expression variability and to identiy potentialtherapeutic targets o the disease.Rettsyndromeistheresultoamonogenicmutation,thatis, the X-linked  MECP󰀲  gene in the overwhelming majority o cases. As R is an X-linked trait and the  MECP󰀲  locus issubject to X inactivation, different patterns o X inactivationmay lead to different phenotypes within a group o patientswho carry the same mutation. Based on these data someauthorsspeculatethattheremightbeagroupoRpatientswith milder phenotypes owing to skewed X inactivation,who have not so ar been identi󿬁ed because o their atypicalphenotypes. Nonetheless, variations in XCI are known toexplain only 󰀱/󰀵 o the variance in severity o the disease [󰀳󰀱],thus not ully accounting or the phenotype severity rangetypically seen in R [󰀳󰀲]. We can saely state that in our patients, as well as in themajority o Rett syndrome patients reported in the literature,Xinactivationwasoundtobebalanced.Tus,itisreasonableto assume that the clinical phenotype o our pairs o sistersappears to be determined mainly by the type and location o the  MECP󰀲  mutations.Statistical analysis, represented by the old changes,revealed in the classical R versus PSV-R comparisona signi󿬁cant overexpression o proteins involved in APR including AMBP, HP, FIBB, A󰀱A, and CO󰀳 [󰀱󰀶, 󰀱󰀸, 󰀲󰀳, 󰀲󰀶]. Although little is known about the APR and the requency o inections in R [󰀳󰀳], our 󿬁ndings evidenced a lack o some key APR components. Possible explanations or these󿬁ndings may include a continuous stimulation o cytokine-mediated liver protein synthesis, an accelerated turnover o APRproteins,oracombinationoboth.TeevidencethattheR patients present chronic terminal bronchiolitis and anincreaseinintestinalmicrobiomeduetoconstipationsuggestthe coexistence o recurrent inections [󰀳󰀴, 󰀳󰀵]. Evidence o  the involvement o in󿬂ammatory events in R, was mainly represented by the signi󿬁cant variations o AMBP and A󰀱A(󿬁fh and sixth comparative groups in able 󰀳), two serineprotease inhibitors linked to the acute phase reaction, whichlimit the damage caused by activated neutrophils and theirenzymeelastase[󰀱󰀶,󰀲󰀳].Amajorroleortheimmunesystem in R pathogenesis has been previously documented by theact that transplantation o wild-type bone marrow restoreswild-type microglia and arrests pathology in a mouse modelo R [󰀳󰀶]. Te other 󿬁nding on a partial de󿬁cit in an oxygentransport HBB [󰀲󰀲] could be compatible with our prior󿬁nding o a subclinical hypoxia with an altered redox statusin R patients with the classical phenotype [󰀳󰀷].Interestingly, RFE was signi󿬁cantly underexpressed inR as compared to healthy controls, con󿬁rming the asso-ciation previously reported in autism [󰀳󰀸]. Alterations inthe RFE levels may lead to abnormal iron metabolismin R; it has been suggested or autism [󰀳󰀸]. On theother hand, RFE is also a negative APR protein whoseexpressionlevelsdecreaseduringin󿬂ammation[󰀲󰀵].Tustheunderexpression o RFE in R suggests once again thatin󿬂ammatoryprocessmayplayakeyroleinthepathogenesiso the disease.More intriguing is the 󿬁nding o an overexpressed CLUS,which may re󿬂ect a counterbalanced response to exces-sive proteins accumulation, namely, the unolded proteinresponse [󰀱󰀷]. Abundant evidences demonstrated that CLUSexpression is increased during cellular stress [󰀱󰀷]. Te chap-erone action o CLUS could be cytoprotective in either or
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