High Frequency of Nonrecurrent MECP2 DuplicationsAmong Brazilian Males with Mental Retardation

Mário Campos Jr. & Sarah M. Churchman &

Cíntia Barros Santos-Rebouças & Frederique Ponchel &Márcia Mattos Gonçalves Pimentel

Received: 30 June 2009 /Accepted: 14 September 2009 /Published online: 6 October 2009# Humana Press 2009

Abstract Structural variations that affect the copy numberof the MECP2 gene were shown to cause mental retardationin males by driving the overexpression of this gene. Toaccess the impact of these rearrangements in males withunexplained mental retardation, we have performed aquantitative real-time polymerase chain reaction assayusing SYBR Green I chemistry to quantify MECP2 genecopy number in 145 Brazilian males with mental retarda-tion of unknown cause. Three patients carrying MECP2duplications (~2%) were identified. The analysis of addi-tional markers flanking the MECP2 region showed that theduplications observed are nonrecurrent. Expression studiesin two of these patients revealed the overexpression of theMECP2 gene compared to the expression level observed incontrols. These findings corroborate other recent reports inthe literature and highlight that the overexpression ofMECP2 caused by duplications involving this gene is arelatively frequent genetic cause of mental retardation inmales, highlighting the importance of MECP2 gene dosagefor diagnostic purposes in such cases.

Keywords Duplication .MECP2 . Mental retardation .

XLMR .Xq28

The MeCP2 protein was originally identified as a methyl-CpG binding protein that promotes histone deacetylationand, consequently, transcriptional repression, linking DNAmethylation to control of expression (Nan et al. 1997).Further studies identified other possible mechanisms bywhich MeCP2 functions, pointing to a more complex rolefor this protein as a modulator of chromatin structure(Georgel et al. 2003; Young et al. 2005). This protein isexpressed in many tissues, but at its highest in the brain,suggesting a primary neuronal role (Lasalle et al. 2001).

Duplications of the MECP2 region leading to over-expression were reported as a significant cause of X-linkedmental retardation (MR) in males, uncovering an additionalassociation between MECP2 and neurological disorders(Van Esch et al. 2005). In addition, it has been reported intransgenic mice that overexpression of the MeCP2 proteinresults in abnormal neurological phenotypes with similar-ities to those observed in mice models with loss-of-functionmutations, and the dosage in which MeCP2 is increased isdirectly related to the severity of the phenotypes presented,but only the modest excess of endogenous MeCP2 levelscan cause a progressive neurological phenotype supportingthe need for tight control of MECP2 expression in vivo(Collins et al. 2004; Luikenhuis et al. 2004).

Duplications of the MECP2 gene were also identified infemales, however in the absence of any abnormal neuro-logical symptoms, maybe due to the protection conceded byskewed X chromosome inactivation (Meins et al. 2005; VanEsch et al. 2005; Friez et al. 2006). These findings broughta new prospective study of MECP2 gene association withMR in males since the frequency of MECP2 pointmutations is low; however, the sequencing technique usedto screen these mutations is not able to detect duplications.

In the present study, we applied a quantitative real-timepolymerase chain reaction assay (qPCR) to determine the

M. Campos Jr. : C. B. Santos-Rebouças :M. M. G. Pimentel (*)Serviço de Genética Humana, Departamento de Genética,Instituto de Biologia Roberto Alcantara Gomes,Universidade do Estado do Rio de Janeiro,Rua São Francisco Xavier, 524, PHLC—sala 500, Maracanã,20550-013 Rio de Janeiro, Rio de Janeiro, Brazile-mail: pimentel@uerj.br

S. M. Churchman : F. PonchelLeeds Institute of Molecular Medicine, University of Leeds,St James’s University Hospital,Leeds LS9 7TF, UK

J Mol Neurosci (2010) 41:105–109DOI 10.1007/s12031-009-9296-2

copy number of the MECP2 gene in a cohort of 145 malepatients with idiopathic MR from the Human GeneticsService of the University of the State of Rio de Janeiro,Brazil. The appropriate Ethics Committee approved theresearch protocols, and informed consent was obtainedfrom the parents of the studied children. These patients hadpreviously been tested for cytogenetic abnormalities (usingG-banding), FRAXA and FRAXE expansions, and MECP2point mutations (using sequencing); all of them returnednormal results.

Genomic DNA from patients and from control individ-uals was isolated from peripheral blood. A qPCR assayusing SYBR Green I chemistry and 7900HT real-time PCRsystem (Applied Biosystems, Warrington, UK) wasdesigned to detect MECP2 gene copy number variations.A previously described set of primers was used (Meins etal. 2005). Calculation of the relative gene copy number wasachieved using ratios to a reference gene of invariable copynumber (Ponchel et al. 2003). A primer pair for a referenceamplicon within the ALB gene, in 4q11, was used. Allreactions were performed in triplicate and a melting curveanalysis was used to ensure specificity of each PCRproduct. We validated our assay by conducting a blindedstudy screening MECP2 copy patterns in a cohort of 14healthy volunteers correctly identifying six males (onecopy) and eight females (two copies).

In order to determine the extent of the duplication in theindividuals that exhibited a double copy pattern and whetherthere was variability between patients, four genes—L1CAM,IRAK1, TKTL1, and GDI1—located on either side ofMECP2 were tested using previously described primers(Meins et al. 2005; Van Esch et al. 2005). The same methodof assay optimization and calculation of gene copy numberused for theMECP2 gene (including the use of the ALB geneas a reference amplicon) was applied also for the study ofthese four additional regions.

Total RNA was extracted from blood samples frompatients with MECP2 duplications and also from healthymale and female controls. Relative dosage of MECP2expression was performed by real-time PCR following thesame optimization and calculation of relative quantityprocedures described for the genomic analysis. However,we have used a set of primers specific for the MECP2messenger RNA (ctg gcc gct ctg ctg g—forward; gcc tac cttttc gaa gta cgc a—reverse) and a previously describedprimer set for the glyceraldehyde-3-phosphate dehydroge-nase messenger RNA (reference amplicon; Ali et al. 2001).

In the present study, we identified three patients (2%) withresults similar to the copy number found in healthy femalecontrols, suggesting duplications in Xq28 including the geneMECP2 (Fig. 1). Analysis was repeated three times for thesepatients (P58, P431, P597), and results confirmed thepresence of two copies of the MECP2 gene in all of them.

All three patients were born from nonconsanguineousparents and, besides being affected by MR, their clinicaldescription varies. Patient P58 is a 13-year-old boy,with apparently normal neurodevelopment during thefirst months of life as reported by his parents. By11 months of age, he had a clear motor developmentdelay, and after that, he developed stereotyped handmovements, hypotonia, MR, and autistic behavior; healso developed epilepsy by 6 years of life. Patient P431is a 14-year-old boy, with clinical features includingabsence of speech, hyperactivity, long face, prominentears, autistic behavior, and MR; his parents reportednormal motor development, but one event of seizure.Patient P597 is an 18-year-old boy with MR, long face,and prominent ears.

Analysis of MECP2 copy number in the mothers of thepatients revealed that the mother of the patient P58 is acarrier of the duplication. A threefold increase in MECP2copy number was observed in this mother in relation to acontrol male (data not shown). Since most of the duplica-tion cases reported in the literature are inherited and themothers apparently protected by an extremely skewed Xinactivation pattern (Meins et al. 2005; Van Esch et al.2005; Del Gaudio et al. 2006), it was likely that this wouldbe the same in our study, accounting for the presence of theMECP2 duplication in this female without neurologicalabnormalities. Surprisingly, the duplication in the patientP431 is de novo; for that reason, we may not rule out thepossibility of gametic mosaicism, but since the patient isaffected with MR, we believe that if the patient is a mosaic,some or all of the cells that require specific MECP2expression levels for normal neurological function mighthave been affected as well as the white blood cells used forthe molecular analysis. Unfortunately, we were unable toreestablish contact with patient P597’s family in order toperform the copy number or MECP2 expression levelanalysis.

Figure 1 Results of the screening for MECP2 duplications. Therelative MECP2 copy number analysis of healthy males and femalesused as controls and all 145 patients. Normalized values of 1 indicatea single MECP2 gene copy while values of 2 indicate two copies. Thethree patients with the MECP2 duplication are identified by a triangle

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Array-comparative genomic hybridization (CGH) hadbecome the most preferred method for detection of micro-deletions and microduplications due to its high-resolutionand genome-wide analysis capability; it has successfullydetected MECP2 copy number variations in several cases inthe literature (Van Esch et al. 2005; Froyen et al. 2007).Another commonly used method to quantify copy numbersis multiplex ligation-dependent probe amplification that cananalyze up to 50 genomic regions in a single multiplexreaction and has also been previously applied in thescreening of duplications of the MECP2 gene (Friez et al.2006; Lugtenberg et al. 2006). Those methods are powerfultools, able to identify novel and/or multiple submicroscopicaberrations; however, real-time PCR is a cost–effectivemethod that offers advantages in routine diagnosis ofknown genomic rearrangements within specific regions,including speed of acquisition of results and high sensitivity(Ponchel et al. 2003; Truong et al. 2008).

Duplications involving the X chromosome have becomeincreasingly relevant in mental disorders. Individuals withduplication in the Xq28 region were reported in a studycomparing 17 patients (15 males and two females) carryingX chromosome functional disomy with two new cases (onemale and one female; Sanlaville et al. 2005). The authorsdescribed functional X chromosome disomy as a rare and/or usually lethal event, yet a possible cause of neurologicalabnormalities. Subsequently, using a real-time PCR ap-proach, a case report of another male with a MECP2duplication was reported (Meins et al. 2005). The patientpresented with clinical features similar to Rett syndrome.Analysis of the duplication revealed that at least 12 othergenes were located within the duplicated region; however,MECP2 presented the most prominent association withneuronal function. These data indicated for the first timethat not only loss-of-function mutations but also duplicationof MECP2 could lead to MR in males. The significance ofthis finding was highlighted further by later studies.Another male with a MECP2 duplication was foundfollowing a genome-wide screening study using arrayCGH (Van Esch et al. 2005). Furthermore, after specificselection of 17 families of affected males with the samephenotype, three additional duplications were identified,suggesting that increased MECP2 gene copy number,caused by submicroscopic duplications, may occur fre-quently in males with severe MR. The frequency of theseevents was further evaluated in a cohort of 122 patientsspecifically referred for MECP2 duplication/deletionanalysis and rated at 1.6% of patients (Del Gaudio et al.2006).

A more recent study has shown that the frequency ofduplications of theMECP2 gene can be of approximately 1%in unexplained cases of X-linked mental retardation in malesand can be as high as 2% in cases of severe and progressive

mental retardation, being a significant cause among genesassociated with mental retardation (Lugtenberg et al. 2009).The frequency achieved in our study is substantial consid-ering other genetic causes of MR. It also represents a higherfrequency compared to MECP2 loss-of-function pointmutations previously observed (Campos et al. 2007).

To investigate the effect of these duplications on theexpression of MECP2, we obtained fresh blood samplesfrom patients P58 and P431 and used 11 healthy males andfemales as controls. We observed the same level ofexpression in both male and female controls. X inactivationin females is suspected to account for this observation(Meins et al. 2005; Van Esch et al. 2005). However, the twopatients analyzed exhibited an approximately tenfoldincrease in relation to controls in association with thepresence of another copy of the gene MECP2. These datasuggested that this second copy of the gene is functional atleast at the transcriptional level, resulting in increasedmRNA levels in patients. This agrees with observationsmade by other groups despite a greater increase detected(Meins et al. 2005; Van Esch et al. 2005) and shows thatthese duplications cause loss of control over MECP2

Figure 2 Top, representation of the MECP2 gene locus andsurrounding regions (obtained from ENSEMBL), the tested regionsare underlined; bottom, results of the regional gene copy numberanalysis performed on the three MR patients showing the extent of theduplication on each side of the MECP2 gene

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expression which may in turn lead to neurologicalmanifestations. Considering the association of these rear-rangements with MR, we also believe that mutationsaffecting cis-acting elements (long distance enhancer forexample) or other regulatory genes that are able to, directlyor indirectly, modulate MECP2 expression and its resultingfunction could also cause neurological abnormalities. Thisis further supported by the identification of two patientswith autistic spectrum disorder, without any detectablemutation in the MECP2 coding region, but exhibiting asignificant increase in expression of this gene (Samaco etal. 2004). These data suggest that putative genetic alterationoutside the coding sequence may also result in clinicalmanifestations by altering MECP2 expression. Further-more, in a large cohort study of males with autism,screening of the MECP2 3′ untranslated region regionshowed variations within the conserved region suggestingthat the presence of MECP2 cis-acting regulatory sequencesresult in reduced MECP2 expression in four patients(Coutinho et al. 2007). Given that little is known aboutthe molecular regulation of MECP2 expression in vivo, it isplausible that mutations in such regions, as well asduplications and deletions, could lead to human mentaldisorders.

Our duplication analysis was extended to the regionsflanking the MECP2 gene in order to estimate the length ofthe duplication and the variability between the patients.Copy number analysis of the regions L1CAM, IRAK1,TKTL1, and GDI1 revealed that the MECP2 was entirelyduplicated in all three patients. The smallest duplicationwas found in patient P58 and included only IRAK1 andTKTL1 in addition to MECP2. The longest duplication,observed in patient P431, contained all of the markerstested, including L1CAM gene, which has a role in neuronalfunction (Vits et al. 1994). The number of regions testedwas not enough to infer the exact size of the duplications;however, it was possible to determine that in each patient,the genomic rearrangement is unique (Fig. 2). Thisobservation corroborates previous reports showing thenonrecurrent nature of duplications in this region (Meinset al. 2005; Van Esch et al. 2005; Del Gaudio et al. 2006).From the literature, it seems that the minimum duplicationrequired for neurological abnormalities leading to MRencompasses the MECP2 gene itself (Del Gaudio et al.2006). Since the patients of our study share clinicalsimilarities and also differences, we cannot exclude thepossibility that the other genes present in the duplicationsmay be influencing the resultant phenotype.

Our findings, together with other data (Van Esch et al.2005; Del Gaudio et al. 2006; Froyen et al. 2007;Lugtenberg et al. 2009), reinforce that duplications involv-ing the MECP2 gene leading to its overexpression are asignificant cause of MR in males. Thus, we would

recommend the use of MECP2 gene copy analysis in maleswith idiopathic MR, for diagnostic purposes and geneticcounseling. We have also validated the application of thereal-time PCR method for this objective, and we believethat it offers a reliable and cost–effective option for routineMECP2 duplication screening.

Acknowledgments We are grateful to CAPES, CNPQ, CEPUERJ,and FAPERJ for the financial support and to the patients and theirfamilies for their cooperation. We also thank Dr. Teresa de SouzaFernandes and her group for their technical help with RNA.

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