Original article. 1. Introduction

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1 Brain & Development 23 (2001) S246 S250 Original article MECP2 gene mutation analysis in the British and Italian Rett Syndrome patients: hot spot map of the most recurrent mutations and bioinformatic analysis of a new MECP2 conserved region Marcella Vacca a,1, Francesco Filippini b,1, Alberta Budillon a,1, Valeria Rossi b,1, Floriana Della Ragione a,1, Maria Luigia De Bonis a,1, Grazia Mercadante a,1, Elisa Manzati c,1, Francesca Gualandi c,1, Stefania Bigoni c,1, Cecilia Trabanelli c,1, Giorgio Pini d,1, Elisa Calzolari c,1, Alessandra Ferlini c,1, Ilaria Meloni e,1, Giuseppe Hayek f,1, Michele Zappella f,1, Alessandra Renieri e,1, Michele D Urso a,1, Maurizio D Esposito a,1, *, Fiona Macdonald g,2, Alison Kerr h,2, Seema Dhanjal i,2, Maj Hulten i,2,3** a International Institute of Genetics and Biophysics, CNR, Via Marconi 10, 80125, Naples, Italy b Dipartimento di Biologia, Università di Padova, Padova, Italy c Dipartimento di Medicina Sperimentale e Diagnostica, Università di Ferrara, Ferrara, Italy d Servizio di Neuropsichiatria Infantile, U.O., Viareggio, Italy e Genetica Medica, Policlinico Le Scotte, Università di Siena, Siena, Italy f Neuropsichiatria Infantile, Policlinico Le Scotte, Siena, Italy g Regional Genetics Services, Birmingham, B15 2TG, UK h Academic Centre, Glasgow University, Department of Psychological Medicine, Gartnavel Royal Hospital, 1055 Great Western Road, Glasgow G12, UK i Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Abstract Rett syndrome (RTT) is an X-linked dominant neurological disorder, which appears to be the most common genetic cause of profound combined intellectual and physical disability in Caucasian females. This syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of unknown target genes. We report a detailed mutational analysis of a large cohort of RTT patients from the UK and Italy. This study has permitted us to produce a hot spot map of the mutations identified. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, conserved among brain-specific regulatory factors. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Rett syndrome; MECP2; Mutation analysis; Recurrent mutations; Bioinformatics; Fork head 1. Introduction Rett syndrome (RTT, MIM ) is a neurological disorder that affects females almost exclusively [1,2]. After an early period of apparently normal or near normal development (until 6 18 months of life), this disorder results in profound mental disability, reduction in speech * Corresponding author (Group 1). Tel.: ; fax: address: desposit@iigbna.iigb.na.cnr.it (M. D Esposito). 1 Group 1. 2 Group 2. 3 ** Corresponding author (Group 2). Tel.: ; fax: address: mhulten@bio.warwick.ac.uk (M. Hulten). and purposeful hand movements and reduced brain growth. The RTT locus has been recently mapped to Xq28 [3], where the MECP2 gene is localized [4]. This allowed subsequent identification of mutations in the MECP2 gene in a proportion of RTT cases [5 10]. MeCP2 belongs to a family of heterogeneous methyl-binding proteins [11]; it contains at least two functional domains, the methyl-binding domain (MBD) and the transcriptional repression domain (TRD). These domains interact respectively with methylated CpGs and transcriptional co-repressors, like msin3a, to repress transcription from methylated promoters and mediate interaction with histone deacetylases [12]. The gene contains a very long 3 0 untranslated region, well conserved throughout the evolution, which could have a structural and/or functional role [13]. The MBD of the /01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 M. Vacca et al. / Brain & Development 23 (2001) S246 S250 S247 Fig. 1. The MECP2 gene exon intron structure and mutation positions are shown. The 3 0 untranslated region is not to scale. Novel mutations are highlighted in a bold oval. Specific protein domains are boxed and their amino acid positions are reported at the bottom. The vertical lines box shows a new putative domain rich in histidine and proline residues. MeCP2 protein has been analyzed by NMR [14], and the MeCP2 protein has been isolated from numerous species [5,15]. We have analyzed 65 Rett patients, 31 British and 34 Italian, for mutations in the coding region of the MECP2 gene. All patients but two show classical symptoms of RTT, according to the accepted clinical criteria [16]. The atypical Rett cases, both Italian, represent preserved speech variants (PSVs) [17]. Only one of these variants shows a mutation. The majority of mutations found are restricted to the functional MBD and TRD domains of the MECP2 gene (Fig. 1). Data collection from this work and from previous reports emphasizes the recurrence of seven mutations, which account for two-thirds (66.57%) of the total. Bioinformatic analysis of the mutations leads us to postulate the existence of a new functional domain in the 3 0 region of the MeCP2 protein, conserved among brain-specific regulatory factors belonging to the FKH (fork head) gene family. 2. Patients Fifty-nine sporadic cases of classic RTT have been analyzed: the onset of regression was between 6 and 24 months. The majority (.80%) show deceleration of head growth, and all show hand stereotypy and breathing dysrhythmia. With respect to other typical clinical characteristics [16], all these patients conformed to the criteria for RTT. In addition, we analyzed four British cases (N4, P26, S11 and 37) which deviate from these classic criteria only concerning head circumference and two Italian atypical RTT patients belonging to the PSV, as defined by Zappella [17]. 3. Materials and methods 3.1. DNA extraction and single-strand conformation polymorphism/conformation sensitive gel electrophoresis analysis DNA was extracted using standard procedures from blood of probands and, where available, parents and siblings. MECP2 coding regions have been studied by both direct sequencing and by single-strand conformation polymorphism (SSCP) and conformation sensitive gel electrophoresis (CSGE) analysis followed by direct sequencing of shifted and/or heteroduplex fragments. In total, eight different primer pairs (from GENSET, Evry, France) have been used: exon 1, F1, 5 0 -GTTATG- TCTTTAGTCTTTGG-3 0 ; Ex1R, 5 0 -TCAATGGGGGCT- TTCAACTTAC-3 0 ; exon 2, R2, 5 0 -GGGGTCATCATA- CATGGGTC-3 0 ; F2, 5 0 -CCTGCCTCTGCTCACTTGTT- 3 0 ; R3bis, 5 0 -TTTCAAGCACACCTGGTCTC-3 0 ; F3 ter, 5 0 -AAAGAAGAGAAAGAGGGCAA-3 0 ; exon 3, R4, CTTCCCAGGACTTTTCTCCA-3 0 ; F4, 5 0 -TTTGTCAG- AGCGTTGTCACC-3 0 ; R10, 5 0 -TTTCCCGCTCTTCTC- ACCGA-3 0 ; Ex3seq2R, 5 0 -GGGAGCGGCACCACGA- GACC-3 0 ; R6, 5 0 -TGAGTGGTGGTGATGGTGGTGG-3 0 ; F6 bis, 5 0 -CCGCCGAGGCCAAAAAGAAA-3 0 ; R9, 5 0 -G- CTCCTCTCTGTTTGGCCTT-3 0 ; F9, 5 0 -CTCAGAGTC-

3 S248 M. Vacca et al. / Brain & Development 23 (2001) S246 S250 Table 1 Mutations in British and Italian patients in the coding region of MECP2 Proband ID NT a AA a Domain/exon RE and gel analysis b New Recurrence c British patients M23 C301T P101S MBD/2 / 1 1 M16 C316T R106W MBD/2 NlaIII (1) 2 1 N4; N19, N24 C397T R133C MBD/3 / 2 3 N35 C401G S134C MBD/3 / 2 1 N10, Y14N, Y26N C473T T158M MBD/3 NlaIII (1) 2 3 N6, N8, N14, N17 C502T R168X Exon 3 HphI (1) 2 4 L21 C622T Q208X TRD/3 / 1 1 L20, Y33L C763T R255X TRD/3 / 2 2 L25 803delG Frameshift, stop at 288 TRD/3 NlaIV (2) 2 1 P26 C880T R294X TRD/3 / 2 1 P9 C916T R306C TRD/3 HhaI (2) 2 1 S del41 Frameshift, stop at 389 Exon 3 * 2 1 Y32Z 1152del44 Frameshift, stop at 389 Exon 3 * 2 1 S del44 Frameshift, stop at 389 Exon 3 * 2 1 Italian patients 124.GS 42ins8 (AGTCAGAA) Frameshift, stop at 34 Exon 2 / 1 1 D2 A64T K22X Exon 2 MaeI (1) 1 1 A062 C401G S134C MBD/3 / 2 1 N1 C423G Y141X MBD/3 RsaI (2) 2 1 A080 C455G P152R MBD/3 / MM A472G T158A MBD/3 BstUI (1) 1 1 A070, A111, 73A, 80A C473T T158M MBD/3 NlaIII (1) 2 4 A050 C502T R168X Exon 3 HphI (1) 2 1 A delG Frameshift, stop at 209 Exon 3 / 1 1 A020, 87.BV, 101.DE, C763T R255X TRD/3 / A, 83A 143.CA, A3, 72A C808T R270X TRD/3 NlaIV (2) A, 94A C880T R294X TRD/3 / CM 1116del86 Frameshift, stop at 375 Exon 3 * PL 1163del26 Frameshift, stop at 395 Exon 3 * 2 1 a Nucleotide and amino acid position of the mutation, numbered from the ATG initiator codon. b Restriction enzyme sites created (1), abolished (2), or not altered (/) by the mutation. An asterisk (*) indicates the presence of a double band due to deletion and visible on agarose gel. c Number of affected probands in this study. CCCAAAGGCCC-3 0 ; Ex3seq3F, 5 0 -TGTCAGAGCCCT- ACCCATAA-3 0 ; F8, 5 0 -GGAGAAGATGCCCAGAGGA- G-3 0 ; or Ex3seq3R, 5 0 -GTGAGAAGAGCGGGAAAGGA PCR conditions and sequencing Standard PCR reactions (948C for 2 min for one cycle; 948C for 30 s, C for 45 s, 728C for 45 s for 35 cycles) were carried out using Perkin-Elmer (USA) AmpliTaq DNA polymerase with buffer recommended by the manufacturers. Amplified fragments were purified using a QIAquick PCR purification kit (QIAGEN, Germany) and sequenced on both strands by use of the same PCR primers with fluorescent-dye terminators on an ABI377 automatic sequencer. For the same mutations (i.e. 3 0 ZCOOH deletions) products have been amplified and cloned using a TOPO TA cloning kit (Invitrogen, The Netherlands) and subsequently sequenced. Restriction analysis was performed using New England Biolabs (USA) restriction enzymes. 4. Results We have used direct sequencing to analyze the MBD and TRD; the remainder of the coding region was analyzed using SSCP and CSGE followed by direct sequencing of the shifted and heteroduplex fragment. Among the patients analyzed, 46 (71%) have been found to have MECP2 mutations (see Table 1). All the mutations reported here are heterozygous, and they are all de novo, none of the parents being a carrier of the mutation. Among the point mutations, all but five are C-T transitions. Three cases (S134C, T141X and P152R) are C-G transversions, one case is an A-T transversion (K22X), and another case is an A-G transition (T158A). Eight cases are frameshift mutations, due either to deletions localized in exon 3 (seven

4 M. Vacca et al. / Brain & Development 23 (2001) S246 S250 S249 Fig. 2. Multiple alignment of the 3 0 region of MeCP2 proteins with members of the FKH/BF-1 family. His, green; Pro, brown; Gln, magenta; negatively charged (Asp, Glu), blue; positively charged (Arg, Lys), red. Residues identical or similar to those present in hmecp2 are shadowed in grey. cases) or to an insertion in exon 2. For some mutations, restriction sites were created or lost, thus making it possible to carry out a rapid family analysis (see Table 1). The majority of mutations found are restricted to the functional MBD and TRD domains of the MECP2 gene (Fig. 1). Data collection from this work and from previous reports emphasizes the recurrence of seven mutations, which account for two-thirds (66.57%) of the total (data not shown). One of the most interesting findings of this study concerns the identification of terminal deletions outside the MBD and TRD domains, in particular those located in a region 3 0 with respect to the TRD. The patients S18, S22 and Y32Z, 85.CM and 106.PL have deletions of 41, 44, 86 and 26 bp, respectively, in a closely linked region of the gene. These deletions, located adjacent to polyhistidine and polyproline stretches, abolish the ZCOOH terminus of the protein in a domain not before implicated in any of the cellular functions of the MeCP2 protein. This region of MeCP2 shows a homology of 35% identity and 50% positivity in a 75 amino acid stretch with two brain-specific factors, brain-specific factor 1 (BF-1) [18] and fork head 4 (FKH4) [19], members of the fork head gene family (Fig. 2). Both genes show expression restricted to the neurons of the developing telencephalon. This subregion partly overlaps with a portion of the ZCOOH terminus of the MeCP2 protein (from aa 404 to 467), which has been shown to facilitate MeCP2 binding to DNA, both when naked and in the nucleosome [20]. Further analysis is required to fully understand the relevance of such subclasses of MECP2 mutations. Interestingly, a mutation in this region (C1216T) has been recently associated with X- linked mental retardation and progressive spasticity in males [21]. Acknowledgements The authors would like to acknowledge the continuous support of the British Rett Syndrome Association (RSAUK) and the Associazione Italiana Rett (AIRETT). This research has been supported by grants to M.D E. from the Telethon n.e869, to M.H. from The Mental Health Foundation and the Patrick Berthoud Charitable Trust, and to A.R. from the Emma and Ernesto Rulfo Foundation. E.M. is a recipient of a fellowship from ANGBSR. References [1] Rett A. On a unusual brain atrophy syndrome in hyperammonemia in childhood. Wien Med Wochenschr 1966;116: [2] Hagberg B, Aicardi J, Dias K, Ramos O. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett s syndrome: report of 35 cases. Ann Neurol 1983;4: [3] Sirianni N, Naidu S, Pereira J, Pillotto RF, Hoffman EP. Rett syndrome: confirmation of X-linked dominant inheritance, and localization of the gene to Xq28. Am J Hum Genet 1998;63: [4] D Esposito M, Quaderi NA, Ciccodicola A, Bruni P, Esposito T, D Urso M, et al. Isolation, physical mapping, and northern analysis of the X-linked human gene encoding methyl CpG-binding protein, MECP2. Mamm Genome 1996;7: [5] Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-cpg-binding protein2. Nat Genet 1999;23: [6] Wan M, Lee SS, Zhang X, Houwink-Manville I, Song HR, Amir R, et al. Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations at CpG hotspots. Am J Hum Genet 1999;6: [7] Cheadle JP, Gill H, Fleming N, Maynard J, Kerr A, Leonard H, et al. Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location. Hum Mol Genet 2000;9: [8] De Bona C, Zappella M, Hayek G, Meloni I, Vitelli F, Bruttini M, et al. Preserved speech variant is allelic of classic Rett syndrome. Eur J Hum Genet 2000;5: [9] Van den Veyver IB, Zoghbi HY. Methyl-CpG-binding protein 2 mutations in Rett syndrome. Curr Opin Genet Dev 2000;3: [10] Dragich J, Houwink-Manville I, Schanen C. Rett syndrome: a surprising result of mutation in MECP2. Hum Mol Genet 2000;9: [11] Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-cpg binding proteins. Mol Cell Biol 1998;11: [12] Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. Transcriptional repression by the methyl-cpg-binding protein MECP2 involves a histone deacetylase complex. Nature 1998;393: [13] Coy JF, Sedlacek Z, Bachner D, Delius H, Poustka A. A complex pattern of evolutionary conservation and alternative polyadenylation within the long 3 0 -untranslated region of the methyl-cpg-binding protein 2 gene (MECP2) suggests a regulatory role in gene expression. Hum Mol Genet 1999;7:

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