ABSTRACT. Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/27/18. For personal use only.

Transcription

1 GENETIC TESTING Volume, Number, 00 Mary Ann Liebert, Inc. Spectrum of MECP Mutations in Rett Syndrome The French Consortium for MECP Gene Analysis: THIERRY BIENVENU, LAURENT VILLARD, NICOLAS DE ROUX, VIOLAINE BOURDON, MICHEL FONTES, CHERIF BELDJORD, MARC TARDIEU, PHILIPPE JONVEAUX, and JAMEL CHELLY Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 0/7/. For personal use only. ABSTRACT Mutations in the MECP (Methyl-CpG-binding protein) gene recently have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. We have collected the results of MECP analysis conducted in four laboratories in France. A total of 0 RTT alleles have been analyzed, demonstrating a total of different mutations so far observed and accounting for % of MECP genes in RTT patients living in France. RX (.%) is the most common of MECP mutations, followed by RX (0.%), R70X (0.%), TM (7.%), and R0C (.%). Only 0 mutations had a relative frequency.%. A total of mutations were found in a small number of RTT alleles (from to ). These data demonstrate the high allelic heterogeneity of RTT in France and provide information relevant to the development of strategies for molecular diagnosis and genetic counseling in RTT families. INTRODUCTION RETT SYNDROME [RTT (MIM 70)] is a progressive encephalopathy that appears to affect females only. It was first described by Rett in (Rett, ). RTT is characterized by neurodevelopmental dysfunction in which a period of stagnation, typically 7 months, is followed by regression of development in young girls (Hagberg et al., ). Other manifestations are frequently observed: severe mental retardation, autism, gait apraxia, hypotonia, disturbance of sleep and breathing, seizures, stereotypical hand movements, and deceleration of head growth. Its prevalence is estimated at :0,000,000 female births. More than % of cases are sporadic, but rare observations of familial recurrence have been reported. Previous exclusion mapping studies using rare RTT families mapped the locus to Xq (Webb et al., ). Xq is a very generich region, and more than one syndrome with mental handicap and neurological signs and symptoms has already been identified within it. However, using a systematic gene screening approach, Zoghbi and colleagues have identified mutations in the gene MECP encoding the X-linked methyl-cpg-binding protein as the cause of some cases of RTT (/ sporadic patients and / familial patients) (Amir et al., ). Numerous studies have since found various mutations (missense, nonsense, and frameshifts) in the coding region of MECP in patients with RTT. Mutations were identified in approximately 0% of patients; the remaining 0% may have mutations in other regions of this gene, such as regulatory elements and noncoding regions, but this remains to be determined (Amir and Zoghbi, 000). The MECP gene spans 7 kb in Xq and lies between the IRAK (IL- Receptor-Associated Kinase) and red opsin loci, a region of conserved synteny with mouse ChrX. The gene is composed of four exons that are transcribed from telomere to centromere, with the -nucleotide coding sequence in exons (exon from bp to, exon, bp7 to 77, and exon, bp7 to ). The MeCP polypeptide contains a methylbinding domain ( AA from AA77 to ), a transcriptional repression domain (AA07 0), and two putative nuclear localization, signals (AA7 ; and a region lying within the TRD around AA 70) (Lewis et al., ; Nan et al., ). Most mutations lie within the methyl-cpg-binding (MBD) or transcriptional-repression (TRD) functional domains. In addition, a number of etions have been identified within a limited region, which suggests a potential recombinational hotspot in the last exon of the gene. INSERM U-ICGM, Faculté de Médecine Cochin, 70 Paris, France. Laboratoire d Hormonologie et de Biologie Moléculaire, CHU Bicêtre, 7 Le Kremlin Bicêtre, France. INSERM U, Faculté de la Timone, Marseille Cedex, France. Département de Pédiatrie, Service de Neurologie, CHU Bicêtre, 7 Le Kremlin Bicêtre, France. Laboratoire de Génétique, Hôpitaux de Brabois, Vandoeuvre Les Nancy, France.

2 It is noteworthy that MECP mutations have also been found to cause a variety of phenotypes other than classic RTT, ranging from very mild mental retardation to severe neonatal encephalopathy in boys born into kindreds with RTT (Meloni et al., 000; Orrico et al., 000; Couvert et al., 00). France has a population of about 0 million persons. The incidence for RTT of one in 0,000 newborns is generally accepted. About individuals (out of 70,000 births) would be newly diagnosed each year. We have collected the results of MECP mutational analysis concerning 0 RTT patients. The analysis was conducted in four laboratories. The objective of this study was to determine the nature, frequency, and distribution of mutations responsible for RTT in France. The determination of the spectrum of MECP mutation is important for a molecular diagnosis strategy and for genetic counseling in RTT families. Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 0/7/. For personal use only FRENCH CONSORTIUM MATERIALS AND METHODS Four French genetic laboratories, all of which perform MECP mutation analysis in public hospitals and/or Medical Schools, were invited to fill in a form about the genotypes they identified in RTT. Genomic DNA extracted from peripheral blood and then amplified by PCR was the starting material for MECP mutation analysis. Three laboratories screened their panel of RTT chromosomes by indirect scanning techniques, such as denaturing gradient gel electrophoresis (DGGE) (Paris) (Bienvenu et al., 000), single-stranded conformation polymorphism (SSCP) (Marseille), or conformation-sensitive gel electrophoresis (CSGE) (Nancy) (Bourdon et al., 00), followed by the sequencing of PCR products displaying abnormal migration. For samples that are negative by screening analysis and to increase sensitivity and reduce the risk of false negatives, direct sequencing of the entire MECP 0 Missense mutations Nonsense mutations Frameshift mutations Large etions Splicing mutations FIG.. Types of MECP mutations detected in RTT individuals. The commonest MECP mutations were nonsense mutations (%), followed by missense mutations (%) and large etions (%). Splicing mutations are rare (%). FIG.. Summary of all MECP mutations identified in this study and distribution of these mutations in the four French genetic laboratories. (A) Missense mutations: the commonest missense mutations were R0W, TM, and R0C. (B) Nonsense mutations: RX and RX have been identified in all laboratories. (C) Splicing mutations, etions, and insertions. : & 7 ; : 70 ; : 0; : 0 ; : 0 ; : ; 7: 0 ; : ; : 7; 0: ; : 7 ; : 7 ; : ; : 0 ; : ; : ; 7: ; : 7; SM : 7- T-G; DUP : 77 dupl CGCCC; rea : gross rearrangements.

3 A 7 Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 0/7/. For personal use only. B C Marseille Nancy Paris Marseille Nancy Paris Kremlin-Bicêtre 0 W0R R0G R0W R0Q P7L RC RH RG KE DG PR DE TM GW T7M A0V PR P0R P0L R0C PA SP X7C Marseille Nancy Paris Kremlin-Bicêtre 0 Kremlin-Bicêtre SX RX RX Marseille Nancy Paris Kremlin-Bicêtre 7 RX 0 R70X RX Q70X 7 RX sm YX Marseille Nancy Paris Kremlin-Bicêtre dup rea Marseille Nancy Paris Kremlin-Bicêtre FIG..

4 Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 0/7/. For personal use only. FRENCH CONSORTIUM coding region was performed by the Marseille group, along with Southern blot analysis in the Nancy laboratory, and long-pcr of coding exon in the Paris laboratory. The direct sequencing of the three coding exons as the primary testing tool was used by only one laboratory (Kremlin-Bicêtre). RESULTS Mutational genotype data were collected for 0 patients with RTT. Mutations responsible for RTT in France A total of 0 patients have been studied. The majority of patients were females with classical sporadic RTT. However, some sporadic females with some features of RTT, but in whom the diagnosis was clinically uncertain, were also included in this study. In total, different mutations were identified in cases with RTT (/0;.%). No mutation was identified in % (Marseille) to % (Kremlin-Bicêtre) (% in Paris and % in Nancy) of the RTT patients. Types of mutations Sixty-nine different molecular defects were identified in this study, including missense mutations, small insertions or etions, nonsense mutations, mutations that affect splicing, and large etions (Fig. ). These mutations consist of missense, nonsense, small insertions or etions, large etions, small duplication, and splice-site mutation, and large molecular rearrangements. During this study, 0 neutral polymorphisms and variants (-C R T; SS C R T; TT 7 C R T; S7S 070 C R T; E7K G R A; SS C R T; 777G R C; 77G R C; 7-insG; inst) were also identified. Distribution of mutations according to frequencies According to the mutation frequencies, we can distinguish four groups (A D). In group A (most common mutations), 7 mutations had relative frequencies.% (R0W, TM, RX, RX, R70X, RX, and R0C). The group B comprised mutations whose respective frequencies ranged from % to % (RC, PR, and 0G). Altogether, these 0 mutations accounted for % of RTT mutation alleles. The third group, group C, comprised mutations which were identified twice (R0Q, DE, P0R, X7C, 0insA, inst,,, and 7-T R G). Finally, 0 mutations included in group D and accounting for % of the RTT mutations were identified only once. By analyzing data on the area of residence of families, we found no significant regional differences in the relative frequencies of the most common RTT mutations. Distribution of mutations within the MECP gene A total of 0.% (7/ patients bearing a MECP mutation) of French RTT alleles were mutated in exon (third coding exon), encoding for part of the MBD and the TRD. Few mutations were located in exon (second coding exon) (/) and no mutation has been identified in exon (first coding exon). DISCUSSION The purpose of this study was to determine the respective frequencies of RTT mutations in France and to compare the results with other European and American reports. Heterogeneity of mutations responsible for RTT in the French population Updated as of February, 00, a total of different mutations have been reported in RTT all over the world (Amir and Zoghbi, 000; Bienvenu et al., 000; Cheadle et al., 000; Dragich et al., 000; Hampson et al., 000; Hupkke et al., 000; Kim and Cook, 000; Obata et al., 000; Wan et al., ; Bourdon et al., 00; Laccone et al., 00). A high proportion of the mutations (7%) involved C R T transitions at CpG mutation hot spots; all of them were also reported in unrelated patients, reflecting the hypermutability of these sites (Amir and Zoghbi, 000). In the great majority of reports, the most common mutation is RX (Amir and Zoghbi, 000; Cheadle et al., 000; Dragich et al., 000; Obata et al., 000; Hampson et al., 000; Huppke et al., 000; Wan et al., ). For Laccone and colleagues (00) in German population, the most frequent mutation was RX. The most frequent mutation in an American cohort (Buyse et al., 000) was the missense mutation TM. Deletions and insertions of multiple nucleotides in the carboxyl terminus of MECP account for % of disease-causing mutations (Amir and Zoghbi, 000). In our large French cohort, different mutations have been identified in RTT patients, and have never been observed by other European, American, or Japanese groups (Fig. A C). In our study, the most common mutation is the nonsense mutation RX (.%), followed by two other nonsense mutations, RX (0.%) and R70X (0.%). The most frequent missense mutations are TM (7.%) and R0C (.%). All of the most frequent mutations identified in our study involved C R T transitions at CpG mutation hot spots. All of these mutations (except RX) were detectable by PCR-restriction fragment length polymorphism (RFLP), which represents a more rapid alternative to direct sequencing. In this study, we identified 0 patients with etions (.7%) and in the majority of these RTT patients (%), the proximal etion breakpoint is located in a restricted GC-rich region of 0 bp between 0 and. The MECP gene is not involved in the etiology of all cases of RTT After scanning of the whole coding and flanking sequences of MECP, there is still a substantial fraction of patients who have no identifiable abnormalities in the MECP gene ( %). Some mutations may be undetected, because they are located in regions of the gene that have not been analyzed [for instance, and untranslated regions (UTRs) or intronic regions], or because they cannot be detected through PCR-based techniques in females (a etion encompassing one or more exons, intragenic inversions). For example, Southern blot analysis performed by the molecular group from Nancy identified two gross DNA rearrangements that

5 MECP IN RETT SYNDROME could not be detected by classical PCR approach (Bourdon et al., 00). These observations suggest that Southern blot analysis should be systematically performed when no MECP mutation has been identified after the whole scanning analysis of the MECP gene by the PCR approach. Despite the fact that RTT samples have not been completely evaluated for complex rearrangements or for noncoding region mutations, the high proportion of patients with no MECP mutation detected (0 %) and the absence of identification of an MECP mutation in a few familial RTT cases suggests that there might be more than one gene for RTT (Xiang et al., 000; Villard et al., 00). Prenatal diagnosis Following requests from several families in which a MECP mutation was identified in each index case, French laboratories have performed prenatal tests. The de novo status of the MECP mutation was confirmed by DNA analysis of the mother in each family. Prenatal diagnosis and direct sequencing performed on amniotic fluid and cultured amniocytes was negative for the mutation in all cases. In view of these preliminary results as well as the very rare familial and germline mosaicism cases, we believe that prenatal diagnosis should not be encouraged in any family with a sporadic case of RTT syndrome. However, if the mutation is inherited from a healthy mother, the family should be informed about the high risk of occurrence in further generations and appropriate genetic counseling should be provided to the family. Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 0/7/. For personal use only. In conclusion, different screening approaches using a combination of scanning methods and direct sequencing identified MECP mutation in approximately % of RTT subjects. Although diverse strategies were used, none of them did allow us to detect a mutation in all cases. Although clinical heterogeneity could not be excluded, altogether these data and the absence of mutations in families not linked to Xq (Villard et al., 00) suggest the presence of at least a second gene involved in Rett syndrome. In this first large cohort of 0 French Rett patients, different mutations have been identified, never observed by other groups. The most frequent mutations are nonsense mutations located in coding exon : RX, RX, and R70X. The determination of this spectrum of MECP mutation provides information relevant to genetic counseling in RTT families. ACKNOWLEDGMENTS This work was supported in part by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), the Association Française contre les Myopathies (AFM), and the Association Française du syndrome de Rett (ASFR). The authors thank the Genethon bank and the colleagues from the Société Française de Neuropédiatrie who participated to the diagnosis evaluation and allowed us to study their patients. REFERENCES AMIR, R.E., and ZOGHBI, H.Y. (000). Rett syndrome: methyl-cpg- Binding protein mutations and phenotype-genotype correlations. Am. J. Hum. Genet. 7, 7. AMIR, R.E., VAN DEN VEYVER, I.B., WAN, M., TRAN, C.Q., FRANCKE, U., and ZOGHBI, H.Y. (). Rett syndrome is caused by mutations in X-linked MECP, encoding methyl-cpg-binding protein. Nature Genet.,. BIENVENU, T., CAZENEUVE, C., KAPLAN, J.C., and BELDJORD, C. (). Mutation heterogeneity of cystic fibrosis in France: Screening by denaturing gradient gel electrophoresis using psoralenmodified oligonucleotide. Hum. Mutat.,. BIENVENU, T., CARRIE, A., DE ROUX, N., VINET, M.C., JON- VEAUX, P., COUVERT, P., VILLARD, L., ARZIMANOGLOU, A., BELDJORD, C., FONTES, M., TARDIEU, M., and CHELLY, C. (000). MECP mutations account for most cases of typical forms of Rett syndrome. Hum. Mol. Genet., 77. BOURDON, V., PHILIPPE, C., LABRUNE, O., AMSALLEM, C., ARNOULD, C., and JONVEAUX, P. (00). A detailed analysis of the MECP gene: prevalence of recurrent mutations and gross DNA rearrangements in Rett syndrome patients. Hum. Genet. 0, 0. BUYSE, I.M., FANG, P., HOON, K.T., AMIR, R.E., ZOGHBI, H.Y., and ROA, B.B. (000). Diagnostic testing for Rett syndrome by DHPLC and direct sequencing analysis of the MECP gene: identification of several novel mutations and polymorphisms. Am. J. Hum. Genet. 7,. CHEADLE, J.P., GILL, H., FLEMING, N., MAYNARD, J., KERR, A., LEONARD, H., KRAWCZAK, M., COOPER, D.N., LYNCH, S., THOMAS, N., HUGHES, M., RAVINE, D., SAMPSON, J.R., and CLARKE, A. (000). Long-read sequence analysis of the MECP gene in Rett syndrome patients: correlation of disease severity with mutation type and location. Hum. Mol. Genet.,. COUVERT, P., BIENVENU, T., AQUAVIVA, C., POIRIER, K., MORAINE, C., GENDROT, C., VERLOES, A., ANDRES, C., LE FEVRE, A.C., SOUVILLE, I., STEFFANN, J., DES PORTES, V., ROPERS, H.H., YNTEMA, H.G., FRYNS, J.P., BRIAULT, S., CHELLY, J., and BELDJORD, C. (00). MECP is highly mutated in X-linked mental retardation. Hum. Mol. Genet. 0,. DRAGICH, J., HOUWINK-MANVILLE, I., and SCHANEN, C. (000). Rett syndrome: a surprising result of mutation in MECP. Hum. Mol. Genet., 7. HAGBERG, B., AICARDI, J., DIAS, K., and RAMOS, O. (). A progressive syndrome of autism, dementia, ataxia and loss of purposeful hand use in girls: Rett s syndrome: report of cases. Ann. Neurol., 7 7. HAMPSON, K., WOODS, C.G., LATIF, F., and WEBB, T. (000). Mutations in the MECP gene in a cohort of girls with Rett syndrome. J. Med. Genet. 7, 0. HUPPKE, P., LACCONE, F., KRAMER, N., ENGEL, W., and HANE- FELD, F. (000). Rett syndrome: analysis of MECP and clinical characterization of patients. Hum. Mol. Genet., 7. KIM, S.J., and COOK, E.H., JR. (000). Novel de novo nonsense mutation of MECP in a patient with Rett syndrome. Hum. Mutat. # 07 on line. LACCONE, F., HUPPKE, P., HANEFELD, F., and MEINS, M. (00). Mutation spectrum in Patients with Rett syndrome in the German population: evidence of hot spot regions. Hum. Mutat. 7, 0. LEWIS, J.D., MEEHAN, R.R., HENZEN, W.J., MAURER-FOGY, I., JEPPESEN, P., KLEIN, P., and BIRD, A. (). Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell, 0. MELONI, I., BRUTTINI, M., LONGO, I., MARI, F., RIZZOLIO, F., D ADAMO, P., DENVRIENDT, K., FRYNS, J.P., TONIOLO, D., and RENIERI, A. (000). A mutation in the Rett syndrome gene, MECP, causes X-linked mental retardation and progressive spasticity in males. Am. J. Hum. Genet. 7,. NAN, X., MEEHAN, R.R., and BIRD, A. (). Dissection of the methyl-cpg binding domain from the chromosomal protein MeCP. Nucleic Acids Res.,.

6 Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 0/7/. For personal use only. FRENCH CONSORTIUM OBATA, K., MATSUISHI, T., YAMASHITA, Y., FUKUDA, T., KUWAJIMA, K., HORIUCHU, I., NAGAMITSU, S., IWANAGA, R., KIMURA, A., OMORI, I., ENDO, S., MORI, K., and KONDO, I. (000). Mutation analysis of the methyl-cpg-binding protein gene (MECP) in patients with Rett syndrome. J. Med. Genet. 7, 0 0. ORRICO, A., LAM, C.W., GALLI, L., DOTTI, M.T., HAYE, K.G., TONG, S.F., POON, P.M.K., ZAPELLA, M., FEDERICO, A., and SORRENTINO, V. (000). MECP mutation in male patients with non-specific X-linked mental retardation. FEBS Lett. 0,. RETT, A. (). Ueber ein eigenartiges hirnatrophisches syndrom bei hyperammoniamie in kindesalter. Wien. Med. Wschr., 7 7. VILLARD, L., LEVY, N., XIANG, F., KPEBE, A., LABELLE, V., CHEVILLARD, C., ZHANG, Z., SCHWARTZ, C.E., TARDIEU, M., CHELLY, J., ANVRET, M., and FONTES, M. (00). Segregation of a totally skewed pattern of X-chromosome inactivation in four damilial cases of Rett syndrome without MECP mutation: implications for the disease. J. Med. Genet. (in press). WAN, M., LEE, S.S., ZHANG, X., HOUWINK-MANVILLE, I., SONG, H.R., AMIR, R.E., BUDDEN, S., NAIDU, S., PEREIRA, J.L., LO, I.F., ZOGHBI, H.Y., SCHANEN, N.C., and FRANCKE, U. (). Rett syndrome and beyond: recurrent spontaneous and familial MECP mutations at CpG hotspots. Am. J. Hum. Genet.,. WEBB, T., CLARKE, A., HANEFELD, F., PEREIRA, J.L., ROSEN- BLOOM, L., and WOODS, C.G. (). Linkage analysis in Rett syndrome families suggests that there may be a critical region at Xq. J. Med. Genet., XIANG, F., BUERVENICH, S., NICOLAO, P., BAILEY, M.E.S., ZHANG, Z., and ANVRET, M. (000). Mutation screening in Rett syndrome patients. J. Med. Genet. 7, 0. Address reprint requests to: Dr. Thierry Bienvenu INSERM U CHU Cochin 7 rue du Faubourg Saint Jacques 70 Paris, France bienvenu@cochin.inserm.fr Chelly@cochin.inserm.fr Received for publication May, 00; accepted December 0, 00.