Abstract Mutations in the coding region of the methyl-CpG-binding protein 2 (MECP2) gene cause Rett syn-drome and have also been reported in a number of X-linked mental retardation syndromes. Furthermore, suchmutations have recently been described in a few autisticpatients. In this study, a large sample of individuals withautism was screened in order to elucidate systematicallywhether specific mutations in MECP2 play a role in autism.The mutation analysis of the coding sequence of the genewas performed by denaturing high-pressure liquid chro-matography and direct sequencing. Taken together, 14 se-quence variants were identified in 152 autistic patientsfrom 134 German families and 50 unrelated patients fromthe International Molecular Genetic Study of Autism Con-sortium affected relative-pair sample. Eleven of these vari-ants were excluded for having an aetiological role as theywere either silent mutations, did not cosegregate withautism in the pedigrees of the patients or represented knownpolymorphisms. The relevance of the three remaining mu-tations towards the aetiology of autism could not be ruledout, although they were not localised within functionaldomains of MeCP2 and may be rare polymorphisms. Tak-ing into account the large size of our sample, we concludethat mutations in the coding region of MECP2 do not playa major role in autism susceptibility. Therefore, infantileautism and Rett syndrome probably represent two distinctentities at the molecular genetic level.

Introduction

Autistic disorder (AD; MIM 209850) is a complex neu-rodevelopmental disorder that is characterised by socialdeficits, communication impairments and patterns of repet-itive and stereotyped behaviours and interests, with onsetwithin the first three years of life. The population preva-lence of core autism was for many years reported to be inthe range of 4–5 per 10,000 births (Smalley et al. 1988),although recent surveys indicate a prevalence of 17 in10,000 (Chakrabarti and Fombonne 2001). Twin and fam-ily studies indicate a strong genetic component (Folsteinand Rutter 1977; Ritvo et al. 1985; Steffenburg et al.1989; Bailey et al. 1995). Autism is 3–4 times more com-mon in males than females; one explanation for the pre-dominance of male patients could be the involvement ofan X-linked gene (Skuse et al. 2000). Moreover, genomescreens have identified several, but non-overlapping, re-gions with small linkage scores on the X chromosome,mostly in Xq (Folstein and Rosen-Sheidley 2001).

Rett Syndrome (RTT; MIM 312750) is also a pervasivedevelopmental disorder, occurring almost exclusively infemales. Numerous studies have demonstrated that themajority of patients with RTT have mutations in the cod-ing region of the X chromosomal gene MECP2 (Xq28; forreview, see Amir and Zoghbi 2000; Buyse et al. 2000;Bourdon et al. 2001). Heterozygous mutations in the genehave been identified in up to 80% of Rett syndrome cases.The wide spectrum of phenotypic variability in RTT iscorrelated with mutation type and location in the MECP2gene (Wan et al. 1999; Cheadle et al. 2000) and the pat-tern of X-inactivation (Amir et al. 2000; Zappella et al.2001). Recently, investigation of males with severe andmild mental retardation (Meloni et al. 2000; Orrico et al.2000; Couvert et al. 2001; Yntema et al. 2002; Klauck etal. 2002) and preserved speech variants (PSV) of Rettsyndrome (Zappella et al. 2001) has identified variousmutations within the coding sequence of the gene sug-gesting that MECP2 is a strong candidate for non-specificX-linked mental retardation (XLMR) and PSV.

Kim S. Beyer · Francesca Blasi · Elena Bacchelli ·Sabine M. Klauck · Elena Maestrini · Annemarie Poustka ·International Molecular Genetic Study of Autism Consortium (IMGSAC)

Mutation analysis of the coding sequence of the MECP2 gene in infantile autism

Hum Genet (2002) 111 :305–309DOI 10.1007/s00439-002-0786-3

Received: 4 March 2002 / Accepted: 11 June 2002 / Published online: 14 August 2002

ORIGINAL INVESTIGATION

The International Molecular Genetic Study of Autism Consortium(IMGSAC) may be reached at http://www.well.ox.ac.uk/~maestrin/iat.html or care of Wellcome Trust Centre for Human Genetics, Oxford, OX3 7BN, UK.

K.S. Beyer · S.M. Klauck · A. Poustka (✉)Department of Molecular Genome Analysis (H0600), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germanye-mail: a.poustka@dkfz.de, Tel.: +49-6221-424742, Fax: +49-6221-423454

F. Blasi · E. Bacchelli · E. MaestriniDepartment of Biology, University of Bologna, Bologna, Italy

© Springer-Verlag 2002

The resemblance between the phenotypes for autismand Rett syndrome and the finding that 70% of individu-als with autism show some degree of mental retardationraises the question of whether specific mutations withinthe coding region of MECP2 are involved in the aetiologyof infantile autism. To date two groups have identifiedmutations in this gene in sporadic cases of autism (Lam etal. 2000; Carney et al. 2001), whereas no mutations havebeen found by another group in a sample of 59 autistic in-dividuals (Vourc’h et al. 2001). The aim of this study hasbeen to test a large and well-characterised sample of autis-tic individuals for mutations in the coding region of theMECP2 gene in order to evaluate its possible role in theaetiology of infantile autism.

Materials and methods

Nomenclature

Gene mutation nomenclature used in this article follows the rec-ommendations of den Dunnen and Antonarakis (2001). Gene sym-bols used in this article follow the recommendations of the HUGOGene Nomenclature Committee (Povey et al. 2001).

Patients

Mutation screening was conducted on 152 patients from 134 Ger-man singleton and multiplex families (117 singleton families, 16families with 2 affected individuals, one family with 3 affectedchildren) and on 50 unrelated patients from the International Mol-ecular Genetic Study of Autism Consortium (IMGSAC) affectedrelative-pairs collection. Autistic patients were recruited and diag-nosed as described previously (Klauck et al. 1997; IMGSAC 1998,2001) according to the criteria of the autism diagnostic interview-revised (Lord et al. 1994; German version: Poustka et al. 1996)and autism diagnostic observation schedule (ADOS; Lord et al.1989) or ADOS-generic (Lord et al. 2000). The individuals werealso psychologically tested for an IQ≥35 (average IQ: 70, standarderror: 1.8) and neurologically examined. Of the 202 probands, 154were male and 48 were female; 149 patients fulfilled the “case type

1” criteria and 53 fulfilled the “case type 2” criteria (described inIMGSAC 2001). Blood samples for DNA extractions were takenfrom patients and available first-degree relatives.

Mutation screening

Genomic DNA was extracted from peripheral blood lymphocytesaccording to standard protocols. Mutation analysis for the 154 Ger-man patients was performed by using denaturing high-pressure liq-uid chromatography (DHPLC; WAVE DNA fragment analysissystem, DNASep column; Transgenomic; O’Donovan et al. 1998).The nine partially overlapping polymerase chain reaction (PCR)products from male patients were analysed after admixture of anequal amount of a control PCR product. The same PCR productsfrom female patients were analysed with and without admixture ofwildtype product. Mutations were confirmed by direct sequencingof the corresponding PCR fragment from the patient and all avail-able first-degree relatives.

The 50 unrelated individuals from IMGSAC affected relative-pair families were screened for mutations by direct sequencing ofseven PCR products covering the coding region of MECP2. Of the50 IMGSAC patients, 38 were selected from affected sib-pairssharing the same maternal Xq27-q28 region, as determined fromthe inheritance of microsatellite markers (DXS998 and DXS1108)that had been previously genotyped for the IMGSAC genome scan(IMGSAC 1998). This information was not available for the re-maining 12 families. All primer sequences and DHPLC conditionsare available upon request.

Results and discussion

In this study, 14 sequence variants within the coding ex-ons and flanking intron sequences were identified in 202autistic patients from 184 families (Table 1). The screen-ing of a control sample was omitted because of the previ-ously published extensive studies in RTT patients andtheir controls. To our knowledge, seven of these variantshave not been previously described. Each variant wasonly detected in one family, with one family inheritingtwo sequence variations (C984T, 1161delCCC).

306

Table 1 Identified MECP2mutations

aPosition 1 corresponds to nu-cleotide 85 in the mRNA refer-ence sequence (accession no.X99686; start codon)bP Polymorphism attributableto silent mutation, no cosegre-gation with autism in this studyor previously described as apolymorphism in Rett studies.? Definition as causative muta-tion for autism or as polymor-phism not possible from thisstudycBoth mutations inheritedwithin one family

cDNAa Protein Varianttypeb

Reference

377+22CÆG – P Lam et al. 2000377+95GÆA – P This studyC542T A181V ? This studyC879T 299T P Cheadle et al. 2000,

Couvert et al. 2001,Bourdon et al. 2001

C984Tc 328L P Buyse et al. 2000C1126T P376S ? This studyC1137T 379P P This study1161delCCCc 388delP P This studyG1189A E397K P Wan et al. 1999

German sample(152 patients from134 families)

G1315A A439T P Cheadle et al. 2000

378-17delT – P Trappe et al. 2001C582T 194S P Amir et al. 2000,

Buyse et al. 2000C1199T 399P P This study

IMGSAC sample(50 patients from50 affected sib-pairfamilies)

C1207T P402L ? This study

Three mutations were located in the intron between ex-ons 3 and 4 (377+22C→G, 377+95G→A, 378-17delT) anddo not influence the splicing process according to databaseanalyses (Spliceview: http://l25.itba.mi.cnr.it/~webgene/wwwspliceview.html). Five variants were synonymous pointmutations (C582T, C879T, C984T, C1137T, C1199T).The two non-synonymous mutations G1189A (E397K)and G1315A (A439T) have been previously described asrare polymorphisms (Wan et al. 1999; Cheadle et al. 2000)and therefore could be excluded from causing autism.

Four additional non-synonymous changes were identi-fied in this study for the first time. In family 257, variant1161delCCC leading to the deletion of one of five succes-sive proline residues (388delP) was identified as beinglinked to the previously published rare polymorphismC948T (328L) (Fig.1A). The affected daughter (II-1) andher non-affected sister (II-2) inherited both variants fromtheir hemizygous non-autistic father (I-2). Since the vari-ants were not transmitted to the affected son II-3, thenovel variant probably represents a rare polymorphism.

The segregation of the C1207T (P402L) variant withinfamily 167 is shown in Fig.1B. It is present in a pair of af-fected cousins (III-1 and III-3) but also in III-4 who doesnot have autism; the two sisters III-3 and III-4 inheritedthe variant from their hemizygous father. Therefore, thisvariant is unlikely to have a pathogenic role.

The two non-synonymous mutations C542T (A181V)and C1126T (P376S) segregate with the autistic pheno-type within the available nuclear families 71 and 226, re-spectively (Fig.1C, D). Because of the lack of genetic ma-

terial and clinical information from other family mem-bers, further analyses could not be performed to clarifywhether these mutations have a causal role in autism orrepresent very rare polymorphisms. Family 226 is of spe-cial interest, since the affected mother of a more severelyaffected son showed no language delay but met the crite-ria for infantile autism. The homozygosity of the affectedmother for the C1126T change implies that both her par-ents inherited the sequence variation, except in the un-likely possibility of maternal uniparental disomy. Infor-mation about the genotype and clinical details of the ma-ternal grandfather would be helpful in elucidating the roleof this sequence variation. Furthermore, the contributionof certain variants in MECP2 to intellectual disability inprobands with autism could be considered. This possibil-ity cannot be ruled out completely in the patients fromboth families 71 and 226 as they show mild mental retar-dation (performance IQs: family 71, II-1, IQ=75; family226, I-1, IQ=93, II-1, IQ=64).

Each of the four non-synonymous changes was identi-fied in only one family in our sample. Consensus proteinstructure prediction (http://npsa-pbil.ibcp.fr) did not re-veal any changes for A181V and P376S and only a ques-tionable loss of a short alpha helix around position 412 ofthe protein for P402L and 388delP. In addition, all fourvariants are not localised in a known functional domain ofMeCP2 (the methyl-binding-domain and the transcriptionrepression domain) and therefore probably do not dramat-ically alter the function of the gene product. It is interest-ing to note, however, that three of the four variants

307

Fig.1A–D Pedigrees of fourfamilies with segregation of in-dicated variants in the MECP2gene. The mutated nucleotideis in bold (open squares unaf-fected males, closed squaresaffected males, open circlesunaffected females, closed cir-cles affected females, wt wild-type, del deletion). Families226, 71 and 257 belong to theGerman patient sample; family167 is an IMGSAC relative-pair family

(P376S, 388delP and P402L) are located in a putativefunctional COOH domain showing homology to brain-specific members of the fork-head gene family (Vacca etal. 2001). Mutations in this domain have been reported inassociation with both RTT (Vacca et al. 2001) and XLMR(Meloni et al. 2000; Couvert et al. 2001); however, thepathogenic role of missense mutations in this domain re-mains questionable (Moncla et al. 2002). The develop-ment of functional assays will be needed to clarify the rel-evance of the non-synonymous changes identified.

In conclusion, the results of our screening in a largewell-characterised patient sample indicate that mutationsin the coding sequence of MECP2 are unlikely to play amajor role in the aetiology of autism. No common RTTmutations (truncations and missense mutations within thetwo functional domains of the gene) have been identifiedin these autistic patients. Therefore, infantile autism andRett syndrome probably represent two distinct entities atthe molecular genetic level. Uncertainty remains for thosepre-diagnosed RTT cases, where no mutations in the cod-ing region have been identified so far. In future, it may benecessary to extend the mutation screening of RTT casesto non-coding regions of the gene and to include genesregulating the expression of the MECP2 gene. To date, theanalysis of the non-coding and regulatory regions of theMECP2 gene have been omitted in the published studiesof RTT, mental retardation and autistic patients. Varia-tions in the long 3’-untranslated region harbouring severalpolyadenylation signals that are differentially used (Coyet al. 1999) could be of especial relevance for eitherautism or Rett syndrome.

Acknowledgements We thank all the patients and their familiesfor their participation in this study and the many referring profes-sionals. We are also grateful to Tatjana Kraus, Sabine Epp and themembers of the sequencing unit of the Department of MolecularGenome Analysis (DKFZ Heidelberg, Germany) for excellenttechnical assistance. This study is funded in part by the DeutscheForschungsgemeinschaft, Telethon-Italy (grant E.1007) and ECFifth Framework (grant QLG2-CT-1999-0094).

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