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

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, such mutations have recently been described in a few autistic patients. In this study, a large sample of individuals with autism was screened in order to elucidate systematically whether specific mutations in MECP2 play a role in autism.

The mutation analysis of the coding sequence of the gene was performed by denaturing high-pressure liquid chro- matography and direct sequencing. Taken together, 14 se- quence variants were identified in 152 autistic patients from 134 German families and 50 unrelated patients from the 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 they were either silent mutations, did not cosegregate with autism in the pedigrees of the patients or represented known polymorphisms. The relevance of the three remaining mu- tations towards the aetiology of autism could not be ruled out, although they were not localised within functional domains of MeCP2 and may be rare polymorphisms. Tak- ing into account the large size of our sample, we conclude that mutations in the coding region of MECP2 do not play a major role in autism susceptibility. Therefore, infantile autism and Rett syndrome probably represent two distinct entities at the molecular genetic level.

Introduction

Autistic disorder (AD; MIM 209850) is a complex neu- rodevelopmental disorder that is characterised by social deficits, communication impairments and patterns of repet- itive and stereotyped behaviours and interests, with onset within the first three years of life. The population preva- lence of core autism was for many years reported to be in the range of 4–5 per 10,000 births (Smalley et al. 1988), although recent surveys indicate a prevalence of 17 in 10,000 (Chakrabarti and Fombonne 2001). Twin and fam- ily studies indicate a strong genetic component (Folstein and 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 of an X-linked gene (Skuse et al. 2000). Moreover, genome screens 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 pervasive developmental disorder, occurring almost exclusively in females. Numerous studies have demonstrated that the majority of patients with RTT have mutations in the cod- ing region of the X chromosomal gene MECP2 (Xq28; for review, see Amir and Zoghbi 2000; Buyse et al. 2000;

Bourdon et al. 2001). Heterozygous mutations in the gene have been identified in up to 80% of Rett syndrome cases.

The wide spectrum of phenotypic variability in RTT is correlated with mutation type and location in the MECP2 gene (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 and mild mental retardation (Meloni et al. 2000; Orrico et al.

2000; Couvert et al. 2001; Yntema et al. 2002; Klauck et al. 2002) and preserved speech variants (PSV) of Rett syndrome (Zappella et al. 2001) has identified various mutations within the coding sequence of the gene sug- gesting that MECP2 is a strong candidate for non-specific X-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

DOI 10.1007/s00439-002-0786-3

Received: 4 March 2002 / Accepted: 11 June 2002 / Published online: 14 August 2002 O R I G I N A L I N V E S T I G AT I O N

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, Germany e-mail: [email protected],

Tel.: +49-6221-424742, Fax: +49-6221-423454 F. Blasi · E. Bacchelli · E. Maestrini

Department of Biology, University of Bologna, Bologna, Italy

© Springer-Verlag 2002

The resemblance between the phenotypes for autism and Rett syndrome and the finding that 70% of individu- als with autism show some degree of mental retardation raises the question of whether specific mutations within the coding region of MECP2 are involved in the aetiology of infantile autism. To date two groups have identified mutations in this gene in sporadic cases of autism (Lam et al. 2000; Carney et al. 2001), whereas no mutations have been found by another group in a sample of 59 autistic in- dividuals (Vourc’h et al. 2001). The aim of this study has been to test a large and well-characterised sample of autis- tic individuals for mutations in the coding region of the

aetiology 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 HUGO Gene 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, 16 families with 2 affected individuals, one family with 3 affected children) and on 50 unrelated patients from the International Mol- ecular Genetic Study of Autism Consortium (IMGSAC) affected relative-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 were also psychologically tested for an IQ≥35 (average IQ: 70, standard error: 1.8) and neurologically examined. Of the 202 probands, 154 were male and 48 were female; 149 patients fulfilled the “case type

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

Mutation screening

Genomic DNA was extracted from peripheral blood lymphocytes according 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 analysis system, 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 an equal amount of a control PCR product. The same PCR products from female patients were analysed with and without admixture of wildtype product. Mutations were confirmed by direct sequencing of 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 of seven PCR products covering the coding region of MECP2. Of the 50 IMGSAC patients, 38 were selected from affected sib-pairs sharing the same maternal Xq27-q28 region, as determined from the 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 conditions are available upon request.

Results and discussion

In this study, 14 sequence variants within the coding ex- ons and flanking intron sequences were identified in 202 autistic 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 and their controls. To our knowledge, seven of these variants have not been previously described. Each variant was only detected in one family, with one family inheriting two sequence variations (C984T, 1161delCCC).

Table 1 Identified MECP2 mutations

aPosition 1 corresponds to nu- cleotide 85 in the mRNA refer- ence sequence (accession no.

X99686; start codon)

bP Polymorphism attributable to silent mutation, no cosegre- gation with autism in this study or previously described as a polymorphism in Rett studies.

? Definition as causative muta- tion for autism or as polymor- phism not possible from this study

cBoth mutations inherited within one family

cDNA^a Protein Variant

type^b

Reference

377+22CÆG – P Lam et al. 2000

377+95GÆA – P This study

C542T A181V ? This study

C879T 299T P Cheadle et al. 2000,

Couvert et al. 2001, Bourdon et al. 2001

C984T^c 328L P Buyse et al. 2000

C1126T P376S ? This study

C1137T 379P P This study

1161delCCC^c 388delP P This study

G1189A E397K P Wan et al. 1999

German sample (152 patients from 134 families)

G1315A A439T P Cheadle et al. 2000

378-17delT – P Trappe et al. 2001

C582T 194S P Amir et al. 2000,

Buyse et al. 2000

C1199T 399P P This study

IMGSAC sample (50 patients from 50 affected sib-pair families)

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) and do not influence the splicing process according to database analyses (Spliceview: http://l25.itba.mi.cnr.it/~webgene/

wwwspliceview.html). Five variants were synonymous point mutations (C582T, C879T, C984T, C1137T, C1199T).

The two non-synonymous mutations G1189A (E397K) and G1315A (A439T) have been previously described as rare 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, variant 1161delCCC leading to the deletion of one of five succes- sive proline residues (388delP) was identified as being linked to the previously published rare polymorphism C948T (328L) (Fig. 1A). The affected daughter (II-1) and her non-affected sister (II-2) inherited both variants from their hemizygous non-autistic father (I-2). Since the vari- ants were not transmitted to the affected son II-3, the novel variant probably represents a rare polymorphism.

The segregation of the C1207T (P402L) variant within family 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 does not have autism; the two sisters III-3 and III-4 inherited the variant from their hemizygous father. Therefore, this variant 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 clarify whether these mutations have a causal role in autism or represent very rare polymorphisms. Family 226 is of spe- cial interest, since the affected mother of a more severely affected son showed no language delay but met the crite- ria for infantile autism. The homozygosity of the affected mother 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 role of this sequence variation. Furthermore, the contribution of certain variants in MECP2 to intellectual disability in probands with autism could be considered. This possibil- ity cannot be ruled out completely in the patients from both families 71 and 226 as they show mild mental retar- dation (performance IQs: family 71, II-1, IQ=75; family 226, 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 protein structure 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 of the protein for P402L and 388delP. In addition, all four variants are not localised in a known functional domain of MeCP2 (the methyl-binding-domain and the transcription repression 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

families with segregation of in- dicated variants in the MECP2 gene. The mutated nucleotide is in bold (open squares unaf- fected males, closed squares affected males, open circles unaffected females, closed cir- cles affected females, wt wild- type, del deletion). Families 226, 71 and 257 belong to the German patient sample; family 167 is an IMGSAC relative- pair family

(P376S, 388delP and P402L) are located in a putative functional COOH domain showing homology to brain- specific members of the fork-head gene family (Vacca et al. 2001). Mutations in this domain have been reported in association with both RTT (Vacca et al. 2001) and XLMR (Meloni et al. 2000; Couvert et al. 2001); however, the pathogenic 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 large well-characterised patient sample indicate that mutations in the coding sequence of MECP2 are unlikely to play a major role in the aetiology of autism. No common RTT mutations (truncations and missense mutations within the two functional domains of the gene) have been identified in these autistic patients. Therefore, infantile autism and Rett syndrome probably represent two distinct entities at the molecular genetic level. Uncertainty remains for those pre-diagnosed RTT cases, where no mutations in the cod- ing region have been identified so far. In future, it may be necessary to extend the mutation screening of RTT cases to non-coding regions of the gene and to include genes regulating the expression of the MECP2 gene. To date, the analysis of the non-coding and regulatory regions of the

of RTT, mental retardation and autistic patients. Varia- tions in the long 3’-untranslated region harbouring several polyadenylation signals that are differentially used (Coy et al. 1999) could be of especial relevance for either autism or Rett syndrome.

Acknowledgements We thank all the patients and their families for their participation in this study and the many referring profes- sionals. We are also grateful to Tatjana Kraus, Sabine Epp and the members of the sequencing unit of the Department of Molecular Genome Analysis (DKFZ Heidelberg, Germany) for excellent technical assistance. This study is funded in part by the Deutsche Forschungsgemeinschaft, Telethon-Italy (grant E.1007) and EC Fifth Framework (grant QLG2-CT-1999-0094).

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