Original article
Deleterious mutations in exon 1 of MECP2 in Rett syndrome

https://doi.org/10.1016/j.ejmg.2005.11.002Get rights and content

Abstract

The MECP2 gene is responsible for 80–85% of typical cases of Rett syndrome with deleterious mutations affecting exons 3 and 4. Recently, an alternate transcript including exon 1 was discovered with a new protein isoform (MeCP2_e1) much more abundant in brain. We screened exon 1 of MECP2 for mutations and for large rearrangements in a panel of 212 typical cases of Rett syndrome and one family case with atypical Rett syndrome. We identified two deleterious mutations (c.48_55dup and c.62+2_62+3del) and four large rearrangements encompassing exon 1 of MECP2. We also identified the c.16_21dup alteration formerly reported as c.3_4insGCCGCC and give additional support to classify this sequence variation as polymorphic. In our large panel of typical Rett, mutations affecting exon 1 of MECP2 represent 1% of the deleterious alleles. This study confirms that mutations in exon 1 of MECP2 are a rare cause of Rett syndrome.

Introduction

Rett syndrome (RTT, MIM # 312750) is a progressive devastating neurological developmental disorder affecting almost exclusively girls, it occurs in 1:15,000 female births and is the most common genetic cause of profound mental retardation in females [17]. Mutations in MECP2 are a frequent cause of RTT [3], [11], [17]. The human MECP2 gene was cloned in 1996 and described first as a three exon gene [6]. Comparative sequence analysis enabled the identification of a fourth 5′ exon initially considered as non coding [15] and until recently, a single MECP2 transcript (MECP2_e2) with all four exons and a start codon in exon 2 was identified. Two studies combining bioinformatics and molecular biology reported a second alternative splice variant (MECP2_e1) with the start codon in exon 1 joined to exon 3 in mature mRNA [10], [12]. The MECP2_e1 open reading frame leads to different MeCP2 terminus with a distinctive 21 amino-acid peptide, MeCP2_e1 is the predominant protein isoform of 498 amino acids in adult human brain [12] where its level is 10 times higher compared to those of the MeCP2_2 isoform of 486 amino acids.

As exon 1 was thought to be non coding, it was therefore not screened for mutation, and deleterious alterations were reported exclusively in exons 3 and 4 ([3], [11], [17], RettBASE). Mutations in exon 2 present only in the transcript generating MeCP2_e2 were never found, this strongly supports a more important functional role for the MeCP2_e1 isoform and suggests that exon 1 has to be analysed in mutation negative RTT patients. So far, few mutations in exon 1 were found, almost exclusively in typical RTT [1], [12], [14], [16]. In this study we assessed the mutation rate in exon 1 of MECP2 in a large cohort of classical RTT by denaturating high pressure liquid chromatography (DHPLC) and quantitative multiplex PCR of short fluorescent fragments (QMPSF).

Section snippets

Patients and DNA samples

We studied 212 patients with classical sporadic Rett diagnosed according to the international criteria [2], [8]. Blood samples were obtained from patients after informed consent. Total genomic DNA was extracted using the Nucleon BACC3 kit (Amersham Life Technologies) according to the manufacturer's protocol.

PCR amplification

Exon 1, a 207 bp long GC-rich amplicon difficult to amplify, was PCR amplified with the Thermoprime Taq polymerase (ABgene) by using 7.5% DMSO, 1 mM MgCl2 and 38 cycles with annealing at

Results

We routinely applied a DHPLC based molecular diagnostic protocol for all four exons of the MECP2 gene. For all RTT patients that did not show any deleterious mutation we searched for large rearrangements of the MECP2 gene by QMPSF. We tested all four exons of the MECP2 gene in a cohort of 212 typical RTT and one family case with atypical RTT. We found 148 deleterious mutations in exons 3 and 4 together with 27 heterozygous gross rearrangements of MECP2 ranging from partial deletions of exon 4

Discussion

Recent studies indicated that mutations in exon 1 are not a common cause of RTT [1], [7], [16]. To our knowledge, before our study a total of five deleterious mutations affecting exclusively the synthesis of MeCP2_e1 were reported in exon 1 of MECP2. The c.38_48del/c.47_57del deletions was reported three times in typical [12], [14], [16] and atypical RTT [1]. Symmetric elements predispose DNA sequences to meiotic microdeletions [9]. The hotspot in exon 1 of MECP2 consists of symmetric elements

Acknowledgements

This study was supported by grant from GIS-Institut des maladies rares. We wish to thank T. Frébourg and M. Tosi for help in implementing the QMPSF protocol for the detection of large rearrangements in the MECP2 gene.

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Databases: OMIM: 312750 (RTT), Genbank: NT_025913 and AY541280.1; RettBASE: http://mecp2.chw.edu.au.

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