Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males

Abstract

Loss-of-function mutations of the MECP2 gene at Xq28 are associated with Rett syndrome in females and with syndromic and nonsyndromic forms of mental retardation (MR) in males. By array comparative genomic hybridization (array-CGH), we identified a small duplication at Xq28 in a large family with a severe form of MR associated with progressive spasticity. Screening by real-time quantitation of 17 additional patients with MR who have similar phenotypes revealed three more duplications. The duplications in the four patients vary in size from 0.4 to 0.8 Mb and harbor several genes, which, for each duplication, include the MR-related L1CAM and MECP2 genes. The proximal breakpoints are located within a 250-kb region centromeric of L1CAM, whereas the distal breakpoints are located in a 300-kb interval telomeric of MECP2. The precise size and location of each duplication is different in the four patients. The duplications segregate with the disease in the families, and asymptomatic carrier females show complete skewing of X inactivation. Comparison of the clinical features in these patients and in a previously reported patient enables refinement of the genotype-phenotype correlation and strongly suggests that increased dosage of MECP2 results in the MR phenotype. Our findings demonstrate that, in humans, not only impaired or abolished gene function but also increased MeCP2 dosage causes a distinct phenotype. Moreover, duplication of the MECP2 region occurs frequently in male patients with a severe form of MR, which justifies quantitative screening of MECP2 in this group of patients.

Figure A1

Figure 1

Pedigrees of the four families (L36, S49, T33, and T88) in which duplications at Xq28 have been identified. Blackened squares indicate affected males, and an asterisk (*) indicates the index patient of each family. Clinical pictures of index patients IV.1 (family L36) at age 1.5 years and III.2 (family S49) at age 12 years are shown. Data from X-inactivation studies are boxed and are shown below each female from whom we could obtain a DNA sample.

Figure 2

Identification of a duplication at Xq28 in family L36. A, X-chromosome array-CGH analysis of DNA (labeled with Cy5) from patient IV.1 mixed with DNA (labeled with Cy3) from a control male. Log₂ normalized ratios are plotted against the position on the X chromosome (in Mb). Except for two polymorphic clones, the ratios for clones in unaffected genomic regions are within the normal interval (−0.36 to 0.36), whereas clones RP11-314B4 and RP11-119A22 at 153 Mb show ratios >0.60 (circled), which is indicative of a duplication. The “gap” at 60 Mb represents the centromeric region for which no clones were available. B, Schematic representation of the Xq28 region (152.20–153.20 Mb), showing the location of the qPCR primer sets, genomic clones, and genes present in this interval for which qPCR primers were designed. The duplicated region, based on the array-CGH and qPCR results, is shown below. C, qPCR data on DNA from the index patient of family L36 (unblackened bars), as well as from two controls (Co1 and Co2 [blackened bars and gray bars, respectively]). The values for the fold differences obtained for each primer set are given above the bars for the L36 patient only.

Figure 3

qPCR data obtained by use of the MECP2a primer set for DNA from two controls (Co1 and Co2 [blackened bars]) as well as 17 patients with XLMR (unblackened bars) who showed a phenotype similar to that presented by the index patient of family L36, who was used as a positive control (gray bar). The values for the fold differences in comparison with controls are given above the bars. A duplication was found in three additional patients from families S49 (patient 11), T33 (patient 16), and T88 (patient 17).

Figure 4

Schematic representation of the positions and sizes of the duplications at Xq28 in the four families with severe MR associated with spasticity. The locations of the qPCR primer sets, genomic clones, and genes present in this interval for which qPCR primers were designed are given. In the lower part, unblackened boxes represent normal regions, and blackened boxes are confirmed duplicated regions. The breakpoints of the duplications are located within the dotted lines. The duplication identified by Meins et al. (2005) is shown at the bottom, as is the common minimal duplicated region in all patients.

Figure 5

Segregation of the duplication with the disease and increased MECP2 expression. A, Segregation was investigated by qPCR performed using the MECP2a primer set on all family members from whom we could obtain a DNA sample. The values for the fold differences in comparison with controls are given above the bars. A blackened bar indicates a male carrier (fold difference ∼2), and a gray bar indicates a female carrier (fold difference ∼1.5). B, Expression of MECP2 mRNA, by use of qPCR primers, in PBLs from the index patients of families L36, T33, and T88 (blackened bars), compared with controls (Co1–Co3 [unblackened bars]). The values for the fold differences in comparison with controls are given above the bars.