Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation

Abstract

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused, in most classic cases, by mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). A large degree of phenotypic variation has been observed in patients with RTT, both those with and without MECP2 mutations. We describe a family consisting of a proband with a phenotype that showed considerable overlap with that of RTT, her identical twin sister with autistic disorder and mild-to-moderate intellectual disability, and a brother with profound intellectual disability and seizures. No pathogenic MECP2 mutations were found in this family, and the Xq28 region that contains the MECP2 gene was not shared by the affected siblings. Three other candidate regions were identified by microsatellite mapping, including 10.3 Mb at Xp22.31-pter between Xpter and DXS1135, 19.7 Mb at Xp22.12-p22.11 between DXS1135 and DXS1214, and 16.4 Mb at Xq21.33 between DXS1196 and DXS1191. The ARX and CDKL5 genes, both of which are located within the Xp22 region, were sequenced in the affected family members, and a deletion of nucleotide 183 of the coding sequence (c.183delT) was identified in CDKL5 in the affected family members. In a screen of 44 RTT cases, a single splice-site mutation, IVS13-1G-->A, was identified in a girl with a severe phenotype overlapping RTT. In the mouse brain, Cdkl5 expression overlaps--but is not identical to--that of Mecp2, and its expression is unaffected by the loss of Mecp2. These findings confirm CDKL5 as another locus associated with epilepsy and X-linked mental retardation. These results also suggest that mutations in CDKL5 can lead to a clinical phenotype that overlaps RTT. However, it remains to be determined whether CDKL5 mutations are more prevalent in specific clinical subgroups of RTT or in other clinical presentations.

Figure 1

Pedigree of family 1, showing results of microsatellite-exclusion mapping. Haplotypes were derived using Cyrillic v2 software (Cyrillic Software) and are shown as differently shaded bars. The black bars represent grandpaternal alleles, the gray bars represent grandmaternal alleles, and the striped bars represent paternal alleles. Candidate regions are indicated by boxes. X-inactivation patterns are shown as ratios below the haplotypes. Microsatellites used in this study and their chromosomal band and map positions are given at the far left of the pedigree. Map distances were derived from the Cedar Genetics Map of the X Chromosome, the Ensembl Human Genome Browser, and the Integrated X Chromosome Database. The approximate positions of each of the candidate genes screened in this family are also indicated.

Figure 2

IVS13-1G→A mutation of CDKL5 in family 2. Chromatograms of the CDKL5 sequence that is mutated in family 2 are shown. A G-to-A transversion involving the intron 13 splice-acceptor site was identified in blood DNA from the subject with an atypical RTT–like phenotype (II:1) but not in her half sister (II:2) with classical RTT or either of her parents.

Figure 3

c.183delT mutation of CDKL5 in family 1. A chromatogram of the CDKL5 sequence that is mutated in family 1 is shown. Nucleotide numbers begin from the A of the translation start codon in exon 2. Individual ID numbers are derived from figure 1. A deletion of nucleotide 183 was observed in the blood DNA of affected individuals (III:1, III:2, and III:3) but not in unaffected family members.

Figure 4

cDNA and translated product of the CDKL5 IVS-1G→A mutation. A, Sequence chromatograms of representative cDNA clones isolated from II:1 (family 2) blood RNA/cDNA. In 5 (16.6%) of 30 clones examined, a c.2047delG CDKL5 cDNA was observed. B, Illustration of the creation of a novel intron 13 acceptor splice site as a result of the IVS13-1G→A mutation in CDKL5 of individual II:1 from family 2. The protein sequence is shown below the genomic sequence.

Figure 5

Schematic representation of the CDKL5 gene. CDKL5 exons are indicated by black boxes, and the last four exons of RS1, a gene overlapping CDKL5, are indicated by white boxes. The alternative-splicing patterns are indicated below the diagram for the known isoforms, I and II. Mutations and associated phenotypes that have been reported to date in association with CDKL5 mutations are given above the diagram, and those described in the present study are shown in bold.

Figure 6

Expression of CDKL5 in the mouse brain. A and B, Expression of Stk9 in the brain of wild-type postnatal week-8 mouse (A, antisense riboprobes; B, sense riboprobes). C, Expression of Mecp2 in the brain of wild-type postnatal week-8 mouse. D, Western-blot analysis showing the absence of the 75-kDa Mecp2 protein in the brain of the Mecp2 mutant male mouse, confirming that a null mutation has been generated by deleting the sequence coding the transcription-repression domain and the C-terminal of the protein (G. J. Pelka, C. Watson, T. Radziewic, M. Hayward, and P. P. L. Tam, unpublished data). E and F, Expression of Stk9/Cdkl5 in the brain of postnatal week-8 Mecp2^−/Y mutant male mouse (E, antisense riboprobes; F, sense riboprobes). In situ hybridization was performed on parasagittal slices of the mouse brain. Abbreviations for brain regions: bg = basal ganglia; cb = cerebellum; cc = cerebral cortex; cp = caudate-putamen; hf = hippocampal formation; ic = inferior colliculi; ob = olfactory bulb; sc = superior colliculi; th= thalamus.