CDKL5 truncation due to a t(X;2)(p22.1;p25.3) in a girl with X‐linked infantile spasm syndrome

To the Editor : X-linked infantile spasm syndrome (ISSX; OMIM 308350) occurs in 2–5 of 10,000 newborns and is characterized by early onset seizures, hypsarrhythmia, and severe mental retardation. Three girls with ISSX and an X;autosome translocation that disrupted the CDKL5 gene have been reported (1–3), whereas ∼50 different CDKL5 mutations have been found in >60 clinically similar patients with normal chromosomes (4–15). We report a girl with severe ISSX and a t(X;2) disrupting the CDKL5 gene in intron 3 and fusing CDKL5 and RPS7 genes. The proposita (Fig. 1a,b) was born to a 35year-old mother and a 45-year-old father. Since the age of 1 month, she presented myoclonic seizures with cyanosis that were partially controlled with several drugs. Her psychomotor development has been profoundly delayed. At the age of 2 years, she exhibited generalized hypotonia, lack of eye contact, poor pupilar reflex, weight of 7300 g [−5.5 standard deviation (SD)], length of 79 cm (−4.5 SD), occipital-frontal circumference (OFC) of 45 cm (−3 SD/−2.5 SD), narrow forehead, frontal hypertrichosis, synophrys, upslanted palpebral fissures, broad nasal bridge, small nose and diminished muscle bulk. Evoked visual and auditory potentials were consistent with central blindness and visual pathway immaturity, but showed normal hearing. Cranial computed tomography scan revealed fronto-temporal cortical atrophy and corpus callosum hypoplasia (Fig. 1c). Repeated electroencephalograms disclosed generalized paroxysmal activity and disorganization. The patient and her mother were karyotyped on GTG-banded chromosomes; the former had a 46,X,t(X;2)(p22.1;p25.3) karyotype assumed to be de novo (Fig. 1d) as the mother was 46,XX and the unlikelihood of the father (not sampled) being a carrier. A preferential inactivation of the normal X was found in 30 RBG-banded metaphases and confirmed by the human androgen receptor assay (HUMARA) (16) that also revealed a maternal origin for this chromosome (data not shown); i.e. the der(X) was the paternal homologue. Genescan analysis confirmed a ∼90% inactivation of the maternal allele (MA) (Fig. 1e). The X-chromosome inactivation in the mother was at random. Fluorescence in situ hybridization (FISH) with clones RP11-558P14 (AL109798) and RP1245G19 (Z92542) revealed that the X-chromosome breakpoint disrupted CDKL5 (Fig. 2a). Reverse transcriptase-polymerase chain reaction (RT-PCR) on patient RNA showed products with CDKL5 primer pairs spanning exons 9–12 (Fig. 2c) and exons 13–18 (data not shown). No product could be obtained using primers located in exons 2 and 8 (Fig. 2c). These results let us hypothesize that the chromosome 2 breakpoint mapped also within a gene and the rearrangement had produced fusion genes. To test this, we have fine mapped the breakpoints by array painting as described previously (17, 18). The results indicated that on der(X) the breakpoint lies within CDKL5 intron 3 (chrX:18,447,901-18,460,135) (UCSC, hg18) and on der(2) in a region that contained the 5′ segment, including exons 1 and 2, of the RPS7 gene (chr2:3,599,632-3,601,126). RT-PCR with CDKL5 exon 2 forward primer and RPS7 reverse primers for amplifying fusion transcripts from the der(2) resulted in products containing CDKL5 exons 2 and 3 spliced to chromosome 2 position 3,600,514 within a single exon EST (expressed sequence tags) (DA710611), followed by RPS7 exons 1–6 (shown in Fig. 2d). This result let us to conclude that the single exon EST, or at least a part of it, presents an extension of the RPS7 5′UTR. Therefore, the ORF of RPS7, which codes for a ribosomal protein of the small subunit, is most likely not affected by the translocation but the CDKL5–RPS7 fusion gene is very likely transcribed from the CDKL5 promoter. For der(X), a similar approach for amplifying