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Purpose: Genetic mutations of the cyclin-dependent kinase-like 5 gene (CDKL5) have been reported in patients with epileptic encephalopathy, which is characterized by intractable seizures and severe-to-profound developmental delay. We investigated the clinical relevance of CDKL5 alterations in both genders.
Methods: A total of 125 patients with epileptic encephalopathy were examined for genomic copy number aberrations, and 119 patients with no such aberrations were further examined for CDKL5 mutations. Five patients with Rett syndrome, who did not show methyl CpG-binding protein 2 gene (MECP2) mutations, were also examined for CDKL5 mutations.
Key Findings: One male and three female patients showed submicroscopic deletions including CDKL5, and two male and six female patients showed CDKL5 nucleotide alterations. Development of early onset seizure was a characteristic clinical feature for the patients with CDKL5 alterations in both genders despite polymorphous seizure types, including myoclonic seizures, tonic seizures, and spasms. Severe developmental delays and mild frontal lobe atrophies revealed by brain magnetic resonance imaging (MRI) were observed in almost all patients, and there was no gender difference in phenotypic features.
Significance: We observed that 5% of the male patients and 14% of the female patients with epileptic encephalopathy had CDKL5 alterations. These findings indicate that alterations in CDKL5 are associated with early epileptic encephalopathy in both female and male patients.
Epileptic encephalopathies are a group of conditions in which neurologic deterioration results mainly from epileptic activity. The clinical and electroencephalography (EEG) characteristics depend on the age of onset and may change over time (Zupanc, 2009). An underlying genetic background has been suggested in patients with epileptic encephalopathy (Nabbout & Dulac, 2008). An X-linked gene coding for cyclin-dependent kinase-like 5 gene (CDKL5; MIM #300203) is one of the genes responsible for epileptic encephalopathy. Kalscheuer et al. (2003) identified de novo balanced X autosome translocations in two female patients with infantile spasms, in whom CDKL5 was disrupted. Since then, the phenotypic spectrum of CDLK5 abnormalities has expanded to include features resembling Rett syndrome (RTT; MIM #312750) with early onset seizures (Evans et al., 2005; Mari et al., 2005). Now, phenotypic features of CDLK5 abnormalities are widely recognized as early infantile epileptic encephalopathy-2 (EIEE-2; MIM #30062) and are characterized as severe epileptic encephalopathy associated with early onset and refractory seizures (Archer et al., 2006; Pintaudi et al., 2008).
Although the consequence of CDKL5 alterations has also been attributed to X-linked dominant infantile spasm syndrome-2 (ISSX2), mutations have been identified not only in female patients but also in some male patients with severe mental retardation and early onset intractable seizures (Elia et al., 2008; Fichou et al., 2009; Sartori et al., 2009). Therefore, we performed a comprehensive analysis for CDKL5 in both female and male patients with epileptic encephalopathy.
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Using aCGH analyses, Erez et al. (2009) identified partial CDKL5 deletions in female patients with early onset intractable epilepsy. Mei et al. (2010) identified four patients who had total or partial deletions in CDKL5. However, those studies included only female patients. In comparison, the aim of our study was to identify candidate genetic causes of early epileptic encephalopathy, and thus we recruited patients of both genders. Genomic copy numbers of whole chromosomes were comprehensively analyzed and submicroscopic chromosomal abnormalities of the CDKL5 region were identified in both genders. The male patient (Patient 1) showed a partial deletion of CDKL5. Patients 2 and 3 showed large deletions in which the four neighboring genes, CDKL5, RS1, PPEF1, and PHKA2, were included. RS1 and PHKA2 are responsible for X-linked diseases, and the function of PPEF1 is unknown. The remaining Patient 4 showed partial deletions of CDKL5 and RS1. Therefore, phenotypic features of Patients 2, 3, and 4 suggest a causal role for CDKL5 deletions in early epileptic encephalopathy. Despite the gender difference and the deleted size differences, the clinical severities of the patients with CDKL5 deletions were similar between genders and similar to those of patients previously reported to have partial or total deletion of CDKL5 (Van Esch et al., 2007; Erez et al., 2009; Bahi-Buisson et al., 2010; Mei et al., 2010).
Previously, CDKL5 mutations were shown to affect mainly female patients, and their frequency has been estimated as approximately 9–28% in female patients with early onset seizures (Bahi-Buisson et al., 2008b; Nemos et al., 2009). However, those studies mainly included female patients. Elia et al. (2008) identified CDKL5 mutations in three male patients with early onset epileptic encephalopathy. Male patients with CDKL5 mutations or deletions have also been reported by others (Fichou et al., 2009; Sartori et al., 2009). In our study, initial identification of CDKL5 deletions in both male and female patients with early epileptic encephalopathy prompted us to analyze CDKL5 nucleotide sequences of both genders, and the results revealed nucleotide changes in two male patients and six female patients. We observed that the clinical severity of the disease did not differ between males and females. Therefore, male as well as female patients with early onset epileptic encephalopathy should be tested for CDKL5 mutations.
Because CDKL5 is located on Xp22.13, genetic traits of CDKL5 alterations have been considered to be X-linked dominant, just as MECP2 mutations are responsible for the majority of RTT cases, a neurologic disorder occurring almost exclusively in females. The rare male patients with MECP2 mutations showed severe mental retardation but no RTT phenotype (Gomot et al., 2003). In comparison, there are no phenotypic differences between male and female patients with CDKL5 mutations or deletions. Bahi-Buisson et al. (2008b) suggested that phenotypic heterogeneity does not correlate with the nature or the position of the mutations or with the pattern of X-chromosome inactivation. Indeed, no clear genotype–phenotype correlation between these factors has been established. Therefore, an important question is why clinical severity is the same between the genders. Based on previous reports, we know that the absence of CDKL5 protein is not lethal in males, and CDKL5 abnormalities result in severe neurodevelopmental delay and early onset epilepsy in both genders (Castren et al., 2011). In this study, the estimated frequencies of CDKL5 abnormalities in patients with epileptic encephalopathy were 5% in male and 14% in female patients. Therefore, the observed difference in the frequency of CDKL5 mutations between male and female patients may simply be a consequence of the fact that female patients have two X chromosomes.
Subjects in our study included five female patients with RTT who did not show MECP2 mutations. However, these female patients did not carry a CDKL5 mutation. Some researchers have found no CDKL5 mutations in patients with RTT (Huppke et al., 2005; Li et al., 2007). Previously, CDKL5 mutations were analyzed in patients with both classic and atypical variants of RTT. However, mutations were identified only in patients with seizure onset before 6 months of age (Evans et al., 2005; Scala et al., 2005; Artuso et al., 2010). In another study, all patients with CDKL5 mutations showed early onset seizures that began before 6 months of age (Erez et al., 2009). These findings suggest that development of early onset seizures is an essential clinical feature in patients with CDKL5 mutations. The onset of epileptic seizures in the first 6 months distinguishes patients with CDKL5 mutations from patients with typical RTT caused by MECP2 mutations (Castren et al., 2011).
All previously reported CDKL5 mutations were sporadic and were identified as de novo. Only a small numbers of mutations were recurrent (Castren et al., 2011). In this study, we observed eight CDKL5 mutations that included six novel and two recurrent mutations. The phenotypic features of the patients with recurrent mutations are similar to those described previously (Sartori et al., 2009; Artuso et al., 2010).
Consistent with the findings of previous studies, we observed polymorphous seizures (i.e., myoclonic seizures, tonic seizures, and spasms) in our study. The clinical course of seizure development was also identical to the proposed three stages reported by Bahi-Buisson et al. (2008a) [i.e., stage I, early onset epilepsy (onset 1–10 weeks); stage II, epileptic encephalopathy with infantile spasms and hypsarrhythmia; stage III, seizure-free in estimated 50% of patients at late infantile period] because our Patient 7 showed good seizure control after 3 years of age. Artuso et al. (2010) reported that patients with CDKL5 mutations showed no abnormalities on brain magnetic resonance imaging (MRI). However, our findings indicated mild frontal lobe atrophy in almost all patients. Therefore, this may be an additional clinical characteristic of patients with CDKL5 mutations.