BACKGROUND: Variants in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been reported as being etiologically associated with early infantile epileptic encephalopathy type 2 (EIEE2). We report on two patients, a boy and a girl, with EIEE2 that present with early onset epilepsy, hypotonia, severe intellectual disability, and poor eye contact. METHODS: Massively parallel sequencing (MPS) of a custom-designed gene panel for epilepsy and epileptic encephalopathy containing 112 epilepsy-related genes was performed. Sanger sequencing was used to confirm the novel variants. For confirmation of the functional consequence of an intronic CDKL5 variant in patient 2, an RNA study was done. RESULTS: DNA sequencing revealed de novo variants in CDKL5, a c.2578C>T (p. Gln860*) present in a hemizygous state in a 3-year-old boy, and a potential splice site variant c.463+5G>A in heterozygous state in a 5-year-old girl. Multiple in silico splicing algorithms predicted a highly reduced splice site score for c.463+5G>A. A subsequent mRNA study confirmed an aberrant shorter transcript lacking exon 7. CONCLUSIONS: Our data confirmed that variants in the CDKL5 are associated with EIEE2. There is credible evidence that the novel identified variants are pathogenic and, therefore, are likely the cause of the disease in the presented patients. In one of the patients a stop codon variant is predicted to produce a truncated protein, and in the other patient an intronic variant results in aberrant splicing.

CLIP Childhood Leukaemia Investigation Prague Department of Paediatric Haematology and Oncology Charles University Prague and University Hospital Motol Prague Czech Republic

Department of Biology and Medical Genetics 2nd Faculty of Medicine Charles University Prague and University Hospital Motol Prague Czech Republic

Department of Paediatric Neurology 2nd Faculty of Medicine Charles University Prague and University Hospital Motol Prague Czech Republic

DNA Laboratory Department of Paediatric Neurology 2nd Faculty of Medicine Charles University Prague and University Hospital Motol Prague Czech Republic

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$a Two Novel Variants Affecting CDKL5 Transcript Associated with Epileptic Encephalopathy / $c J. Neupauerová, K. Štěrbová, M. Vlčková, V. Sebroňová, T. Maříková, M. Krůtová, S. David, P. Kršek, M. Žaliová, P. Seeman, P. Laššuthová,

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$a BACKGROUND: Variants in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been reported as being etiologically associated with early infantile epileptic encephalopathy type 2 (EIEE2). We report on two patients, a boy and a girl, with EIEE2 that present with early onset epilepsy, hypotonia, severe intellectual disability, and poor eye contact. METHODS: Massively parallel sequencing (MPS) of a custom-designed gene panel for epilepsy and epileptic encephalopathy containing 112 epilepsy-related genes was performed. Sanger sequencing was used to confirm the novel variants. For confirmation of the functional consequence of an intronic CDKL5 variant in patient 2, an RNA study was done. RESULTS: DNA sequencing revealed de novo variants in CDKL5, a c.2578C>T (p. Gln860*) present in a hemizygous state in a 3-year-old boy, and a potential splice site variant c.463+5G>A in heterozygous state in a 5-year-old girl. Multiple in silico splicing algorithms predicted a highly reduced splice site score for c.463+5G>A. A subsequent mRNA study confirmed an aberrant shorter transcript lacking exon 7. CONCLUSIONS: Our data confirmed that variants in the CDKL5 are associated with EIEE2. There is credible evidence that the novel identified variants are pathogenic and, therefore, are likely the cause of the disease in the presented patients. In one of the patients a stop codon variant is predicted to produce a truncated protein, and in the other patient an intronic variant results in aberrant splicing.

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$a Sebroňová, Věra $u 2 Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol , Prague, Czech Republic .

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$a Maříková, Tat'ána $u 3 Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol , Prague, Czech Republic .

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$a David, Staněk $u 1 DNA Laboratory, Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol , Prague, Czech Republic .

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$a Kršek, Pavel $u 2 Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol , Prague, Czech Republic .

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$a Seeman, Pavel $u 1 DNA Laboratory, Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol , Prague, Czech Republic .

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$a Laššuthová, Petra $u 1 DNA Laboratory, Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol , Prague, Czech Republic .

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