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A novel transcript of cyclin-dependent kinase-like 5 (CDKL5) has an alternative C-terminus and is the predominant transcript in brain


The X-linked cyclin-dependent kinase-like 5 (CDKL5) gene is an important molecular determinant of early-onset intractable seizures with infantile spasms and Rett syndrome-like phenotype. The gene encodes a kinase that may influence components of molecular pathways associated with MeCP2. In humans there are two previously reported splice variants that differ in the 5′ untranslated exons and produce the same 115 kDa protein. Furthermore, very recently, a novel transcript including a novel exon (16b) has been described. By aligning both the human and mouse CDKL5 proteins to the orthologs of other species, we identified a theoretical 107 kDa isoform with an alternative C-terminus that terminates in intron 18. In human brain and all other tissues investigated except the testis, this novel isoform is the major CDKL5 transcript. The detailed characterisation of this novel isoform of CDKL5 reveals functional and subcellular localisation attributes that overlap greatly, but not completely, with that of the previously studied human CDKL5 protein. Considering its predominant expression in the human and mouse brain, we believe that this novel isoform is likely to be of primary pathogenic importance in human diseases associated with CDKL5 deficiency, and suggest that screening of the related intronic sequence should be included in the molecular genetic analyses of patients with a suggestive clinical phenotype.

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We would like to express our gratitude to the Rett Syndrome Australian Research Fund, the Italian association proRETT ricerca, the Rotary Club of Narellan, the Harrington Park Foundation, and the Country Women’s Association of NSW for additional financial support. This research was funded by National Health and Medical Research Council of Australia project grants 346603 (PT, JC) and 570752 (PT, JC), the E-Rare EuroRETT Network (NL), Fondazione Cariplo 2010-0724 (NL), the Italian Ministry of Research (CKN), and FP7-PEOPLE-ITN-2008 (CKN).

Conflict of interest

The authors have no conflict of interest to declare.

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Correspondence to John Christodoulou.

Electronic supplementary material

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Supplementary Figure 1: CDKL5/cdkl5 genomic structure and splice variants.Graphical representation of the genomic structures and reported CDKL5/cdkl5 splice variants of A) human and B) mouse. Exons found in both species are shaded blue, those exons conserved in primates are shaded grey, while the mouse specific exon 19 is black. Arrows indicate the approximate positions of the PCR primers used, black arrows are those used for end point RT-PCR and the grey arrows are those used for qPCR. Exons are not drawn to scale. (JPG 807 KB)

Supplementary Figure 2: Alignment of the orthologs of CDKL5 ClustalW alignment of the available CDKL5 orthologs: human (Homo sapiens; GenBank accession CAI42485), rhesus monkey (Macaca mulatta; XR_009820), Sumatran orangutan (Pongo abelii; XP_002831484.1), chimpanzee (Pan troglodytes; XR_023224), crab-eating macaque (Macaca fascicularis; AB168731.1), mouse (Mus musculus; NP_001019795), rat (Rattus norvegicus; XP_002727631.1), rabbit (Oryctolagus cuniculus; XP_002719977.1), dog (Canis familiaris; XP_548881), cow (Bos Taurus; XP_002700425.1), horse (Equus caballus; XP_001491126), white-tufted-ear marmoset (Callithrix jacchus; XP_002762761.1), giant panda (Ailuropoda melanoleuca; EFB28542.1), gray short-tailed opossum (Monodelphis domestica; XP_001380717.1), platypus (Ornithorhynchus anatinus; XP_001514328), chicken (Gallus gallus; XP_425571), zebra finch (Taeniopygia guttata; XP_002196986), zebrafish (Danio rerio; BAH24206.1 (A) and NP_001139240.1 (B)), spotted green pufferfish (Tetraodon nigroviridis; CAF95446.1 (A) and CAF99817.1 (B)), tiger pufferfish (Takifugu rubripes AAD28798) and western clawed frog (Xenopus (Silurana) tropicalis; AAI59156.1) (TIFF 10,292 kb)

Supplementary Figure 3: Tissue specific expression of CDKL5/cdkl5.Quantitative PCR of A) total cdkl5/CDKL5, B) cdkl5107/CDKL5107 variant, C) cdkl5105/CDKL5115 variants. The values showing FOLD difference with respect to the brain are calculated using the (ΔΔCT) method. The total RNA input was first normalised to GAPDH/gapdh (ΔCT) and the obtained values calibrated to the whole brain (ΔΔCT). FOLD difference was calculated using the algorithm 2-(ΔΔCT). The error bars show the standard deviation, the calculated p values are based on the comparison to the whole brain (ns = not significant, *p<0.05, ** p<0.01, ***p<0.001). (JPG 944 kb)

Supplementary Figure 4: Amplification of the open reading frames of CDKL5 transcripts. Long range PCR of cDNA made from 2μg of RNA. For each of the samples, the whole 20μl PCR reaction was loaded onto the gels. Samples are 1) NT2 cDNA, 2) SH-SY5Y cDNA, 3) HEK-293 cDNA, 4) HeLa cDNA, 5) NT2 negative RT, 6) SH-SY5Y negative RT, 7) HEK-293 negative RT, 8) HeLa negative RT. The six primer combinations used are exon 1-intron 18, exon 1a-intron 18, exon 1-exon 21, exon 1a-exon 21, exon 2-intron 18 and exon 2-exon 21. The molecular weight marker is the 1KbPLUS (Invitrogen). (TIFF 5,500 kb)

Supplementary Figure 5: Western blot of endogenous CDKL5107. Endogenous CDKL5 was analysed in total cell extracts of murine brain (adult) and N2a cells, human NT2, SH-SY5Y and HEK293 cells with a polyclonal anti-CDKL5 antibody. The migration of exogenously expressed FLAG-tagged CDKL5115 and CDKL5107 is shown to the left. Tubulin was used as loading control. The arrow indicates the band corresponding to CDKL5105/107 isoforms that are sensitive to siRNA of CDKL5. Molecular masses are indicated to the left.(JPG 192 KB) (JPG 148 kb)

Supplementary Figure 6: siRNA knockdown of CDKL5 in NT2 cells.Western blot analysis of murine N2a and human NT2 cells transfected for 72 hours with or without (NT) siRNAs against CDKL5 or as control a non-targeting scrambled sequence (Scr.). CDKL5 levels were analysed in total cell extracts with a polyclonal anti-CDKL5 antibody using tubulin as internal standard. A murine brain extract and FLAG-tagged CDKL5107 and CDKL5115 were used as positive controls. Molecular masses are indicated to the left. (JPG 192 kb)

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Williamson, S.L., Giudici, L., Kilstrup-Nielsen, C. et al. A novel transcript of cyclin-dependent kinase-like 5 (CDKL5) has an alternative C-terminus and is the predominant transcript in brain. Hum Genet 131, 187–200 (2012). https://doi.org/10.1007/s00439-011-1058-x

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  • Splice Variant
  • Pathogenic Mutation
  • Zebra Finch
  • Epileptic Encephalopathy
  • Full Open Reading Frame