CDKL5 variants: Improving our understanding of a rare neurologic disorder

Abstract

Objective: To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene.

Methods: We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity.

Results: The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency.

Conclusions: These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain.

Figure 1. Distribution of exonic CDKL5 variants

A cartoon of the gene structure is given at the top, with exons of hCDKL5_1, the dominant brain transcript isoform, colored blue-green (coding regions) and black (UTRs). Introns are not drawn to scale. In the diagram beneath, variant types are grouped together, and individual variants are plotted according to their location in the gene. Red indicates pathogenic or likely pathogenic variants; green indicates benign or likely benign variants; and amber indicates variants of uncertain significance.

Figure 2. Pathogenic CDKL5 missense variants cluster in the catalytic domain

Functional domains in the CDKL5 protein are color coded. Numbers refer to the positions of amino acids. Variants in red (upper) are pathogenic or likely pathogenic. Variants in black (lower) are benign or likely benign. ^aVariant with a splicing consequence. ^bVariant of uncertain significance. NES = putative nuclear export signal; NLS = putative nuclear localization signal; ST = serine-threonine kinase active site; TEY = conserved Thr-Glu-Tyr motif.

Figure 3. CDKL5 variant c.2152G>A causes skipping of exon 14 in HEK293T cells

(A) Schematic representation of the minigene constructs used in the in vitro splicing assay (not to scale). The pET01 vector contains 5′ and 3′ exons separated by an intron sequence. Minigenes contain wild-type (wt) or mutant (mut; c.2152G>A) CDKL5 exon 14 sequences flanked by portions of their natural introns (thick lines). Primers used for RT-PCR experiments are indicated by arrows. Splicing events, indicated by dashed lines, would result in a 348-bp or a 242-bp product depending on inclusion or otherwise of CDKL5 exon 14. (B) Agarose gel showing RT-PCR results from the splicing assay using pET01 vector and minigenes of wild-type or mutated CDKL5 exon 14. Upper bands (348 bp) indicate the presence of exon 14, whereas lower bands (242 bp) indicate exclusion of exon 14. Marker indicates 100 bp ladder; −RT indicates a negative control without reverse transcriptase.