A Case of CDKL5 Deficiency Due to an X Chromosome Pericentric Inversion: Delineation of Structural Rearrangements as an Overlooked Recurrent Pathological Mechanism

Abstract

CDKL5 deficiency disorder (CDD) is an X-linked dominant epileptic encephalopathy, characterized by early-onset and drug-resistant seizures, psychomotor delay, and slight facial features. Genomic variants inactivating CDKL5 or impairing its protein product kinase activity have been reported, making next-generation sequencing (NGS) and chromosomal microarray analysis (CMA) the standard diagnostic tests. We report a suspicious case of CDD in a female child who tested negative upon NGS and CMA and harbored an X chromosome de novo pericentric inversion. The use of recently developed genomic techniques (optical genome mapping and whole-genome sequencing) allowed us to finely characterize the breakpoints, with one of them interrupting CDKL5 at intron 1. This is the fifth case of CDD reported in the scientific literature harboring a structural rearrangement on the X chromosome, providing evidence for the hypothesis that this type of anomaly can represent a recurrent pathogenic mechanism, whose frequency is likely underestimated, with it being overlooked by standard techniques. The identification of the molecular etiology of the disorder is extremely important in evaluating the pathological outcome and to better investigate the mechanisms associated with drug resistance, paving the way for the development of specific therapies. Karyotype and genomic techniques should be considered in all cases presenting with CDD without molecular confirmation.

Keywords: CDKL5; CDKL5 deficiency disorder (CDD); Xp22.13; epileptic encephalopathy; optical genome mapping; structural rearrangements; whole-genome sequencing.

Figure 1

A brief bilateral tonic seizure, with a diffuse EEG counterpart, is followed by a cluster of periodic myoclonias synchronized with periodic spike-wave complexes on the EEG.

Figure 2

The seizure begins with a single myoclonus associated with a diffuse spike, followed by epileptic spasms associated with periodic high-amplitude slow waves and then again by myoclonias with a spike-wave EEG pattern. Myoclonias and spasms are interspersed.

Figure 3

(A) G-banding karyotype: female euploid karyotype, harboring a pericentric inversion on the X chromosome [46,X,inv(X)(p22.12q11)]. (B) Ideograms of the two X chromosomes showing the inversion breakpoints. (C) FISH assay using Xp11.1q11.1 Alpha Satellite (green) and NYX (orange, Xp11.4) probes characterizing the proximal breakpoint of the inversion. The two green fluorescent signals on the rearranged X chromosome (right) prove that one of the breakpoints involved the pericentromeric region. The NYX orange signal moves distally, between the two green signals. (D) FISH assay using a custom-designed oligonucleotide probe characterizing the distal breakpoint of the inversion, involving CDKL5. The Xp22.13 (green)/CDKL5 (orange) probe signals, normally joining at the extremity of the X chromosome short arm, split on the two arms of the rearranged chromosome.

Figure 4

Genomic analysis of peripheral blood from the patient was performed, characterizing the inversion breakpoints at Xp22.13. (A) The patient’s optical map (blue bar) was aligned and compared with the reference optical map (green bar). Molecular labels are reported as vertical lines on both the patient’s and reference’s maps. The patient’s map only matches the reference’s map in the first gene portion, with the distal breakpoint of the inversion being in the pericentromeric region of Xq, where the high chromatin condensation does not allow for enzymatic labeling. (B) WGS data focusing on CDKL5 (NM_001323289) show a misalignment at the intron 1 level (the alternative sequence is reported as colored segments, representing each nucleotide: green for A, blue for C, red for T, orange for G).

Figure 5

Custom Xp22.13/CDKL5 FISH probe design. A red-labeled probe was designed on the CDKL5 gene sequence (orange segment) while a green-labeled one was located about 589 kb upstream (green segment).