CDKL5 variants

CDKL5 variants in the population.

The number of expected and observed variants in CDKL5 from the recent large-scale analysis of genetic variation by ExAC is described in table 1.⁹ Synonymous variants in CDKL5 are reported to occur at approximately the expected frequency and are found throughout the coding region of CDKL5 (figure 1). By contrast, the number of missense variants observed (n = 157) is lower than expected (table 1). The population sample analyzed in the ExAC study was devoid of severe pediatric disease; therefore, none of these 157 missense variants is, on its own, pathogenic and causative for CDKL5 deficiency. Only 15 (∼10%) of these missense variants are located in the catalytic domain of CDKL5, a 286-amino acid region (∼30% of protein length) crucial for protein function² (figures 1 and 2). No frameshift or splicing variants were identified in ExAC; however, 2 nonsense variants (described as stop-gain in table 1) were identified: p.Arg952Ter and p.Arg970Ter. Both nonsense variants are found in exon 21 and therefore affect only the hCDKL5_5 isoform, which is expressed almost exclusively in the testis.^12,18

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.

Some known exonic coding and untranslated regions (UTRs) of CDKL5, recently identified and present only in minor transcript isoforms,¹² are not included in the exonic regions covered by ExAC. For example, exon 17 (originally termed 16b¹⁹) is present in the hCDKL5_2 isoform and is expressed only at low levels in the developing and adult brain.¹² Only 2 exon 17 variants have been identified, both in dbSNP (rs289269000 and rs181987256), neither of which is associated with a disease phenotype.

CDKL5 missense variants in patients.

Missense variants reported in patients are found throughout the portions of CDKL5 that encode the major brain transcript isoform, hCDKL5_1 (figure 1). Only 1 of the 15 missense variants identified within the catalytic domain in ExAC has been reported in a patient with CDKL5 deficiency: c.719G>C (p.Ser240Thr; figure 2). This exon 9 variant has recently been reported as the first familial case of CDKL5-related disease²⁰ in a heterozygous female, who displayed global psychomotor delay and autistic disturbances but no epilepsy. The same variant was detected in her asymptomatic mother, and a skewed X-chromosome inactivation (XCI) ratio was thought to account for this phenotypic difference. We further identified 2 individuals with this variant (1 in ExAC and 1 in 1000G), neither individual having displayed signs of a neurodevelopmental disorder phenotype, consistent with the inclusion criteria in those studies. It is unlikely that 3 asymptomatic carriers of this variant would all have similarly skewed XCI, suggesting that this variant may not in fact be causative. Current evidence suggests that the p.Ser240Thr should be classified as being of “uncertain significance.”

Combining all known CDKL5 variant data, there are 59 missense variants in the catalytic domain, which we consider pathogenic or likely pathogenic for CDKL5 deficiency based on our assignment criteria (figure 2). This includes 11 novel missense variants identified in the clinical cohorts in this study (table 2). In each case, the patient received a clinical diagnosis of CDKL5 deficiency, and, in most cases, VEP analysis supported the prediction of a pathogenic variant.

Outside the catalytic domain, there are 179 different missense variants (figure 1). Upstream of the catalytic domain, 3 variants (p.Ile3Phe, p.Asn5Asp, and p.Ile6Thr) are known to be present in ExAC and 1000G, not associated with CDKL5 patients and predicted to be benign by VEP analysis. Fifty-one different missense variants have been found in the last 3 exons (20, 21, and 22). Forty of these are present in ExAC, often in hemizygous males. c.2995G>A (p.Val999Met), previously thought to be pathogenic, is found at notable frequency in ExAC (0.01178) and should therefore be classed as a benign polymorphism. A further 10 are present in dbSNP; none of these variants appear to be responsible for a CDKL5-like phenotype, and VEP analysis predicts these variants to be benign. This is consistent with these exons being absent in the primary brain transcript isoform. In our clinical cohorts, a variant in exon 21, c.2980G>A (p.Gly994Arg), was identified in a patient with developmental delay, but was not clinically diagnosed with CDKL5 deficiency (table 2). The variant was inherited from her asymptomatic mother and, combined with VEP analysis, should therefore be classified as benign.

The remaining missense variants are distributed between exons 11 and 19 (figure 1). Ninety-seven of these are found in ExAC, many occurring in hemizygotes. In some cases, such as p.Ile508Thr and p.Thr734Ala, the ExAC allele frequency and the presence of these variants in hemizygotes allow them to be reclassified as likely benign. Additional variants present in dbSNP (but not in ExAC) are not associated with any clinical phenotype. In our clinical cohorts, we identified further missense variants in patients, where a clinical diagnosis of CDKL5 deficiency was not given (table 2). p.His317Arg, p.Asp396Glu, p.Asn748Ser, and p.Asp797Asn are present in ExAC/1000G, and all variants were identified as being inherited from a healthy mother or father (table 2), suggesting that they are all benign variants. In our analysis, there are 7 missense variants outside the catalytic domain that should, at present, be classified as “of uncertain significance” pending more information (figure 2). p.Val351Ile is recorded in dbSNP (rs587783150) and p.Thr538Ala is recorded in the RettBASE,¹³ for which no clinical or screening information is available. p.Leu302Phe and p.Asp422Glu are unique variants that have been associated with seizure phenotypes, although the phenotype of these cases is atypical in comparison to other CDKL5 patients reported in these studies.^21,22 p.Asn399Thr and p.Val793Ala have also been associated with epilepsy phenotypes^4,23; these variants have also been observed as singletons in ExAC.

The most compelling evidence of a pathogenic missense variant outside the catalytic domain concerns the c.2152G>A (p.Val718Met) variant. This variant has been identified in 3 unrelated CDKL5 patients in 3 independent studies.^24,–,26 In each case, the patient presented with symptoms consistent with CDKL5 deficiency and harbored no known variants in other epilepsy-related genes. This variant is not present in the 1000G or the ExAC population databases. VEP analysis of c.2152G>A, which affects the last base of exon 14, predicted a detrimental effect on splicing (MaxEntScan diff: 2.89). We assessed this variant in vitro using a minigene splicing assay, showing that splicing is disrupted, resulting in exon 14 being omitted from the transcript (figure 3). We therefore suggest that the c.2152G>A variant be categorized as a splicing rather than a missense variant. The consequence (r.2047_2152del) is a frameshift variant that would generate a premature stop codon in exon 16. This finding removes evidence supporting the existence of pathogenic missense variants outside the catalytic domain of CDKL5, and we therefore conclude that caution should be applied in interpretations of pathogenicity whenever an apparent missense variant is found outside the catalytic domain.

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.

Nonsense, frameshift, and splicing variants.

Pathogenic nonsense, frameshift, and splicing variants are found throughout the coding region of hCDKL5_1, the predominant brain transcript isoform (figure 1). Efforts have been made to establish genotype-phenotype relationships based on the position of these variants in the gene, by predicting structural and functional consequences.⁸ However, the levels of functional CDKL5 in patients with truncating variants at different points throughout the gene are still not known. Novel nonsense and splicing variants identified in the clinical cohorts in this study are described in table 2, and novel frameshift variants in table e-1, http://links.lww.com/NXG/A2.

In exon 19, the last exon utilized by the hCDKL5_1 transcript, a variant described in dbSNP (rs863225289) has been identified in a patient with early infantile epileptic encephalopathy 2 (MIM: 300672). This variant lies downstream of the internal splice donor site in exon 19 and so would not be expected to affect the protein product made from the testis-specific hCDKL5_5 transcript isoform. However, in relation to hCDKL5_1, the major brain transcript isoform, this is a nonsense variant (c.2176C>T, p.Gln906Ter, with respect to hCDKL5_1). This may trigger nonsense-mediated decay (NMD; due to the presence of a long 3′-UTR²⁷) and result in typical CDKL5 deficiency. Additional observations of this variant would be required to validate this conjecture, but it is consistent with the idea that truncating variants in exon 19 may be pathogenic.

Three nonsense variants have been detected in exon 21, which is specific to isoform hCDKL5_5: c.2854C>T, p.Arg952Ter; c.2908C>T, p.Arg970Ter; and c.2941C>T, p.Arg981Ter. Both p.Arg952Ter and p.Arg970Ter variants have been described in patients with CDKL5-like symptoms.^28,29 However, in both cases, delay in seizure onset and an absence of other features of CDKL5-associated pathology were reported. p.Arg952Ter was identified in the patient's mother, grandmother, and half-sister, and in 6 individuals in a control population.²⁸ A further 12 instances of this allele are present in ExAC, including 3 hemizygotes (allele frequency 0.0001367). A recent case study also identified this variant in an asymptomatic hemizygous male.³⁰ p.Arg970Ter occurs in a single heterozygous female in ExAC (in the case study, the variant was not detected in the mother and the father was deceased²⁹). Together, these findings suggest that variants in these late exons do not cause a CDKL5 deficiency phenotype.

A c.2941C>T, p.Arg981Ter variant was identified in our clinical cohorts (table 2). A girl presented with a phenotype consistent with CDKL5 deficiency—profound cognitive impairment, mild dysmorphic appearance, stereotypical hand wringing, and epilepsy (from age 6 months). Both parents were deceased; therefore, it is unknown whether this variant was inherited or occurred de novo. This variant is not present in ExAC or in 1000G, and the clinical evidence would suggest defining this as a pathogenic variant. However, this variant lies immediately downstream of the 2 nonsense variants described above, both of which have considerable evidence suggesting that they should be classified as benign. We conclude that, at present, p.Arg981Ter should be classified as being of “uncertain significance.”

It is important that no pathogenic frameshift or splicing variants were found affecting exons 20, 21, and 22 (figure 1). A novel splicing variant was identified in our clinical cohorts (table 2): c.2797+2T>C. The patient, a boy, was not clinically diagnosed with CDKL5 deficiency and was found to have inherited the variant from his asymptomatic mother. VEP analysis of this variant predicts the abolition of the splice donor site of exon 20, which would be likely to result in a premature stop codon.

Copy number variations and CDKL5 duplication.

Several studies have reported duplications of Xp22 associated with intellectual disability and autism phenotypes,^31,–,34 but the duplicated regions reported (spanning 8–21 Mb) included as many as 80 genes, and interpretation of gene-specific overexpression effects in such circumstances is problematic. A more recent study described 3 unrelated families with more compact duplication regions incorporating CDKL5.³⁵ Four different duplicated regions were described, ranging from 540 to 935 kb in size. In the case of the 683-kb duplication harbored by three of the patients, the duplicated region does not include exon 1 of CDKL5, so the predominant adult brain isoform, hCDKL5_1, would not be expected to be overexpressed in these individuals. The authors point out that the alternative hCDKL5_5 transcript may be expressed and overexpression effects during fetal development may result in the observed phenotype. Three patients also harbored additional duplicated regions on other chromosomes, and parents harboring these same CDKL5-containing duplications were either phenotypically unaffected or displayed mild intellectual disability.³⁵ Therefore, the pathway leading to the phenotypes in these individuals remains somewhat unclear, as experimental confirmation of effects on CDKL5 transcript and protein levels is not yet available. Here, we report 2 duplications and 1 triplication in our clinical cohorts (table 2). However, in each of these copy number variations, only a part of the gene is duplicated; therefore, it is extremely unlikely that an increased level of CDKL5 would be present in these individuals.

Variants in regulatory and UTRs.

The recent identification of novel exons and transcription start sites at the 5′ end of the gene suggests the presence of multiple promoter regions.¹² However, most brain transcripts are driven by a putative promoter upstream of exon 1. Several variants in the 5′-UTR have been reported, but so far only those associated with the deletion of exon 1 or 2, or the disruption of splicing from exon 1 to exon 2 (c.-162-2A>G³⁶), have been shown to be pathogenic. Only 2 variants in the putative promoter region, c.-440G>T and c.-189C>T, have so far been found in patients with a CDKL5-like phenotype,³ but at present their significance remains uncertain. The large 6.6 kb 3′-UTR of hCDKL5_1 has only recently been defined,¹² so the region has not been analyzed in patients either by targeted sequencing or by exome analysis, and we found no evidence for the presence of clinically relevant variants in the 1000G data.

Incidence of CDKL5 deficiency.

The incidence of CDKL5 deficiency is unknown. We have analyzed data from the International CDKL5 Disorder Database⁸ to provide a lower estimate of birth prevalence in Australia of 0.21 cases per 100,000 live births (95% CI 0.12–0.33) for the years 1982–2014. Although a birth prevalence in this range would indicate that CDKL5 deficiency is an ultra rare disorder, it is likely that this figure will increase, as targeted next-generation sequencing for investigation of early-onset epileptic encephalopathy becomes more common.^7,25

In our analysis, the most frequent CDKL5 deficiency–causing variants are 2 nonsense variants in exon 12: c.1648C>T (p.Arg550Ter) and c.1675C>T (p.Arg559Ter). Each of these variants affects only 3% of all patients, highlighting the high degree of allelic heterogeneity in CDKL5 deficiency.