Clinical Phenotype of 5 Females With a CDKL5 Mutation

Abstract

Mutations in the X-linked cyclin dependent kinase like 5 (CDKL5) gene have been reported in approximately 80 patients since the first description in 2003. The clinical presentation partly corresponds with Rett syndrome, considering clinical features as intellectual disability, hypotonia, and poor visual, language, and motor development. However, these patients do not meet the consensus criteria for Rett syndrome since they lack the clear period of regression. Furthermore, in contrast to Rett syndrome, patients with CDKL5 mutations, have seizures or infantile spasms starting in the first weeks of life. We present clinical phenotype of 5 girls having a mutation in the CDKL5 gene. All mutations are novel and are pathogenic since they either lead to a frameshift in the reading frame or affect a consensus splice site. Four of the mutations are detected de novo in the affected girl.

Keywords

early Rett syndrome early-onset seizures infantile spasms

Since 2003, mutations of the cyclin dependent kinase like 5 (CDKL5) gene, located on chromosome Xp22, are known to cause an epileptic encephalopathy.^1,2 Characteristic clinical features include a severe intellectual disability, infantile spasms or early-onset seizures, and a clinical picture partly resembling Rett syndrome.^3–5 Typical Rett syndrome is characterized by an apparently normal development from birth until ~6 months of age, followed by postnatal deceleration of head growth, regression with loss of acquired skills and speech in combination with gait abnormalities and development of stereotypic hand movements.⁶ A significant proportion (approximately 95%) of typical Rett patients have a mutation in the MECP2 gene.⁷

However, a wide degree of females with Rett is MECP2 mutation negative and is considered clinically having a variant or atypical form of Rett syndrome. These cases show absence of a period with normal development, early onset of seizures, or a relatively mild phenotype with some preserved skills and speech.⁸ In this group of patients, mutations of the CDKL5 gene can be found. In addition to CDKL5, there are other genes associated with atypical Rett, ie, FOXG1, MEF2C, and NTNG1. ⁸ There are only a few male CDKL5 mutation cases described.² Since 2003, approximately 80 patients with CDKL5 mutations have been reported.⁸

We present the clinical phenotype of 5 girls having a CDKL5 mutation and compare this phenotype with previous reports and with Rett syndrome. In our 5 patients, 5 novel mutations in the CDKL5 gene were found.

Patients and Methods

Patients

Five girls with mutations in the CDKL5 gene were identified in the Pediatric Neurology Department and the Department of Human Genetics.

Mutation Analysis

Mutation analysis was performed in a DNA-diagnostic setting and involved sequencing of the entire coding region and splice sites of the CDKL5 gene (NM_003159.2) and MLPA analysis using kit P189B1 (MRC-Holland; Amsterdam, the Netherlands). Primer sequences and polymerase chain reaction conditions are available upon request. GenBank accession number NM_003159.2 was used as the DNA reference sequence. The nomenclature used follows the HGVS mutation nomenclature guidelines (www.hgvs.org). Pathogenicity prediction of previously unreported variants was performed using Alamut version 2.0 (Interactive Biosoftware, Rouen, France).

Results

Patient 1

A 7-week-old girl was referred with myoclonic seizures that occurred dozens of times a day. After a normal course of pregnancy, there was meconium containing amniotic fluid at birth. Apgar scores were 6 and 8 after 5 and 10 minutes, respectively. Family history of the nonconsanguineous parents was unremarkable. Neurological examination revealed poor vision and generalized hypotonia. Laboratory examination, including extensive metabolic analysis and cerebral magnetic resonance imaging (MRI), showed no abnormalities. Interictal electroencephalography (EEG) during wakefulness showed poorly organized background activity, predominantly delta and theta activity, without epileptiform activity. Because of persistent clinical seizure activity, phenobarbital (5 mg/kg daily) was started. The myoclonic seizures persisted, and after several weeks, flexion spasms appeared. Neurological examination at the age of 5 months was unchanged, with poor vision and severe generalized hypotonia. The EEG now showed hypsarrythmia. Pyridoxine (50 mg daily) was given for 2 weeks without any change in seizure activity. Phenobarbital was withdrawn and valproic acid was started (up to a daily dose of 36 mg/kg). Chromosomal microarray analysis was normal, as was mutation analysis of the MECP2 gene. Mutation analysis of the CDKL5 gene revealed an insertion of 2 nucleotides (c.275_276insAA) resulting in a frameshift (p.Glu93fs). This mutation was not present in either parent.

After several switches and combinations of antiepileptic drugs, including levetiracetam, vigabatrin, adrenocorticotropic hormone (ACTH), and nitrazepam, it was concluded that the epilepsy had become intractable.

At the age of 28 months, ketogenic diet was started, which resulted in a moderate decrease in seizure frequency. At the age of 36 months, the patient has poor vision, no language development, and a severe generalized hypotonia with tetraparesis.

Patient 2

This woman was referred at the age of 20 years. After an uneventful pregnancy and birth, she suffered from myoclonic and atonic seizures from the age of 3 weeks. Neurological examination at age 10 weeks revealed poor vision and generalized hypotonia. Cerebral computed tomography (CT) was normal. EEG showed generalized epileptiform activity, both in sleep and wakefulness. Metabolic investigations were normal, and valproic acid (20 mg/kg daily) was started. At the age of 1 year, she was able to sit with support, and had marked feeding difficulties and a poor vision. Seizure types were myoclonic, generalized tonic-clonic, and extension spasms. Because of a poor response to phenytoin, carbamazepine, phenobarbital, and levetiracetam, it was concluded the epilepsy had become intractable.

At referral, she had dozens of seizures a day (myoclonic, tonic-clonic), despite levetiracetam and phenobarbital therapy. Neurological examination revealed severe intellectual disability, no language development, poor vision, and a severe spastic tetraparesis. She also had developed a mild thoracolumbar scoliosis. EEG showed generalized epileptiform discharges. Cerebral MRI at the age of 21 years was normal. Chromosomal microarray analysis was normal, as was mutation analysis of the MECP2 gene. Mutation analysis of the CDKL5 gene revealed a de novo mutation in intron 3: c.99+5G>A, which is predicted to affect correct splicing of the CDKL5 mRNA. However, since RNA studies have not been performed, the effect on the protein level cannot be predicted.

Overview of All Patients

An overview of clinical features and genetic findings of all 5 female patients having a CDKL5 mutation is shown in Table 1 . Age at diagnosis varied from 10 months to 21 years.

Table 1.

Clinical and Genetic Findings of 5 Females With a CDKL 5 Mutation

Patient	1	2	3	4	5
Age at diagnosis (yrs)	10 months	21	7	3	2
Feeding difficulties	yes, PEG	yes	yes, PEG	yes, PEG	yes
Visual development	poor	poor	poor	poor	poor
Language development	poor	poor	poor	poor	poor
Hypotonia and motor signs	yes, severe generalized with tetraparesis	yes, severe generalized with spastic tetraparesis	yes, with tetraparesis	yes, severe with tetraparesis	yes, severe with tetraparesis
Seizure type	myoclonic, infantile spasms	myoclonic, infantile spasms, tonic-clonic	tonic, tonic-clonic	myoclonic	infantile spasms, tonic-clonic
Seizure onset (weeks)	7	3	6	8	4
Intractable epilepsy	yes	yes	yes	yes	yes
Ketogenic diet	moderate decrease in seizure frequency				moderate decrease in seizure frequency
Cerebral MRI findings	normal	normal	normal	normal	normal
Mutation	c.275_276insAA (de novo)	c.99+5G>A (de novo)	c.2105_2106del (de novo)	c.283-3_290del (de novo)	c.1784dup (mother negative, father not tested)
Protein	p.Glu93fs	p.?	p.His702fs	p.?	p.Leu596fs

Abbreviations: PEG, percutaneous endoscopic gastrostomy; p.?, effect on the protein level cannot be predicted; MRI, magnetic resonance imaging.

All patients had seizure onset in the first weeks of life. Seizure types were variable, with patients having a combination of infantile spasms, tonic seizures, tonic-clonic seizures, or myoclonic seizures. In all, seizures became intractable, with 2 patients having moderate decrease of seizure frequency on ketogenic diet. There was only a moderate and brief response to antiepileptic drugs. In all 5 girls, extreme hypotonia was present since birth, together with a tetraparesis, changing into a spastic tetraparesis with increasing age (patient 2). Evident dysmorphic features were absent. There was a global developmental delay, with severe intellectual disability and poor language, visual, and motor development. Because of feeding difficulties, 3 of 5 patients required feeding through a percutaneous endoscopic gastrostomy (PEG) tube. Cerebral MRI was unremarkable in all patients, including the 21-year-old. Autonomic disturbances were absent in all 5 girls.

Mutation analysis of the CDKL5 gene in patient 1 revealed a de novo insertion of 2 nucleotides (c.275_276insAA) resulting in a frameshift (p.Glu93fs). In patient 2, a de novo mutation in intron 3 of the CDKL5 gene was found: c.99+5G>A, which is predicted to affect correct splicing of the CDKL5 mRNA, However, since RNA studies have not been performed, the effect on the protein level cannot be predicted. In patient 3, a de novo deletion of 2 nucleotides (c.2105_2106del) resulting in a frameshift (p.His702fs) was found. In patient 4, the CDKL5 gene showed a deletion of 11 nucleotides containing the splice acceptor site of exon 6 (c.283-3_290del). Since the splice consensus site is not present anymore, this mutation will affect correct splicing of the CDKL5 mRNA and, therefore, affect protein function. Without RNA studies the exact effect on the protein level cannot be predicted. This mutation was also de novo. In patient 5, mutation analysis of the CDKL5 gene revealed a duplication of 1 nucleotide at position 1784 (c.1784dup), leading to a frameshift in the CDKL5 open reading frame (p.Leu596fs). The mother is not a carrier of this mutation; DNA of the father was not available for testing.

Discussion

Clinical phenotype of the presented 5 girls having a CDKL5 mutation is comparable with those already described in literature. As known from published reports, females with a CDKL5 mutation suffer from severe cognitive impairment, marked hypotonia, and severe impairment of visual, language, and motor development.^3–5,9–11 Feeding difficulties, present in all 5 patients, have been reported before in literature, albeit only in a few cases.^3,5 Usually there is poor eye fixation, with avoidance of eye contact; absence of speech or the ability to speak only a few words with limited understanding; and generalized hypotonia with failure to develop motor skills or with regress of motor abilities.^3–5,9–11 Our cases were compatible with the description above, but milder cases have also been described.^3–5,9–11CDKL5 patients commonly have an early seizure onset with a subsequent mixed seizure disorder that becomes intractable, as was shown in our cases.¹⁰ In all of the presented females and in accordance with known cases, seizures started in the first few weeks of life, initially brief with a duration of seconds.¹⁰ With time, frequency and duration of seizures increase, with seizures occurring up to hundreds of times daily. Reaction to various antiepileptic drugs often seems positive at first, but typically proves to be moderate and of short duration.¹⁰ As to results of ketogenic diet, which seemed to lead to a moderate reduction of seizure frequency in 2 of our patients, current literature is less optimistic and generally reports no improvement at all.^2,3,9 Many different seizure types are possible and can also occur within a single patient. Seizure type can change with time. Two of our 5 patients (patients 1 and 2) suffered from periods of status epilepticus. In contrast with the description above, with seizures becoming intractable, there have also been cases described with no seizures at all, which proves the spectrum of the severity of the seizure disorder to be wide.¹⁰ Autonomic disturbances were absent in our population. Literature is not consistent about this feature.¹⁰

The 5 females described above do not meet the consensus criteria for typical or atypical Rett syndrome, since a period of nearly normal development followed by regression with recovery or stabilization is required to make this diagnosis.^6, However, there is overlap with Rett syndrome, with features such as hypotonia, deceleration of head growth, and limited handskills and hand stereotypies.9 Most of these Rett features become more evident in older patients.⁹ For instance, our patient 2 developed hand wringing and had limited handskills. In accordance with most literature, brain MRI findings were unremarkable in the presented cases, although MRI abnormalities have been described, consisting of cortical atrophy and hyperintensities in the posterior white matter and temporal poles.^2,9,10

In patients with a CDKL5 mutation, interictal EEG shows a wide variety of background characteristics. Background activity can range from burst suppression and multifocal epileptiform activity to moderate background slowing with sporadic epileptiform discharges or normal background activity.² According to literature, the different degrees of EEG abnormalities do not seem to match with severity of seizures or clinical phenotype.²

The mutations reported in this study have not been described in literature before, but they either lead to a frameshift in the reading frame or affect a consensus splice site. Therefore, all mutations are predicted to result in nonsense mediated decay of the CDKL5 mRNA and are, therefore, considered to lead to a loss of function of the CDKL5 protein. Furthermore, 4 of the mutations are found to be de novo in the patient.

As the name already suggests, CDKL5 belongs to a class of protein kinases.^4,10 There is conflicting evidence whether there is a direct interaction between MECP2, the gene that is mutated in a significant proportion of typical Rett syndrome and CDKL5. ^4,8,10

The large COOH terminal domain of the CDKL5 protein is required to stabilize interactions with splicing regulatory proteins such as MECP2. ¹⁰ Mutations in the CDKL5 gene, both over-expression as well as down-regulation of CDKL5, are hypothesized to result in loss of kinase activity, affecting the function of these and other regulatory proteins, which subsequently is thought to cause clinical phenotype.¹⁰ This is in accordance with the overlapping phenotypic spectrum of CDKL5 and MECP2, which presumes a common pathogenic process.¹⁰ The wide spectrum of the severity of the disorder is supposed to be caused by the location of the mutations in the gene, their functional consequences for transcription and translation, and varying patterns of X-inactivation.^9,10 The exact function of the protein, however, still remains to be elucidated. CDKL5 mutations can be found in a significant proportion of female patients with severe mental disability, marked hypotonia, and intractable epilepsy, especially with an early onset of seizures or infantile spasms.^9,10 In addition, CDKL5 mutations can be found in girls with Rett-like symptoms and early onset of seizures.⁹ Girls with clinical features as described above should be analyzed for having CDKL5 mutations. Identification of more patients will result in a better knowledge of this disorder, especially of the clinical course. The recognition of more patients perhaps can lead to more effective treatment options of the intractable epilepsy.

Footnotes

Acknowledgments

This work would not have been possible without the participation of our colleagues C. Marcelis, T. Kleefstra, and M. Kets, who all work at the Department of Human Genetics. This work was presented at the American Academy of Neurology 63rd Annual Meeting, April 9 to April 16, 2011, Honolulu, Hawaii.

All authors participated in the writing of the case report: X. Stalpers as main author, A Verrips as senior author. L. Spruijt and H. Yntema focused on the parts with genetics expertise. X. Stalpers wrote the first draft of the manuscript.

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The authors received no financial support for the research, authorship, and/or publication of this article.

Institutional ethical approval was not indicated for this article and review of published literature.

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