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Pediatric Neurology 48 (2013) 367e377

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Pediatric Neurology

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Original Article

CDKL5 and ARX Mutations in Males With Early-Onset Epilepsy

Ghayda M. Mirzaa MDa,*, Alex R. Paciorkowski MDb, Eric D. Marsh MD c,d,Elizabeth M. Berry-Kravis MD, PhD e, Livija Medne MS c, Art Grix MD f, Elaine C. Wirrell MD g,Berkley R. Powell MDh, Katherine C. Nickels MDg, Barbara Burton MD i, Andrea Paras MS i,Katherine Kim MS i, Wendy Chung MD j, William B. Dobyns MDa, Soma Das PhDk

aDivision of Genetic Medicine, Department of Pediatrics, University of Washington and Center for Integrative Brain Research, Seattle Children’s ResearchInstitute, Seattle, WashingtonbDepartments of Neurology, Pediatrics, and Biomedical Genetics, Center for Neural Development and Disease, University of Rochester Medical Center,Rochester, New YorkcDivision of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PennsylvaniadDepartments of Neurology and Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaeDepartments of Pediatrics, Neurological Sciences, and Biochemistry, Rush University Medical Center, Chicago, IllinoisfDepartment of Genetics, Permanente Medical Group, Roseville, CaliforniagDepartment of Neurology, Divisions of Epilepsy and Child and Adolescent Neurology, Mayo Clinic, Rochester, MinnesotahChildren’s Hospital Central California, Madera, CaliforniaiChildren’s Memorial Hospital and Northwestern University Feinberg School of Medicine, Chicago, IllinoisjDivision of Molecular Genetics, Department of Pediatrics, Columbia University, New York, New YorkkDepartment of Human Genetics, University of Chicago, Chicago, Illinois

article information

Article history:Received 7 September 2012Accepted 31 December 2012

a

Mo

* Communications should be addressedChildren’s Research Institute; Center for Int1900 Ninth Avenue Mailstop C9S-10; Seattle,

E-mail address: gmirzaa@uw.edu

0887-8994/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.pediatrneurol.2012.12.030

bstract

utations in CDKL5 and ARX are known causes of early-onset epilepsy and severe devel-pmental delay in males and females. Although numerous males with ARX mutations

associated with various phenotypes have been reported in the literature, the majority ofCDKL5 mutations have been identified in females with a phenotype characterized by early-onset epilepsy, severe global developmental delay, absent speech, and stereotypic handmovements. To date, only 10 males with CDKL5 mutations have been reported. Our retro-spective study reports on the clinical, neuroimaging, and molecular findings of 18 maleswith early-onset epilepsy caused by either CDKL5 or ARX mutations. These 18 patientsinclude eight new males with CDKL5 mutations and 10 with ARX mutations identifiedthrough sequence analysis of 266 and 346 males, respectively, at our molecular diagnosticlaboratory. Our large dataset therefore expands on the number of reported males withCDKL5 mutations and highlights that aberrations of CDKL5 and ARX combined are animportant consideration in the genetic forms of early-onset epilepsy in boys.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Early infantile epileptic encephalopathy (Ohtaharasyndrome), early myoclonic epilepsy, infantile spasms, andLennox-Gastaut syndrome are age-dependent epilepsysyndromes that show overlap in phenotype and,

to: Dr. Mirzaa; Seattleegrative Brain Research;WA 98101.

ll rights reserved.

increasingly, in genotype [1]. Thesedisorders are categorizedas “developmental epilepsy” and even though this latterterm is not a part of the International League AgainstEpilepsy classification, we feel it best captures the demon-strated biological kinship among these disorders of early-onset epilepsy [1,2]. Moreover, “developmental epilepsy” isan improvement over the term “epileptic encephalopathy”because, although the epilepsy is a key feature, it is morelikely that the genetic lesion when present is the primarydriver of developmental outcome in these children [3]. Theetiology of these developmental forms of epilepsy include

Table 1. Summary of the clinical, neuroimaging, and molecular findings in boys with CDKL5 mutations (n [ 8)

No. 1 2 3 4

ID# LR03-031a1 LR04-421 LR04-409 LR03-121

Ethnicity Caucasian Caucasian Caucasian Caucasian

Age last assessed 7.5 yr 6 yr 4 yr 14 yr

Seizure onset 4 wk 5.5 wk 9 wk 6 wk

Seizure types ISS, tonic, myoclonic, atonic Tonic, ISS, generalizedtonic clonic

ISS ISS, myoclonic, tonic

Intractable epilepsy þ þ þ þEEG findings HYPS, multifocal bursts of

polyspike activityHYPS HYPS Continuous bihemispheric

epileptiform discharges

Cerebral atrophy � þ � þþAge at last MRI 13 yr 4 yr <4 yr 9 yr

Severe global DD þ þ þ þTone Mixed Mixed Hypotonia Mixed

Cortical visual Impairment þ þ þ þPostnatal microcephaly � þ ND �Abnormal movements þ � � þOutcome Deceased Alive Alive Alive

Mutation c. 578A > G c. 513C > A c.2413C > T c.175C > T

(exon 9) (exon 8) (exon 17) (exon 5)

p.D193G p.Y171X p.Q805X p.R59X

Type Missense Nonsense Nonsense Nonsense

Inheritance Germline mosaicism De novo De novo De novo

Family history Affected maternal half-sister* 2 maternal SAB 2 maternal SAB No known neurologic problems

Abbreviations:DD ¼ Developmental delayEEG ¼ ElectroencephalographicHYPS ¼ HypsarrhythmiaISS ¼ Infantile spasmsLG ¼ Lennox-GastautND ¼ No dataOFC ¼ Occipitofrontal circumferenceSAB ¼ Spontaneous abortionVNS ¼ Vagus nerve stimulator

* Affected maternal half-sister (assessed at 5.5 yrs) had onset of seizures at 6 to 7 mo consisting of eye fluttering and jerky movements of the arms. Initial EEG showedprehypsarrhythmic changes. She subsequently had brief complex partial seizures every 1 to 3 mo that were well-controlled on one antiepileptic medication. She had severedevelopmental delay. She rolled over at 1 yr of age and at 5.5 yr had minimal head control and was unable to sit independently and remained nonambulatory. She vocalizedwithout distinct words. She also has abnormal movements, teeth grinding, and breath-holding spells. The mother of these two siblings is asymptomatic.

y These two patients were also included in a recent report by our co-authors [33].

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377368

specific cortical malformations, metabolic disorders, andmutations of several recently identified genes in the absenceof metabolic or structural brain abnormalities that includeARX, CDKL5, SLC25A22, STXBP1, and SPTAN1, among others[4-12].

Mutations in CDKL5 have been predominantly reportedin females with a phenotype characterized by early-onsetrefractory epilepsy, severe developmental delay, absent orvery limited speech, and additional features such as handstereotypies and postnatal deceleration of head growth insome patients [13-18]. In a large study, CDKL5 mutationswere estimated to occur in approximately 8% of girls withearly-onset seizures (before 9months) and up to 28% of girlswith early-onset seizures and infantile spasms [19]. Muta-tions in males, on the other hand, appear less common,having been reported in 10 boys to date with a more severephenotype characterized by early-onset tonic and

myoclonic seizures, intractable infantile spasms, severeglobal developmental delay, cortical visual impairment,sleep disturbances, and hand stereotypies in some patients[13,20-25].

ARX was the first gene implicated in nonsyndromicearly-onset epilepsy and has been associated with a widespectrum of developmental disorders with and withoutbrain malformations. The former group includes X-linkedlissencephaly with abnormal genitalia, an X-linked lis-sencephaly with abnormal genitalia variant with severehydrocephalus, and Proud syndrome (agenesis of thecorpus callosum with abnormal genitalia). The lattergroup includes X-linked infantile spasms, Partingtonsyndrome (intellectual disability and dystonia), andnonspecific X-linked intellectual disability [8,26-30].Previous studies of ARX mutations in 115 boys withinfantile spasms of unknown cause identified expansions

5y 6y 7 8

LR11-421 LR11-048 LR05-277 LR11-420

Somali Caucasian Caucasian Caucasian

10 mo 2 mo 6 yr 4 yr

6 mo 2 mo 2 wk 5.5 wk

ISS, partial, multifocal Partial, ISS Tonic, tonic clonic, ISS, myoclonic Tonic, ISS

þ þ þ þMultifocal epileptiformdischarges, recordedgeneralized seizures

Slow background(5 Hz), multifocal discharges

Slow R sharp waves;disorganized, slow, sharpwaves, multifocal discharges

Localization-related epilepsy,likely arising from the Rfrontocentral area

� � þþ þND 2 mo 6 wk 2 yr

þ þ þ þHypotonia Severe hypotonia Severe hypotonia Severe hypotonia

þ þ þ þ� � � �þ � þ �Alive Alive Alive Alive

c.2593C > T Ex 10-15del Ex 3del c.62A > G

(exon 18) (exon 2)

p.Q865X p.E21G

Nonsense Deletion Deletion Missense

De novo ND De novo De novo

No known neurologic problems No known neurologic problems No known neurologic problems No known neurologic problems

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377 369

of the first polyalanine (polyA) tract in six boys (5.2%). Allhad infantile spasms by 6 months of age, developmentaldelay, and generalized dystonia leading to a severemovement disorder [28].

We studied two cohorts of 266 and 346males referred toour clinical molecular laboratory for CDKL5 and ARX muta-tion analyses, respectively. The indications for testingincluded early-onset, medically refractory epilepsy (earlyinfantile epileptic encephalopathy, infantile spasms, earlymyoclonic epilepsy), global developmental delay, or both.We identified CDKL5 mutations in eight males and ARXmutations in 10. Here we report these 18 boys and discusstheir phenotypic features. Analysis of these genes in thesetwo large cohorts of boys provides further evidence thatmutations in CDKL5 and ARX combined account for animportant fraction of the genetic causes underlying early-onset epilepsy in males.

Materials and Methods

Patient ascertainment

Between 2002 and 2011, 266 boys were referred to the clinicalmolecular diagnostic laboratory at the University of Chicago for mutationanalysis of CDKL5 and 346 for mutation analysis of ARX for the indica-tions mentioned previously. Patients with brain malformations wereexcluded from our study. Retrospective clinical, neuroimaging andelectroencephalographic (EEG) data were obtained on patients in whoma mutation was identified. The Institutional Review Board at TheUniversity of Chicago approved this study.

Molecular methods

Genomic DNA was extracted from peripheral blood samples usingeither the Puregene kit, Magnapure, or Autogen systems following themanufacturers’ recommendations. PCR amplification was performedwith 50 ng of genomic DNA using Taq DNA polymerase (Applied

Table 2. Summary of the clinical, neuroimaging, and molecular findings in boys with ARX mutations (n [ 10)

No. 1 2 3 4

ID# LR04-386 LR04-272 LR03-309 LR02-457

Ethnicity Caucasian Caucasian Asian ND

Age at last assessment 17 yr 6 yr 21 mo 19 yr

Seizure onset Neonatal 6 wk 13 wk Neonatal

Seizure types Tonic ISS, complex partial ISS, myoclonic, complex partial ISS, partial

Intractable epilepsy þ þ þ þ

EEG findings Generalized atypical spike,polyspike, slow wavedischarges, multifocal

HYPS, frequentelectrodecrementalresponses

Multifocal epileptiformdischarges, disorganizedbackground

HYPS (6 wk), Lennox-Gastautsyndromeelike pattern (17 yr)

Cerebral atrophy þ þ þ þþAge at last MRI 4 yr ND ND 19 yr

Severe global DD þ þ þ þTone Spasticity Hypotonia Severe hypotonia Mixed

Postnatal microcephaly � ND � ND

Abnormal movements þ þ � þOutcome Alive Alive Alive Alive

Mutation c.333_335dup(GGC)7(exon 2)

c. 333_335dup(GGC)7(exon 2)

c. 333_335dup(GCG)7(exon 2)

c. 333_335dup(GCG)7(exon 2)

Type In-frame; insertion7 polyalanine repeats

In-frame; insertion7 polyalanine repeats

In-frame; insertion7 polyalanine repeats

In-frame; insertion7 polyalanine repeats

Inheritance ND ND De novo De novo

Family history ND ND Febrile seizures No known neurologic problems

For abbreviations, see Table 1.* This patient had PAID syndrome (paroxysmal autonomic instability with dystonia), dystonic posturing and choreoathetosis, and bilateral clonus.y This patient’s mother is asymptomatic and has never had a brainMRI. A maternal cousin has a history of autism-spectrum disorder, seizures, and developmental delay. A

maternal half-aunt had seizures at 13 to 14 years and a maternal uncle had seizures secondary to head trauma during a motor vehicle accident.

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377370

Biosystems, Foster City, CA). Primers were used to amplify the coding andflanking noncoding regions of CDKL5 and ARX. Optimized conditionswere used for the GC-rich exon 2 of the ARX gene, as previouslydescribed [27]. The coding regions of the CDKL5 (exons 2-21) and ARX(exons 1-5) genes were targeted for mutation analysis performed bySanger sequencing using standard methods.

Magnetic resonance imaging and EEG studies

Brain magnetic resonance imaging (MRI) was performed on allsubjects and images were evaluated for structural and maturationalabnormalities by the investigators. Pretreatment EEG studies were per-formed in all subjects and were evaluated for epileptiform activity andabnormalities in background rhythm. A consensus definition of hypsar-rhythmia (voltage > 200 mV, interhemispheric asymmetry, lack ofanteroposterior gradient, multifocal sharp waves) was used [31]. Theinvestigators were not blinded to the mutation status of the individualsat the time of review.

Results

The clinical, molecular, and neuroimaging findings in theeight CDKL5 mutation-positive and 10 ARX mutation-positive males are summarized in Tables 1 and 2.

Molecular findings

CDKL5The CDKL5 mutations identified in our cohort included

four nonsense mutations (p.Y171X, p.Q805X, p.R59X,p.Q865X), two missense mutations (p.E21G and p.D193G),and two intragenic deletions (of exon 3 and exons 10-15).Maternal testing of six patients revealed that mutationswere de novo in all six. Patient LR03-031 and his maternalhalf-sister had the same missense mutation (p.D193G),whereas maternal testing was negative, suggestingmaternal germline mosaicism. Figure 1A illustrates theseidentified CDKL5 mutations.

ARXThe ARXmutations identified included seven expansions

of the first polyalanine tract [c.333_335dup(GGC)7 in sixpatients and c. 333_335dup(GGC)8 in one patient], oneexpansion of the second polyalanine tract (429_452dup24),a missense mutation (p.T333S), and a 103-bp insertion atthe intron 4-exon 5 junction (IVS4-82_e x 5 1469ins103)resulting in frameshift and premature termination of amino

5 6 7 8 9 10

LR11-422 LR11-425 LR11-423 LR02-310 LR04-427 LR03-413

ND Mixed European Native American/European

Asian Caucasian/African-American

Eastern European(Jewish)

3 yr 16 yr 20 mo ND ND 13 mo

4 mo 2 mo 10 days 3 mo 24 days 6 mo

ISS ISS, complex partial ISS, complex partial,partial

Tonic, tonic spasms Tonic, tonic spasms ISS, myoclonic

þ þStatus post VNS placementat 12 yr

þ � þ þ

HYPS Focal sharps atfrontoparietal areas, slowbackground (16 yr)

HYPS HYPS Burst suppression,generalized polyspikes

HYPS

þ � þ � � þND 12 yr 14 mo 2 yr Infancy 1 yr

þ þ þ þ þ þAxial hypotonia Hypotonia Axial hypotonia with

opisthotonic posturingHypotonia ND Hypotonia

� � � ND þ �þ þ þ* � � �Alive Alive Alive Alive Alive Alive

c. 333_335dup(GGC)7(exon 2)

c. 333_335dup(GGC)7(exon 2)

c.333_335dup(GGC)8(exon 2)

c.429_452dup24(exon 2)

IVS4-82_ex51469ins103(intron 4/exon 5)

c. 998C > G(p.T333S)(exon 2)

In-frame; insertion7 polyalanine repeats

In-frame; insertion7 polyalanine repeats

In-frame; insertion8 polyalanine repeats

In-frame; insertion8 polyalanine repeats

Insertion 103 bp Missense

ND ND Maternally inheritedy ND Maternally inherited De novo

ND No known neurologicproblems

See below ND ND 2 healthy siblings,1 SAB

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377 371

acid sequence. Mutation analysis in the mothers of fourpatients revealed that two were carriers (LR11-423 andLR04-427), whereas the other two had de novo mutations(LR03-309 and LR02-457). Figure 1B illustrates these iden-tified ARX mutations.

Frequency of CDKL5 and ARX mutations in males with early-onsetepilepsy

Analysis of our two cohorts revealed a mutationfrequency of 3% and 2.8% in the CDKL5 and ARX genes,respectively, in boys with early-onset intractable epilepsyreferred to our laboratory for genetic testing. A total of 205boys were analyzed for both genes, and included the 18mutation-positive boys (8.7%).

Clinical findings

CDKL5 cohortBoys with the CDKL5 mutations ranged from 2 months

to 14 years of age at the time of evaluation. Infantile

spasms, defined as epileptic spasms with ictal electro-decrement and interictal hypsarrhythmia, occurred atsome point in all eight patients [31]. Other seizure typesinclude tonic, myoclonic, and partial seizures. Seizureonset occurred between 2 weeks and 6 months of age.The characteristic three-stage evolution of epilepsy,characterized by early epilepsy (stage 1), infantile spasms(stage 2), and multifocal refractory epilepsy (stage 3) wasseen in at least six patients on whom detailed clinicaldata were available [17]. All eight patients had profounddevelopmental delay with minimal or no language andmotor skills, severe tone abnormalities, and corticalvisual impairment. Abnormal involuntary movementsseen in four patients include hand stereotypies, dystonicmovements, and head stereotypies resembling spasmusnutans (Table 1). Other significant medical problemsinclude feeding difficulties with severe gastroesophagealreflux and secondary respiratory insufficiency, requiringgastrostomy (G-tube) placement, in at least sevenpatients.

Figure 1. The identified mutations in CDKL5 (A) and ARX (B). (A) The mutations indicated below the gene are the previously reported CDKL5 mutations,whereas those indicated above the gene are the identified mutations in our cohort that include four nonsense mutations, two missense mutations, and twointragenic deletions. (B) The identified ARX mutations including eight polyalanine repeat expansions, one insertion mutation in an intron/exon boundary,and one missense mutation. The p.D30Y mutation was identified in our laboratory in the carrier state in a mother whose son was tested elsewhere.

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377372

Patient LR03-031 had a maternal half-sister with thesame missense mutation in CDKL5. She had severeglobal developmental delay, hand stereotypies, hypotonia,cortical-visual impairment, and breath-holding spells, yether epilepsy was milder than that of her brother as she didnot have infantile spasms or refractory epilepsy at any pointduring her epilepsy course. Further details regarding herepilepsy phenotype are in Table 1.

ARX cohortThe 10 boys with ARX mutations were 13 months to 19

years of age at the time of assessment. All had severeinfantile-onset refractory epilepsy. Seizure types includeinfantile spasms, tonic, myoclonic and partial seizures. Ofnote, five had early-onset seizures that evolved to infantilespasms and a pattern similar to the three-stage seizureevolution seen with CDKL5 mutations. All 10 patients hadsevere global developmental delay. Involuntary move-ments were seen in six patients and consisted predomi-nantly of chorea and/or dystonia. Interestingly, patientLR11-423 had paroxysmal autonomic instability with dys-tonia (PAID) syndrome characterized by severe dystoniaand choreoathetosis of the face and extremities in associ-ation with fever, tachycardia tachypnea, and pupillarydilatation [32]. He was treated with baclofen and clona-zepam (Table 2).

Neuroimaging findings

CDKL5 cohortAlthough early brain MRIs were normal in several boys,

later imaging in at least three boys showed progressivecerebral and cerebellar atrophy with increased extra-axialspaces, widened sulci, ventriculomegaly, and white matterthinning. Cerebral and cerebellar atrophy were evident onbrain MRI as early as 6 weeks of age in patient LR05-277.Representative brain MRI findings are illustrated in Fig 2.

ARX cohortBrain MRI studies in boys with ARX mutations showed

the characteristic T2 signal intensities in the globus pallidusand putamen in four patients [27,28]. Serial imagingdemonstrated progressive diffuse cerebral atrophy withventriculomegaly as early as 1 year of age in at least sevenpatients (Fig 3).

Epilepsy and EEG findings

CDKL5 cohortAll eight patients with CDKL5 mutations in our cohort

developed infantile spasms at some point in their epilepsycourse, with some patients exhibiting very early onset ofthis form of epilepsy. Patient LR03-031a1 had onset of

Figure 2. Brain magnetic resonance images of three males with CDKL5mutations. T1-weighted mid-sagittal, parasagittal and axial, and T2-weighted coronalimages of patient LR04-421 (A-D), T1-weighted mid-sagittal, parasagittal and coronal, and T2-weighted axial images of patient LR03-121 (E-H) and patientLR11-048 (I-L). Note widening of the cortical and cerebellar sulci consistent with atrophy, mildly increased extra-axial space, and thin corpus callosum.

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377 373

epileptic spasms in clusters at 1 month of age with hyp-sarrhythmia on EEG. There was partial response to pheno-barbital, carbamazepine, and vigabatrin, but the child wasnever seizure-free. At last follow-up at 7 years, he continuesto have up to eight seizures a day characterized by tonicstiffening of the extremities with eye deviation. EEGscontinue to show periodic burst suppression and discon-tinuous background. Subject LR04-421 had tonic seizures at5.5 weeks of life that evolved into infantile spasms refrac-tory to adrenocorticotropin (ACTH), multiple antiepileptics,and the ketogenic diet. EEG at 5 months showed hypsar-rhythmia. Subject LR03-121 began to have extensorposturing and apnea at 6 weeks of life with normal EEG. By8 weeks, clinical extensor epileptic spasms were observed.At 4 months, the EEG showed continuous bihemisphericepileptiform discharges but characteristics of hypsar-rhythmia were not noted. Subsequent EEG at 23 monthsshowed high voltage and frequent focal sharp discharges.Epilepsy semiology evolved from epileptic spasms thatresponded briefly to treatment with ACTH at 4 months,until tonic and myoclonic seizures emerged at 8 monthsthat were refractory to 17 antiepileptic medications insuccession as well as the ketogenic diet. At age 2 years, hebegan to have atonic seizures as well as tonic andmyoclonicseizures. EEG at 3 years showed continuous bioccipitalepileptiform activity, very slow background, and lack ofelectrographic response to stimulation.

ARX cohortThe boys with ARX mutations similarly had early-onset

epilepsy. Subject LR04-272 exhibited flexor epileptic

spasms at 6 weeks of age combined with right-sided tonicseizures and behavioral arrest. EEG initially demonstratedburst suppression that evolved into hypsarrhythmia withfrequent electrodecrement with and without epilepticspasms by 4 months of age. Subject LR04-427 began havingtonic seizures at 21 days of age, increasing in frequency toevery 15 minutes. There was initial response to pyridoxalphosphate and then to carbamazepine. By 3 months,seizures were tonic spasms with burst suppression andgeneralized polyspikes on EEG. Subject LR02-457 hadseizures in the first days of life with multifocal spikes andpolyspike and wave with frequent electrodecrement. By 6weeks, EEG showed high voltage (100-150 mV) with bilat-eral spikes and waves with electrodecrement. Clear infan-tile spasms emerged at 6 months. Epilepsy was ultimatelylifelong and intractable, with EEG and clinical featuresmeeting criteria for Lennox-Gastaut syndrome. SubjectLR11-423 had his first seizures at 10 days of life, withrecurrent rhythmic movements of the upper extremitiesthat were initially thought to be benign myoclonic move-ments of infancy. These evolved into flexor epileptic spasmsleading to a diagnosis of infantile spasms by 3 months thatwere subsequently unresponsive to ACTH, vigabatrin, andthe ketogenic diet. EEGs showed hypsarrhythmia. Infantilespasms continued at last assessment at 20 months.

Discussion

Common clinical features of the developmental epilep-sies (early infantile epileptic encephalopathy, earlymyoclonic epilepsy, infantile spasms, and Lennox-Gastaut

Table 3. Summary of the clinical and molecular data of published males with CDKL5 mutations (n [ 10)

Reference [13](pt III.3)*

[20](pt 1/8)

[20](pt 2/8)

[20](pt 3/8)

Age at assessment 16 yr 13 yr 9 yr 3.5 yr

Seizure onset Neonatal 3 mo 8 mo 2 mo

Seizure types ISS, LG ISS, myoclonic tonic,complex partial

Complex partial, ISS,tonic, myoclonic

Tonic, ISS, complexpartial

Tone Spastic quadriparesis Hypotonia ND Hypotonia

Postnatal microcephaly þ ND ND ND

Severe global DD þ þ þ þOther neurologic findings Cortical blindness,

hyperventilation� Pyramidal signs,

ataxia, echolalia�

Outcome Deceased at 16 y(aspiration pneumonia)

Alive Alive Alive

Mutation/deletion c.183delT (exon 5) c.872G > A (p.C291Y) c.863C > T (p.T288I) c.872G > A (p.C291Y)

Type Frameshift Missense Missense Missense

Inheritance De novo De novo De novo De novo

Family history 2 affected sisters (twins) Nonconsanguineousparents; 1 healthy sibling

Nonconsanguineousparents; first child

Nonconsanguineousparents; 2 SAB

* This patient’s two sisters (twins; 19 yr) had the same mutation. III.1 had profound DD, decreased OFC (50% to 98% at birth; 2% to 50% at 19 yr), epileptic encephalopathy(seizure onset 9 wk; ISS, later had generalized tonic-clonic, myoclonic, and absence seizures), generalized spasticity, scoliosis, constipation. III.2 had mild DD (IQ w 70),autistic features. She had no seizures or tone abnormalities. Family history is also remarkable for two stillbirths and one miscarriage.

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377374

syndrome) include drug-resistant seizures, profound neu-rodevelopmental delay, and, for most affected children,poor neurological outcome. Although the developmentalepilepsies are often associated with structural brainabnormalities, inborn errors of metabolism, and othergenetic disorders, the underlying etiology for many remainsto be identified.

CDKL5

Mutations in CDKL5 have been reported extensively infemales with epileptic encephalopathy and a severe devel-opmental syndrome in which multiple neurofunctionaldomains are affected [13-18]. Some of the phenotypicfeatures observed in females with CDKL5 mutations,particularly hand stereotypies, limited hand use, decelera-tion of head growth, and absent or limited speech, resemblethose seen in Rett syndrome, and have thus been confus-ingly termed “atypical Rett syndrome.”

Reports of males with CDKL5 mutations have beeninfrequent, suggesting possibly early male lethality and X-linked inheritance. The 10 reported males had a moresevere phenotype characterized by early-onset intractableepilepsy, predominantly infantile spasms, and profounddevelopmental delay with lack of motor skills, stereotypicmovements, and cortical visual impairment [18] (Table 3).The CDKL5 mutations identified to date include fivemissense mutations, two frameshift mutations, one splicingmutation (in a somatic mosaic form), one nonsense muta-tion in a male with Klinefelter syndrome (47,XXY), anda deletion of exon 1 [13,20-23,25]. Larger aberrations

involving the CDKL5 gene have been reported in twopatients with microdeletions of the X chromosome andexpanded syndromes: (1) a large 2.8-Mb deletion ofXp22.2-Xp22.13 including exons 1-11 of CDKL5 and the NHS(Nance-Horan syndrome) gene in a boy with tetralogy ofFallot, microphthalmia, microcornea, congenital cataracts,and dysmorphic features and (2) a mosaic 128-kb deletionthat includes exons 1-3 of CDKL5 in a male with severerefractory epileptic encephalopathy [24,34].

Here, we report eight males with CDKL5 mutations, twoof whom have been included in a separate publication [33].These patients shared many phenotypic features of thosepreviously reported in the literature and demonstrate thatmales with CDKL5 mutations have a severe neuro-developmental profile characterized by refractory neonatalor infantile-onset epilepsy (with early infantile epilepticencephalopathy or infantile spasms occurring in most),severe global developmental delay with minimal acquisi-tion of skills, cortical visual impairment, tone abnormalities,and hand stereotypies. As expected with most forms ofdevelopmental epilepsy, serial neuroimaging showednonspecific features such as progressive cerebral and cere-bellar atrophy with progressively enlarging extra-axialspaces and ventriculomegaly (Fig 2). EEGs were all poorlyorganized and showed multifocal epileptiform abnormali-ties in addition to many showing hypsarrhythmia. PatientsLR05-277 and LR11-048 of our cohort had intragenic dele-tions of CDKL5 and are phenotypically similar to boys withpoint mutations.

All boys reported to date, including our cohort, hadintractable epilepsy, severe developmental delay, and poor

[21] [22] [23] [25](pt 1)

[25](pt 6)

[25](pt 9)

4 yr 4 yr 2 yr 8 mo 6 mo 1 yr, 9 mo 2 yr

2 mo 15 days 2 mo 1 mo 2 wk 3 mo

ISS, tonic, myoclonic ISS, tonic generalized tonicclonic, myoclonic

ISS, tonic, partial ISS Spasms ISS

Hypotonia Hypotonia Hypotonia No hypotonia Hypotonia Hypotonia

ND þ � � � �þ þ þ þ þ þ� Breath-holding spells � ND ND ND

Alive Alive Alive ND ND ND

c.191T > C(p.L64P)

c.1675C > T(p.R559X)(47,XXY)Balanced Xi(50%-50%)

c.99þ5G > A(somatic mosaicism)

Exon1 deletion c.533G > A(p.R178Q)

c.1079delT(p.L360Fs >367X)

Missense Nonsense Splicing; frameshift Deletion Missense Frameshift

ND ND Somatic mosaicism ND De novo ND

Nonconsanguineousparents; 1 healthy sibling

Nonconsanguineousparents; first child

Nonconsanguineousparents

ND ND ND

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377 375

neurocognitive outcome. The brother-sister pair reportedhere illustrates milder epilepsy severity in girls. In additionto these siblings, germline mosaicism for a CDKL5 mutationhas been identified in at least one other family [13]. Thispreviously reported family included a severely affectedbrother and his identical twin sisters, one of whomhadmilddevelopmental delay with autistic features, but no seizures.This also possibly highlights the difference in the severity ofphenotypes between boys and girls with CDKL5 mutations,and both families illustrate the importance of consideringgermline mosaicism in recurrence risk counseling.

ARX

Mutations in ARX have been reported in a series ofoverlapping phenotypes, divided broadly into those with orwithout brain malformations, with a clear distinction inphenotypes between affectedmales and females and stronggenotype-phenotype correlation [27]. The mildest pheno-types of nonsyndromic developmental delay generallyoccur with expansions of the first and second polyA tractsand missense mutations, whereas severe brain malforma-tions occur with more severe mutations predicted to lead tocomplete loss of ARX function such as splicing, nonsense,and insertion and deletion mutations [27]. Moreover, singlebase pair substitutions leading to missense changes in thehomeodomain region and the region before the aristalessdomain also lead to very severe phenotypes, suggestingthese residues are critical to ARX function [26-30].

It is estimated that aberrations in ARX may account forup to 5.2% of the genetic etiologies in males with early

epileptic encephalopathy or X-linked infantile spasms.Expansions within the first or second polyA tracts are themost common events. Expansions of the first polyA tract arebelieved to be more common than expansions of thesecond, except perhaps in rare multiplex families [28]. Noreported mutations or expansions of the third and fourthtracts exist to date. The severity of the clinical presentationgenerally increases with increasing length of the expansion[27]. Consistent with these data, seven of our patients hadpolyA repeat expansions. One patient had an expansion ofeight GGC trinucleotides instead of the more commonlyreported seven trinucleotides. To date, this is the firstpatient identified with this c.333_335dup(GGC)8 mutation.Notably, this patient had the earliest onset of seizures (at 10days of age), suggesting amore severe phenotype comparedwith patients with the common c.333_335dup(GGC)7mutation. Previously, two boys presenting with earlyinfantile epileptic encephalopathy were reported with denovo 33-bp duplications, c.298_300dup(GCG)9, expandingthe first polyA tract and suggesting that longer expansionsare consistent with earlier onset and more severe pheno-types [35].

Several of our ARX mutations are novel and includea missense mutation within the homeodomain (p.T333S),a 103-bp insertion at the intron 4-exon 5 junction (IVS4-82_e x 5 1469ins103), and a missense mutation within theoctapeptide domain of the ARX region (p.D30Y). The largeinsertion at the intron 4eexon 5 region is predicted to affectnormal splicing, similar to one other mutation reported inthis region [35]. Of our twomaternally inherited mutations,one was an expansion of the first polyA tract and the other

Figure 3. Brain magnetic resonance images of males with ARX mutations. T1-weighted mid-sagittal and two T2-weighted axial images of patient LR11-423(A-C) and patient LR02-457 at 5 years (D-F) and 19 years of age (G-I). Note the characteristic T2 signal intensities in the basal ganglia bilaterally (E and H,arrows), cortical and cerebellar atrophy, ventriculomegaly, and thinning of the corpus callosum.

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377376

was the splicing mutation. Studies have shown that carrierfemales of expansions are asymptomatic, whereas carrierfemales of exon 1-4 truncations and homeodomain muta-tions are often symptomatic. This is suggestive of loss of ARXfunction secondary to these mutations [36].

Movement disorders have been reported with severalARX mutations. Examples include hand dystonia in Part-ington syndrome resulting from expansion of the secondpolyA tract, status dystonicus in 3 of 115 males withinfantile spasms and expansions of the first polyA tract, andmild dystonia in familial cases of syndromic and non-syndromic X-linked developmental delay in associationwith the common 24-bp duplication of the second polyAtract [26-28,37,38]. Four patients with expansions of thefirst polyA tract in our cohort had abnormal movements,including one who had PAID syndrome, a poorly under-stood phenomenon of episodic central dysautonomia con-sisting of hyperthermia, hypertension, tachycardia, intensehyperhidrosis, and dystonia typically seen in associationwith various cerebral insults [32,39] (Table 2). To our

knowledge, this is the first report of PAID syndromewith anARX mutation.

The clinical phenotypes of both ARX- and CDKL5-positivemales appear similar with a severe neurodevelopmentalcourse characterized by early-onset refractory epilepsy(infantile spasms being the most common seizure type),severe global developmental delay, severe tone abnormal-ities, and a poor neurocognitive outcome. As would be ex-pected with the seizure types these patients presentedwith, the EEG abnormality frequently observed was a burstsuppression pattern evolving into hypsarrhythmia. We didnot appreciate any features allowing clinical distinction tobe made between ARX and CDKL5 in boys in our cohort.

In conclusion, our findings describe the clinical featuresof eight males with CDKL5 mutations and report severalnovel ARX mutations. Mutations in either the CDKL5 or ARXgenes accounted for 8.7% in our study cohort. Given ascer-tainment bias, and that it is unknown if the boys in thiscohort were screened for other known genetic etiologies ofearly onset epilepsy, it is not possible to estimate the true

G.M. Mirzaa et al. / Pediatric Neurology 48 (2013) 367e377 377

frequencies of CDKL5 and ARX mutations in all boys withthis phenotype. However, our data support other large-scalestudies suggesting that both CDKL5 and ARX play animportant role in the pathogenesis of severe infantile-onsetepilepsy in boys [25,40].

We extend our thanks to the families and their physicians for their contribution tothis study.

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