JAMANeurology | OriginalInvestigation ......ASD, stereotypies, and hand-wringing Normal at 9 mo At...

Neurodevelopmental Disorders Caused by De Novo Variantsin KCNB1 Genotypes and PhenotypesCarolien G. F. de Kovel, PhD; Steffen Syrbe, MD, PhD; Eva H. Brilstra, MD, PhD; Nienke Verbeek, MD, PhD;Bronwyn Kerr, PhD; Holly Dubbs, MD; Allan Bayat, MD, PhD; Sonal Desai, MGC, CGC; Sakkubai Naidu, MD;Siddharth Srivastava, MD; Hande Cagaylan, MD; Uluc Yis, MD, PhD; Carol Saunders, PhD; Martin Rook, PhD;Susanna Plugge, MSc; Hiltrud Muhle, MD; Zaid Afawi, MD, PhD; Karl-Martin Klein, MD, PhD;Vijayakumar Jayaraman, MSc; Ramakrishnan Rajagopalan, PhD; Ethan Goldberg, MD, PhD; Eric Marsh, MD, PhD;Sudha Kessler, MD, MSCE; Christina Bergqvist, MD; Laura K. Conlin, PhD; Bryan L. Krok, PhD;Isabelle Thiffault, PhD; Manuela Pendziwiat, MSc; Ingo Helbig, MD; Tilman Polster, MD, PhD;Ingo Borggraefe, MD, PhD; Johannes R. Lemke, MD, PhD; Marie-José van den Boogaardt, MD, PhD;Rikke S. Møller, MSc, PhD; Bobby P. C. Koeleman, PhD

IMPORTANCE Knowing the range of symptoms seen in patients with a missense orloss-of-function variant in KCNB1 and how these symptoms correlate with the type of variantwill help clinicians with diagnosis and prognosis when treating new patients.

OBJECTIVES To investigate the clinical spectrum associated with KCNB1 variants and thegenotype-phenotype correlations.

DESIGN, SETTING, AND PARTICIPANTS This study summarized the clinical and geneticinformation of patients with a presumed pathogenic variant in KCNB1. Patients wereidentified in research projects or during clinical testing. Information on patients frompreviously published articles was collected and authors contacted if feasible. All patientswere seen at a clinic at one of the participating institutes because of presumed geneticdisorder. They were tested in a clinical setting or included in a research project.

MAIN OUTCOMES AND MEASURES The genetic variant and its inheritance and informationon the patient's symptoms and characteristics in a predefined format. All variants wereidentified with massive parallel sequencing and confirmed with Sanger sequencing in thepatient. Absence of the variant in the parents could be confirmed with Sanger sequencingin all families except one.

RESULTS Of 26 patients (10 female, 15 male, 1 unknown; mean age at inclusion, 9.8 years; agerange, 2-32 years) with developmental delay, 20 (77%) carried a missense variant in the ionchannel domain of KCNB1, with a concentration of variants in region S5 to S6. Three variantsthat led to premature stops were located in the C-terminal and 3 in the ion channel domain.Twenty-one of 25 patients (84%) had seizures, with 9 patients (36%) starting with epilepticspasms between 3 and 18 months of age. All patients had developmental delay, with 17 (65%)experiencing severe developmental delay; 14 (82%) with severe delay had behavioralproblems. The developmental delay was milder in 4 of 6 patients with stop variants and ina patient with a variant in the S2 transmembrane element rather than the S4 to S6 region.

CONCLUSIONS AND RELEVANCE De novo KCNB1 missense variants in the ion channel domainand loss-of-function variants in this domain and the C-terminal likely cause neurodevelop-mental disorders with or without seizures. Patients with presumed pathogenic variants inKCNB1 have a variable phenotype. However, the type and position of the variants in theprotein are (imperfectly) correlated with the severity of the disorder.

JAMA Neurol. 2017;74(10):1228-1236. doi:10.1001/jamaneurol.2017.1714Published online August 14, 2017.

Author Affiliations: Authoraffiliations are listed at the end of thisarticle.

Corresponding Author: Bobby P. C.Koeleman, PhD, Department ofGenetics, University Medical CenterUtrecht, PO Box 85500, 3508 GAUtrecht, the Netherlands([email protected]).

Research

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E arly infantile epileptic encephalopathies (EEs) form agroup of disorders that are characterized by epileptic sei-zures starting during the first year of life. Patients havedevelopmental delay (DD) or even regression to which the epi-leptic discharges are presumed to contribute. The seizures areoften unresponsive to treatment. Many of those epilepsies havea genetic disposition.1

In the past few years, several new genes have been iden-tified that can cause EE when mutated. One of the more re-cently discovered genes is KCNB1 (potassium voltage-gatedchannel subfamily B member 1 or delayed rectifier potassiumchannel 1 [DRK1]) (OMIM 616056), which acts in a dominantmanner. Variants are described by Torkamani et al,2 who iden-tified a de novo KCNB1 variant when exome sequencing a fam-ily with a child with EE. They subsequently found 2 other in-dependent patients with EE and KCNB1 variants. One of thosepatients had earlier been described in the Epi4K effort3 as hav-ing Lennox-Gastaut syndrome. Subsequently, more patientswith variants in KCNB1 were described.4-10

The KCNB1 gene encodes the Kv2.1 pore-forming, voltage-sensing α-subunit of a delayed rectifier potassium channel. It isexpressed in various neuronal cells in the brain.11 In Ensembl(GRCh38.p3), only 1 transcript is known (ENST00000371741).It consists of only 2 exons and contains 4 known domains(Figure): the T1 domain, which is involved in multimeriza-tion; the ion_trans domain, which contains the transmem-brane elements; and 2 intracellular Kv2 channel–specific do-mains (Pfam; http://smart.embl-heidelberg.de/).12,13 Theprotein has 6 transmembrane elements in the ion_trans do-main; the first 4 form the voltage-sensor domain, with S4 beingthe actual voltage sensor, whereas S5 and S6 form the poredomain of the protein. The extracellular loop between S5and S6 contains the selectivity filter (TVGYG amino acids;UNIPROT.org). This structure is similar to other voltage-gated potassium channels, such as KCNQ2 (OMIM 602235) and

KCNQ3 (OMIM 602232).14 The protein forms homotetramersand heterotetramers with various other voltage-gated potas-sium channel proteins, such as KCNB2 (OMIM 607738), andproteins from the KCNG (Kv6), KCNE, and KCNS (Kv9)families.15-17

MethodsIn a recent screen of 358 children with EEs, we identified 3 novelKCNB1 variants (approximately 1%), all of which were foundto be de novo.18 We subsequently collected data from 13 ad-ditional patients from research and diagnostic sources whowere reported to carry a de novo novel KCNB1 variant not pres-ent in public databases. In this article, we describe the phe-notypic and genetic characteristics of these patients. In addi-tion, we collected more information about 10 patients who weredescribed previously. For 3 of the 10 patients in previously pub-lished articles, the authors have provided updated informa-tion for the current article. We present the available informa-tion on 26 patients for a broad overview of the phenotypic andgenetic variability and similarities. Statistical tests were per-formed using χ2 2 × 2 contingency tables; the significance ofthe enrichment of de novo mutation was calculated using a

Key PointsQuestion How does the phenotype of patients with a KCNB1mutation correlate with the type of mutation?

Findings This study found that patients with a de novo mutationin KCNB1 present a variable phenotype of neurodevelopmentaldisorder.

Meaning De novo mutations in KCNB1 are associated with avariable neurodevelopmental disorder.

Figure. Distribution of Pathogenic Variants in KCNB1

Extracellular i ii iii iv v vi

Selectivity filter

Cytoplasmic

Milder

Developmental delay

No seizures

430

220

160

Pathogenic missense variantPathogenic LoF variantBTBKv2-specific domain

850

Schematic view of the Kv2.1 (KCNB1) protein in the membrane, showing pathogenic missense and loss-of-function (LoF) variants. BTB indicates broad-complex,tramtrack, and bric-brac domain.

Neurodevelopmental Disorders Caused by KCNB1 De Novo Variants Original Investigation Research

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http://omim.org/entry/616056http://smart.embl-heidelberg.de/http://omim.org/entry/602235http://omim.org/entry/602232http://omim.org/entry/607738http://www.jamaneurology.com/?utm_campaign=articlePDF%26utm_medium=articlePDFlink%26utm_source=articlePDF%26utm_content=jamaneurol.2017.1714

χ2 goodness-of-fit test. A 2-sided χ2 test with P < .05 determinedstatistical significance. Collection of data procedures agreedwith the local ethics guidelines at the different institutes.

ResultsDescription of the VariantsWe identified 16 patients with KCNB1 variants who were pre-viously not described and collected information about 10 pre-viously described patients (10 female, 15 male, 1 unknown;mean age at inclusion, 9.8 years; age range, 2-32 years).2-9 Inthese 26 patients, we identified 23 different variants; 3 vari-ants were seen twice each. Twenty patients (77%) carried mis-sense variants, and 6 (23%) carried nonsense or frameshift vari-ants leading to a premature termination codon (Table 1). Allthe missense variants are located within the Pfam ion_trans do-main (ie, the 6 transmembrane regions and their extracellu-lar and intracellular connecting loops) (Figure). Two variantsin 4 patients are gating charge residues in voltage sensor S4.19

Three variants are in the selectivity filter between S5 and S6.Three of the loss-of-function (LoF) variants are in the first Kv2channel–specific domain in the intracellular C-terminus of theprotein. The other 3 LoF variants are in the S1 to S2 linker andin the S5 to S6 P-loop. The gene has 2 exons, and all variantsare in the second (last) exon. All nonsense or frameshift vari-ants are expected to result in a truncated protein rather thannonsense-mediated decay because they are located in the ter-minal part of the gene. No variants were detected in the cyto-plasmic N-terminal part of the gene. All missense variants al-ter strongly conserved amino acids and are predicted to behighly deleterious by SIFT (Sorting Intolerant From Toler-ant), Polyphen2, and Combined Annotation Dependent Deple-tion (CADD) scores.20-22 The lowest CADD score was 23.3. Inall individuals except one (patient 18), for whom parentalsamples were not available for segregation analysis, the vari-ant was confirmed to be de novo. The variant not confirmedto be de novo, R312H, was seen as de novo in another patientand was absent from the Exome Aggregation ConsortiumExAC Browser (http://exac.broadinstitute.org/)23; thus, weinterpreted it to be pathogenic and included patient 18 in ourseries. An overview of characteristics of the patients and theirvariants is given in Table 1.

Description of the PatientsThe observed phenotypes in patients are summarized in Table 2and described in detail in this section. All 26 patients pre-sented with primary DD in the first year of life, preceding on-set of epilepsy. First signs of DD were unspecific, with mus-cular hypotonia or hypertonia being recurrently reported(11 of 23 individuals [48%] with available neurologic exami-nation findings). Twenty-one of 25 patients (84%) had intrac-table epilepsy, with seizure onset in infancy or early child-hood (median age at onset, 12 months; range, 3 months to4 years). Epileptic spasms alone or as part of West syndromewere observed in 9 patients (36%), with a median onset of sei-zures of 12 months (range, 3-18 months). The only patient whodeveloped spasms before 6 months of age was patient 10, who

carried the prominent variant G381R within the selectivity fil-ter of the P-loop.7 In most patients with epilepsy, the semio-logic features developed over time into multiple seizure types,including tonic, focal-clonic, myoclonic, and atypical ab-sences. Three patients did not have epileptic seizures (pa-tients 1, 3, and 24), 1 reported only infantile spasms (patient2), and no data were available for 1 (patient 8). The patients withLoF variants in the C-terminal region had no seizures or hadinfantile spasms only; in addition, 1 patient with a variant inthe S1 to S2 linker reported no seizures. However, one patient(patient 3) with LoF variant in the C-terminus reported se-vere DD. Of the patients with LoF variants, 2 were reported tohave severe DD and 4 had moderate or moderate-severe DD(1 unspecified). In contrast, the only patient with a missensevariant for whom moderate DD was reported was patient 23,who had a missense variant in the S1 to S2 linker. In contrast,all patients with severe epilepsy harbored variants withinthe voltage sensor (S4) and the pore-forming domains (S5,P-loop, and S6) of KCNB1. These observations suggest an as-sociation between location of mutation and phenotype andseverity of disease.

In general, the epilepsy in most patients was highly re-fractory to diverse and multiple antiepileptic drugs. Anecdot-ally, partial efficacy has been reported for ketogenic diet, whichresulted in a significant decrease in seizure burden in 2 pa-tients (patients 14 and 18) but seemed ineffective in 2 others(patients 11 and 20). Improvement was also observed in 1 pa-tient who received steroid pulses (patient 17), whereas intra-muscular adrenocorticotropic hormone therapy for epilepticspasms was reported as not being of value in 3 patients (pa-tients 2, 11, and 14). Vagus nerve stimulation and corpus cal-losotomy surgery were performed in 1 patient (patient 9) with-out substantial improvement of epilepsy. Brain biopsy in thispatient revealed minimal cortical dysplasia consisting of ex-cessive but not dysplastic neurons, a finding that argues againsta role of KCNB1 in disorders of neuronal migration.

Electroencephalographic (EEG) data were reported for 21patients. The EEG displayed features of EE in 20 of 21 pa-tients (95%), including hypsarrhythmia and general slowingof background activity together with multifocal spikes andpolyspikes, and 9 of 21 patients (43%) with abnormal EEG re-sults had a combination of focal and generalized epileptic dis-charges, with 1 being mainly focal. Five patients (24%) had con-tinuous spike and wave during slow-wave sleep (CSWS) orelectrical status epilepticus during slow-wave sleep symp-toms. Patients 14, 18, and 19 had CSWS on EEG, and patients21 and 25 had a history of electrical status epilepticus duringslow-wave sleep.

For 13 patients, more detailed information on motor func-tion was available. Motor performance was poor, with lim-ited ability to walk freely and significant signs of upper motorneuron disease (brisk reflexes, contractures, clonus, and posi-tive plantar response) or ataxia in 10 patients (77%), all withmissense variants within vital channel domains (S4-S6). Theremaining 3 patients had normal neurologic examination find-ings and carried variants in S2 or the C-terminal.

For 14 of 17 patients (82%) with available data, behavioralproblems were reported. Autism spectrum disorder was

Research Original Investigation Neurodevelopmental Disorders Caused by KCNB1 De Novo Variants

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Tabl

e1.

Clin

ical

Phen

otyp

esof

Prev

ious

lyD

escr

ibed

and

New

Patie

ntsW

ithH

eter

ozyg

ousD

eN

ovo

and

Puta

tive

De

Nov

oKC

NB1

Mut

atio

ns

Patie

ntN

o.M

utat

ion

(hg1

9;N

M_0

0497

5.2)

Chan

nel

Dom

ain

Age,

y/Se

x/Ag

eat

Seiz

ure

Ons

etEp

ileps

yDD

Beha

vior

MRI

Find

ings

EEG

Find

ings

Neu

rolo

gic

Exam

inat

ion

Find

ings

Oth

er

1ch

r20:

4799

0350

G>Ap

.Arg

583*

C-te

rmin

al11

/F/N

ANo

seiz

ures

Mod

erat

e-se

vere

(abl

eto

spea

kan

dw

alk)

Seve

rebe

havi

oral

prob

lem

s,AD

HD,a

ndAS

D

Norm

alat

age

10y

Notp

erfo

rmed

NANA

2ch

r20:

4799

0498

G>Tp

.Y53

3*C-

term

inal

32/M

/5m

oIn

fant

ilesp

asm

sUn

spec

ified

,wal

king

at2

yNA

cCT

norm

alHy

psar

rhyt

hmia

NANA

3ch

r20:

4799

0593

T>TA

p.K5

02fs

C-te

rmin

al10

/M/N

ANo

seiz

ures

Seve

reSe

vere

beha

vior

alpr

oble

ms,

ADHD

,and

ASD

Chia

ri1

mal

form

atio

nat

2y

Notd

one

NANA

4ch

r20:

4799

0849

G>Cp

.F41

6LS6

18/F

/42

mo

Toni

c,m

yocl

onic

,and

atyp

ical

abse

nces

Seve

reNA

Periv

entr

icul

arw

hite

mat

ter

abno

rmal

ities

Slow

back

grou

ndac

tivity

and

shar

p-sl

ow-w

ave

disc

harg

esat

2-2.

5Hz

alte

rnat

ing

with

rapi

ddi

scha

rges

Hype

rton

ia,

dyst

onia

Gast

rotu

befe

edin

g

5ch

r20:

4799

0849

G>Cp

.F41

6LS6

15/F

/14

mo

Toni

c-cl

onic

,sta

tus

epile

ptic

us,a

ndst

imul

usev

oked

Seve

reSt

ereo

typi

esan

dha

nd-w

ringi

ng

NAAt

14y,

mul

tifoc

alan

dge

nera

lized

ETP

Atax

iaSc

olio

sis

6ch

r20:

4799

0896

C>Tp

.G40

1RS6

4/M

/17

mo

Clon

ican

dfo

calc

loni

cSe

vere

:no

wor

dsan

dun

able

tosi

tby

4y

NANo

rmal

at9

mo

and

prog

ress

ive

brai

nat

roph

yat

1y

6m

oan

d2

y2

mo

Diff

use

poly

spik

esan

dw

aves

with

inte

rmitt

ent

mul

tifoc

alsp

ikes

at1

y6

mo

Chor

eic

mov

emen

ts,

myo

clon

ia

NA

7ch

r20:

4799

0904

C>Ap

.C39

7FS6

22/M

/10

mo

Toni

c-cl

onic

and

mul

tifoc

alSe

vere

NAAt

2y,

periv

entr

icul

arle

ukom

alac

iaan

dla

tera

lve

ntric

les

NASp

astic

tetr

apar

esis

NA

8ch

r20:

4799

0924

T>Gp

.K39

1NS5

-S6

linke

r(P

-loo

p)NA

NANA

NANA

NAAb

norm

alNA

9ch

r20:

4799

0944

C>Ap

.P38

5TS5

-S6

linke

r(P

-loo

p)17

/M/1

3m

oFo

cald

ysco

gniti

ve,d

rop

atta

cks,

toni

c,no

ctur

nal

toni

c,cl

onic

,and

myo

clon

us

Seve

re:w

alks

only

with

assi

stan

ce,a

taxi

cga

it,an

dno

nver

bal

ASD,

ster

eoty

pies

,an

dha

nd-w

ringi

ng

Norm

alat

6y

and

mild

cere

bella

rvo

lum

elo

ssat

11y

Mul

tifoc

alsh

arps

and

slow

ing

and

diso

rgan

izat

ion

ofth

eba

ckgr

ound

activ

ityat

13y

and

slow

diso

rgan

ized

back

grou

ndw

ithne

arco

ntin

uous

epile

ptifo

rmac

tivity

at16

y

Hype

rton

ia,

atax

ia,t

rem

or,

and

spas

ticity

Scol

iosi

sand

gast

rotu

befe

edin

g

10ch

r20:

4799

0956

C>Tp

.G38

1RS5

-S6

linke

r(s

elec

tivity

filte

r)

8/M

/3m

oSt

artle

epis

odes

at3

mo

evol

ving

toIS

(40/

d),

stim

ulus

-ind

uced

toni

cse

izur

es,f

ocal

dysc

ogni

tive,

and

foca

lm

otor

seiz

ures

Seve

reNA

NAAt

7y:

mul

tifoc

al,

phot

osen

sitiv

e,an

dsl

owHy

pert

onia

,sp

astic

ityPr

ogre

ssiv

em

icro

ceph

aly

(con

tinue

d)





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Tabl

e1.

Clin

ical

Phen

otyp

esof

Prev

ious

lyD

escr

ibed

and

New

Patie

ntsW

ithH

eter

ozyg

ousD

eN

ovo

and

Puta

tive

De

Nov

oKC

NB1

Mut

atio

ns(c

ontin

ued)

Patie

ntN

o.M

utat

ion

(hg1

9;N

M_0

0497

5.2)

Chan

nel

Dom

ain

Age,

y/Se

x/Ag

eat

Seiz

ure

Ons

etEp

ileps

yDD

Beha

vior

MRI

Find

ings

EEG

Find

ings

Neu

rolo

gic

Exam

inat

ion

Find

ings

Oth

er

11ch

r20:

4799

0962

C>Tp

.G37

9RS5

-S6

linke

r(s

elec

tivity

filte

r)

9/M

/8m

oIS

and

late

rmul

tiple

seiz

ure

type

s,in

clud

ing

foca

ldys

cogn

itive

,at

onic

,and

gene

raliz

edto

nic-

clon

ic

Seve

re:m

otor

and

lang

uage

ASD,

ster

eoty

pies

,an

dha

nd-w

ringi

ng

Norm

alat

9m

oAt

15m

o,m

odifi

edhy

psar

rhyt

hmia

Trun

cal

hypo

toni

aan

dbr

isk

refle

xes

Stra

bism

us

12ch

r20:

4799

0964

T>Cp

.V37

8AS5

-S6

linke

r(s

elec

tivity

filte

r)

4/F/

13m

oM

yocl

onic

seiz

ure

follo

wed

bya

toni

cco

mpo

nent

,gen

eral

ized

toni

c-cl

onic

seiz

ure,

and

IS

Seve

re:n

osp

eech

,lim

ited

voca

lizat

ion;

at3

y,fu

nctio

ning

as5-

8m

o;by

3y,

able

tosi

tin

depe

nden

tlybu

tno

stan

ding

orw

alki

ng

NANo

rmal

Lack

ofa

post

erio

r-do

min

ant

rhyt

hm,g

ener

aliz

edsl

owin

g,ge

nera

lized

spik

es,p

olys

pike

sand

shar

pw

aves

,fre

quen

tbu

rsts

ofab

ortiv

efr

onta

l-do

min

ant

gene

raliz

edsp

ikes

,and

poly

spik

esan

dsh

arp

wav

edi

scha

rges

Hypo

toni

aCo

rtic

alvi

sual

impa

irmen

t

13ch

r20:

4799

0976

G>Ap

.T37

4IS5

-S6

linke

r(P

-loo

p)5/

F/6

mo

Toni

c-cl

onic

,ato

nic,

foca

ldys

cogn

itive

atyp

ical

abse

nce,

and

IS

Yes,

unsp

ecifi

edNA

Norm

alUn

spec

ified

Spas

ticity

NA

14ch

r20:

4799

0976

G>Ap

.T37

4IS5

-S6

linke

r(P

-loo

p)11

/F/1

3m

oIS

that

prog

ress

edto

Lenn

ox-G

asta

utsy

ndro

me;

curr

ently

,to

nic

and

abse

nce

seiz

ures

Seve

re:a

t7y

6m

o,co

uld

siti

ndep

ende

ntly

,wal

kw

ith2

hand

sass

iste

d,an

dco

mm

unic

ate

with

20si

gns

ASD,

ster

eoty

pies

,an

dha

nd-w

ringi

ng

T2-h

yper

inte

nsity

of periv

entr

icul

arw

hite

mat

tera

t9

mo

and

norm

alat

4y

At5

y,bi

late

ralC

SWS

with

spik

ean

dw

ave

com

plex

esw

ithsh

iftin

gas

ymm

etrie

s;at

10y,

near

lyco

ntin

uous

epile

ptifo

rmac

tivity

char

acte

rized

byin

depe

nden

tlef

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Tabl

e1.

Clin

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Phen

otyp

esof

Prev

ious

lyD

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vere

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tal

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nce

first

year

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per

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alat

8y

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4799

1181

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apyr

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ent

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4799

1415

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4799

1465

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with

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reported in 10 individuals, with different variants locatedthrough S4 to S6 and the C-terminus. In 4 patients, caregiversnoted specifically nonpurposeful hand-wringing stereoty-pies. In addition, attention-deficit/hyperactivity disorder orhyperactivity with inattention in 7 patients and temper tan-trums in 3 patients were mentioned.

Information on brain imaging was available from 23 of 26patients (22 with magnetic resonance imaging and 1 with com-puted tomography). Results were normal in 19 patients (83%)or revealed unspecific features after infancy, such as Arnold-Chiari malformation type 1 (patient 3), progressive supraten-torial brain atrophy (patient 6, G401R, with infantile epi-lepsy), cerebellar volume loss (patient 8, P385T, infantileepilepsy proceeding to Lennox-Gastaut syndrome), and whitematter abnormalities (patient 14, T374I, West syndrome pro-gressing to Lennox-Gastaut syndrome), corresponding to sei-zure burden in these patients.

DiscussionAll missense variants clustered within the ion_trans domain,in which few natural segregating variants are found. On theexome variants server with data from more than 6500 indi-viduals (http://evs.gs.washington.edu/EVS/), no coding variantswere found in the ion_trans domain. In the ExAC database withmore than 60 000 individuals, 9 coding variants were presentin this domain.

On the basis of the work of Samocha et al,24 we exploredthe frequency of missense variants in the ExAC database in thedifferent regions of the gene relative to the neutral expecta-tion to identify gene regions intolerant to missense variation.The ratio of missense to synonymous variants is 0.26 in the

ion_trans domain; 0.33 in the upstream region, including theT1 domain; and 2.14 in the downstream region, including theKv2 channel–specific domains. This ratio is expected to be 2.3for the whole gene after neutral expectation according toSamocha et al24; therefore, both the ion_trans domain and theupstream region of the gene are intolerant to missense vari-ants (P < 1 × 10−5). Missense variants in those regions thus havea high probability of being pathogenic.24 We assume that allmissense variants discussed in the present study are causal forthe neurodevelopmental disorder in the patient.

We also consider the LoF variants described in the presentstudy to be pathogenic. In the ExAC database, only 2 rare LoFvariants were found: p.R854* (flagged as dubious) andp.K776Qfs*23. These variants are located in the C-terminal partof the gene, closer to the terminal part than were the LoF vari-ants found in the patients and beyond the Kv2 channel–specific domains (Figure). Therefore, it can be expected thatthese variants, if they exist, do not affect normal function ofKv2.1. In contrast, the 3 C-terminal mutations observed in thepatients were in the Kv2.1-specific domain located more up-stream and therefore may be expected to have a functional ef-fect, for example, lead to differences in protein folding or affectformation of tetramers. All LoF variants in the patients were inthe last exon of the gene; thus, we expect that they will lead totruncation rather than nonsense-mediated decay.25 Because theprotein forms (hetero)tetramers, we expect that the effect of themissense and truncating variants is dominant negative unlessthe mutated protein is not incorporated in the membrane.

Because we consider all variants described in this article ascausal for the phenotype, the phenotypic range described islikely to be representative of variants in this gene. There maybe a bias, however, toward patients with epilepsy because ourinitial screen was conducted in a panel of patients with EE andmany colleagues in our network have a special interest in thisphenotype. Torkamani et al2 may also have searched only forpatients with seizures when following up their initial finding.The patients without seizures in this article were from exome-wide diagnostic screening of patients with (neuro)developmen-tal problems and from the Deciphering Developmental Disor-ders program in the United Kingdom, which also was a generalscreening of children with developmental disorders. As de-scribed in the Results section and as seen in Table 1, the rangeof symptoms in patients with variants in KCNB1 was diverse inmany aspects.

Genotype-Phenotype AssociationsMost missense variants were in the S5 to S6 linker, which formsthe pore region, particularly in or near the selectivity filter.Three patients had missense variants in one of the positivelycharged R-residues in voltage sensor S4. As described in theResults section, the distribution of variants in patients overthe domains is different from that in the general population.The variants that we found in the less constrained C-terminuswere LoF variants.

Missense variants within the voltage sensor domain or thepore region of the channel were found in patients with severeepilepsy and global DD, including intellectual disability and of-ten spasticity. Therefore, missense variants in the S4 to S6 re-

Table 2. Major Clinical Characteristics of the Patients Carryinga Presumed Pathogenic KCNB1 Mutation

PhenotypeNo. of Patients/Patients WithData Available (%)

Developmental delay 26/26 (100)

Intractable epilepsy 21/25 (84)

Epileptic spasms 9/25 (36)

EE-EEG 20/21 (95)

Focal-generalized discharges 9/21 (43)

CSWS or ESES 5/21 (24)

Poor motor function 10/13 (77)

Hypotonia 11/23 (48)

Ataxia 4/23 (17)

Abnormal behavior 14/17 (82)

ADHD 7/20 (35)

ASD 10/20 (50)

Hand-wringing 4/20 (20)

Temper tantrums 3/20 (15)

Abnormal brain imaging 4/23 (17)

Abbreviations: ADHD, attention-deficit/hyperactivity disorder; ASD, autismspectrum disorder; CSWS, continuous spike and wave during slow-wave sleep;EE, epileptic encephalopathy; EEG, electroencephalography; ESES, electricalstatus epilepticus during slow-wave sleep.





http://evs.gs.washington.edu/EVS/http://www.jamaneurology.com/?utm_campaign=articlePDF%26utm_medium=articlePDFlink%26utm_source=articlePDF%26utm_content=jamaneurol.2017.1714

gion seem to be correlated with a more severe prognosis. AnLoF variant close to S2 and one in the C-terminus were foundin patients with milder DD, who acquired walking and were ableto communicate. Two of the 3 LoF variants in the C-terminuswere in patients without seizures (at 11 and 32 years of age),whereas a patient with a missense variant in the S1 to S2 linker,close to S1, had no seizures at 4 years of age. The degree of in-tellectual disability in this patient was unspecified.9 The LoFvariant 15 (p.W369*) in the pore region was seen in a patientwith severe DD and epilepsy at the age of 5 years, whereas theLoF variant near that in LoF variant 16 (p.S363fs*) was foundin a patient at 2 years of age with moderate DD and focal sei-zures but no additional reported problems.

No pathogenic variants have been reported thus far in thehighly constrained N-terminal region. A handful of rare mis-sense variants were described for this region in the ExACcontrol population. It remains to be determined whether mis-sense or LoF variants in this region also lead to neurodevel-opmental disorders. The strong constraints on variation in thisregion suggest that many of the possible variants are delete-rious or even fatal. Finally, patients carrying missense muta-tions more often had abnormal neurologic examination re-sults (17 of 19 available [89%]) compared with 1 of 3 LoF carriers(33%) (P = .02, χ2 test for independence). Similarly, normalmagnetic resonance imaging results were reported for all 6 pa-tients carrying an LoF mutation. Overall, patients with KCNB1mutations have a variable phenotype, a finding highlighted bythe observation that even patients carrying the same muta-tion have differences in phenotype.

Functional studies2,8,9 have been performed for several ofthe described variants. In general, the electrophysiologic char-acteristics of the different variants have a variety of func-tional aberrations, such as loss of voltage sensing (p.R306C),reduced current (p.G410R), and suppression of repetitive fir-ing (p.R306C, p.G410R). Of interest, all 3 missense mutationsin the ion-selectivity filter domain have been studied and ren-der Kv2.1, a nonselective cation channel that is not voltage ac-tivated. However, the general loss of channel function anddominant-negative effects seen in most studied variants arein support of the presumed pathogenicity and link to the EEphenotype. Finally, a Kv2.1−/− mouse model was not prone tospontaneous seizures but exhibited hypocampal hyperexcit-ability and epileptiform activity on stimulation.26 Together,these observations further support our conclusion that theKCNB1 variants described here are likely to be pathogenic.

Comparison to KCNQ2KCNB1 forms a gene family with KCNB2, with which it can formheterotetramers. To date, KCNB2 has not been implicated inmonogenic disorders. KCNB1 has a similar structure as otherpotassium channels involved in severe epilepsy, such asKCNQ2. Pathogenic mutations leading to EE in the other po-tassium channels are found in the transmembrane regions. Thisfinding is similar to the mutations in KCNB1 described in thisstudy. Furthermore, 2 of the pathogenic variants found inKCNQ227 were homologous to presumed pathogenic variantsdescribed in this article: the charged residue in the voltagesensor (p.R306C; variant 20) and missense variants 13 and14 (p.T374I) in the pore region. This finding underlines theimportance of these residues for the functioning of theproteins.

LimitationsOur study also has some limitations. Our patients may havebeen selected for certain phenotypes, in particular for epi-lepsy. Our focus on EE may have excluded cases with muchmilder phenotypes. Furthermore, the number of observa-tions remain small, and description of additional cases willfurther elucidate the full clinical spectrum associated withmutations in KCNB1.

ConclusionsMissense and LoF variants in the ion_trans domain of KCNB1 andLoF variants in the C-terminal region can cause neurodevelop-mentaldisorderwithorwithoutepilepticseizures.Missensevari-ants in the C-terminus are less likely to cause developmental dis-orders, whereas the consequences of variants in the N-terminusis not clear. The more severely affected patients consistently har-bored missense variants within vital channel domains of KCNB1and experienced late-onset epileptic spasms that evolved totreatment-refractive epilepsy and severe global DD. The effectof LoF variants was less clear, although a tendency existed to-ward less severe phenotypes. However, more outcome data onpatients with LoF variants are needed to support clinical predic-tions and counseling of such patients. There was a marked ten-dency to develop spastic paraparesis or tetraparesis and ataxiaas well as autistic spectrum disorder. Finally, patients carryingmissense mutations were more likely to have neurologic andmagnetic resonance imaging abnormalities.

ARTICLE INFORMATION

Accepted for Publication: May 18, 2017.

Published Online: August 14, 2017.doi:10.1001/jamaneurol.2017.1714

Author Affiliations: Department of Genetics,University Medical Center Utrecht, Utrecht,the Netherlands (de Kovel, Brilstra, Verbeek,van den Boogaardt, Koeleman); Department ofLanguage and Genetics, Max Planck Institute forPsycholinguistics, Nijmegen, the Netherlands(de Kovel); Division of Child Neurology andInherited Metabolic Diseases, Department ofGeneral Pediatrics, Center for Pediatrics and

Adolescent Medicine, University HospitalHeidelberg, Heidelberg, Germany (Syrbe); Instituteof Evolution, Systems and Genomics, Faculty ofMedical and Human Sciences, University ofManchester, Manchester, England (Kerr);Manchester Centre For Genomic Medicine, CentralManchester University Hospitals National HealthService Foundation Trust, Manchester, England(Kerr); Manchester Academic Health ScienceCentre, Manchester, England (Kerr); Division ofNeurology, The Children's Hospital of Philadelphia,Philadelphia, Pennsylvania (Dubbs, Goldberg,Marsh, Kessler, Bergqvist, Helbig); Department ofPediatrics, University Hospital of Hvidovre,

Copenhagen, Denmark (Bayat); Department ofNeurogenetics, Kennedy Krieger Institute,Baltimore, Maryland (Desai); Department ofNeurology and Pediatrics, Johns Hopkins School ofMedicine, Baltimore, Maryland (Naidu); HugoMoser Research Institute, Kennedy KriegerInstitute, Baltimore, Maryland (Naidu); Departmentof Neurology, Boston Children's Hospital, Boston,Massachusetts (Srivastava); Department ofMolecular Biology and Genetics, BogaziçiUniversity, Istanbul, Turkey (Cagaylan); Division ofChild Neurology, Department of Pediatrics, Schoolof Medicine, Dokuz Eylül University, İzmir, Turkey(Yis); Center for Pediatric Genomic Medicine,





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Department of Pathology and Laboratory Medicine,Children's Mercy Hospital, Kansas City, Missouri(Saunders, Thiffault); Department of Pediatrics,Children's Mercy Hospital, Kansas City, Missouri(Saunders, Thiffault); Pediatric Pathology andLaboratory Medicine, University of Missouri–KansasCity School of Medicine, Kansas City (Saunders);Department of Medical Physiology, UniversityMedical Center Utrecht, Utrecht, the Netherlands.(Rook); Department of Biomedical Sciences,University Medical Center Utrecht, Utrecht,the Netherlands (Plugge); Department ofNeuropediatrics, University Medical CenterSchleswig-Holstein, Christian Albrechts University,Kiel, Germany (Muhle, Pendziwiat); Department ofPhysiology and Pharmacology, Tel Aviv UniversityMedical School, Ramat Aviv, Israel (Afawi);Department of Neurology, Epilepsy CenterFrankfurt Rhine-Main, Center of Neurology andNeurosurgery, University Hospital, Goethe-University Frankfurt, Frankfurt, Germany (Klein);Division of Genomic Diagnostics, Children’s Hospitalof Philadelphia, Philadelphia, Pennsylvania(Jayaraman, Rajagopalan, Conlin, Krok); UniversityMedical Center Schleswig-Holstein, ChristianAlbrechts University, Kiel, Germany (Helbig);Epilepsiezentrum Bethel, Krankenhaus Mara,Kinderepileptologie, Bielefeld, Germany (Polster);Department of Pediatric Neurology, DevelopmentalMedicine and Social Pediatrics Dr. von Hauner’sChildren’s Hospital, University of Munich, Munich,Germany (Borggraefe); Institute of HumanGenetics, University of Leipzig Hospitals and Clinics,Leipzig, Germany (Lemke); Danish Epilepsy Centre,Dianalund, Denmark (Møller); Institute for RegionalHealth Services, University of Southern Denmark,Odense, Denmark (Møller).

Author Contributions: Dr de Kovel had full accessto all the data in the study and takes responsibilityfor the integrity of the data and the accuracy of thedata analysis.Study concept and design: de Kovel, Syrbe, Yis,Koeleman.Acquisition, analysis, or interpretation of data:de Kovel, Syrbe, Brilstra, Verbeek, Kerr, Dubbs,Bayat, Desai, Naidu, Srivastava, Cagaylan, Saunders,Rook, Plugge, Muhle, Afawi, Klein, Rajagopalan,Jayaraman, Goldberg, Marsh, Kessler, Bergqvist,Conlin, Krok, Thiffault, Pendziwiat, Helbig, Polster,Borggraefe, Lemke, van den Boogaardt, Møller,Koeleman.Drafting of the manuscript: de Kovel, Kerr, Plugge,Afawi, Jayaraman, Helbig, Koeleman.Critical revision of the manuscript for importantintellectual content: de Kovel, Syrbe, Brilstra,Verbeek, Dubbs, Bayat, Desai, Naidu, Srivastava,Cagaylan, Yis, Saunders, Rook, Muhle, Klein,Rajagopalan, Goldberg, Marsh, Kessler, Bergqvist,Conlin, Krok, Thiffault, Pendziwiat, Helbig, Polster,Borggraefe, Lemke, van den Boogaardt, Møller,Koeleman.Statistical analysis: de Kovel, Syrbe, Afawi.Obtained funding: Helbig, Koeleman.Administrative, technical, or material support:de Kovel, Syrbe, Brilstra, Verbeek, Dubbs, Bayat,Desai, Naidu, Cagaylan, Yis, Rajagopalan, Goldberg,Kessler, Bergqvist, Conlin, Thiffault, Helbig, Lemke,van den Boogaardt, Koeleman.Study supervision: Afawi, Kessler, Helbig, Koeleman.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was funded by grantG.A.136.11.N from the Dutch Epilepsy Foundation

(Dr Koeleman) and Euroepinomics-RES (Dr Helbig).Dr Helbig was supported by grants HE 5415/5-1 andHE 5415/6-1 from the German Research Foundationand grant U01-HG006546 from the NationalHuman Genome Research Institute of the NationalInstitutes of Health. The Wellcome Trust supportedthe data generation of the Decipher project.

Role of the Funder/Sponsor: The sponsors had norole in the design and conduct of the study;collection, management, analysis, andinterpretation of the data; preparation, review, orapproval of the manuscript; and decision to submitthe manuscript for publication.

Additional Contributions: We acknowledge thecontributions of Nancy Spinner and Ian Krantz, whowere not compensated for their help. This studymakes use of data generated by the Database ofGenomic Variation and Phenotype in Humans UsingEnsembl Resources (DECIPHER) community. A fulllist of centers that contributed to the generation ofthe data is available at http://decipher.sanger.ac.ukand via email from [email protected]. Wethank the patients and their parents forcontributing to this research and all clinicians whoprovided samples from their patients. We thank theExome Aggregation Consortium and the groupsthat provided exome variant data for comparison.A full list of contributing groups can be found athttp://exac.broadinstitute.org/about.

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