Compound and Digenic Heterozygosity Predicts Life

DOI: 10.1161/CIRCGENETICS.113.000288
Compound and Digenic Heterozygosity Predicts Life-Time Arrhythmic
Outcome and Sudden Cardiac Death in Desmosomal Gene-Related
Arrhythmogenic Right Ventricular Cardiomyopathy
Running title: Rigato et al.; Genetic determinants of arrhythmic outcome in ARVC
Ilaria Rigato, MD, PhD1; Barbara Bauce, MD, PhD1; Alessandra Rampazzo, BSc, PhD2;
Alessandro Zorzi, MD1; Kalliopi Pilichou, PhD1; Elisa Mazzotti, MD, PhD1; Federico Migliore,
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
MD, PhD1; Martina Perazzolo Marra, MD, PhD1; Alessandra Lorenzon,, BSc,, PhD2;
Andr
An
drea
dr
ea N
Nava,
ava,
av
a, M
MD
D1;
Marzia De Bortoli, BSc, PhD2; Martina Calore, BSc, PhD2; Andrea
Luciano Daliento, MD1; Dario Gregori, MA, PhD1; Sabino Iliceto, MD
D1; Gaetano
Gaet
Ga
etan
et
anoo Th
an
Thie
Thiene,
iene
ie
n , MD1;
ne
Cristina
Cri
ristin
in
na Basso, MD, PhD1; Domenico
Dom
men co Corrado, MD
meni
MD,
D, PhD1
1
Department
p rtme
par
ment
nt ooff Ca
Card
Cardiac,
dia
iac,
c, T
Thoracic,
hooraaci
cic,
c aand
n V
nd
Vascular
ascu
as
culaar Sc
cu
Scie
Sciences;
ienc
ie
nces
nc
e ; 2De
es
Department
D
part
pa
r me
rt
ment
ntt ooff Bi
Biol
Biology,
olog
ol
o y,
og
University
U iv
Un
iversity
y of Padua,
a, Padua,
Paduaa, Italy
I al
It
ay
Correspondence:
Domenico Corrado, MD, PhD
Inherited Arrhythmogenic Cardiomyopathy Unit
Department of Cardiac Thoracic and Vascular Sciences
University of Padova, Italy
Via N. Giustiniani 2
35121 Padova
Italy
Tel +39 049 8212458
Fax +39 049 8212309
E-mail: [email protected]
Journal Subject Codes: [109] Clinical genetics
1
DOI: 10.1161/CIRCGENETICS.113.000288
Abstract:
Background - Mutations in genes encoding for desmosomal (DS) proteins are the most common
cause of ARVC. We assessed the value of genotype for prediction of life-time major arrhythmic
events and sudden cardiac death (SCD) in DS-gene related ARVC.
Methods and Results - The overall study population included 134 DS-gene mutation carriers [68
males; median age 36 years (22-52)] from 44 consecutive ARVC families undergoing
comprehensive genetic screening. The probability of experiencing a first major arrhythmic event
or SCD during life-time was determined by using date of birth as start point for the time to event
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
analysis and was stratified by gender, DS-genes, mutation types and genotype
yp complexity
p
y ((single
versus multiple mutations). One-hundred-thirteen patients (84%) carried
ed a single
sin
ingl
glee DS-gene
gl
DS--gene
DS
n
mutation in DSP (n=44;39%); PKP-2 (n=38;34%); DSG-2 (n=30;26%)
DSC-2
%) andd DS
DSC
C-22
(n=1;1%),while
compound
w le 221
while
1 ppatients
att ntts (16%) had a complex ggenotype
atient
enotype with comp
en
mpoound heterozygosity in 7
mp
and digenicc he
heterozygosity
median
observation
years,
eterozygosi
sity
si
ty inn 14.
14 Over
Ov a m
ed
diaan obse
servat
ati
tionn pe
pperiod
r odd ooff 399 ((22-52)
ri
2 -52
22
52)) ye
earrs, 222
patients (16%)
families
6%)
6%)
% fr
ffrom
om 20
20 ddifferent
iffferen
en
nt fa
amilies
es had
had arrhythmic
arrrhhythm
hmic
icc events
eveents such
suuch as
as SCD
S D (n=1),
SC
(n=
(n
=1),, aborted
ab
borrtee
SCD due too ventricular fibr
fibrillation
briillaation
br
onn ((n=6),
n=6),
) ssustained
),
u ta
us
tained ventricular
ventricu
ve
ula
l r ta
ttachycardia
ach
c yc
y ar
ardi
d a (n=14)
( =14) and
(n
appropriatee defibrillator interventi
intervention
Multiple
DS-gene
mutations
were
ion ((n=1).
n=1)). Mu
M
ltip
i le D
S-ggene muta
tions
i
andd male ge
ggender
nder w
independent
life-time
Ratio
(95%CI=1.54ntt predictors
pred
pr
edic
ed
icto
ic
tors
rs ooff li
life
fe-ti
fe
time
ti
me arrhythmic
arr
rrhy
hyth
hy
hmi
micc events
even
ev
ents
ts wi
with
th
h a Hazard
H za
Ha
zard
rdd Ra
R
tioo off 3.
ti
3.71
7 (95%CI=
71
(95
95%C
%CI=
%C
I
I=
8.92;p=0.003) and 2.76 (95%CI=1.19-6.41;p=0.02), respectively.
Conclusions - Compound/digenic heterozygosity was identified in 16% of ARVC-causing DSgene mutation carriers and was a powerful risk factor for life-time major arrhythmic events and
SCD. These results support the use of comprehensive genetic screening of DS-genes for
arrhythmic risk stratification in ARVC.
Key words: cardiomyopathy, genetics, risk stratification, sudden cardiac death, ventricular
arrhythmia
2
DOI: 10.1161/CIRCGENETICS.113.000288
Introduction
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable heart muscle disease
characterized by fibrofatty replacement of the right ventricular (RV) myocardium1,2. The natural
history of ARVC is predominantly related to RV electrical instability with ventricular
tachycardia (VT) and ventricular fibrillation (VF) leading to sudden cardiac death (SCD)3-6.
Studies of molecular genetics have provided significant insights in our understanding of the
pathogenesis and pathophysiology of ARVC, showing that in its pure form it is a genetic
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
disorder of intercellular junctions resulting from mutations of genes which encode for
desmosomal (DS) proteins such as plakoglobin (JUP), desmoplakin (DSP),
DSP),
) pplakophilin-2
lakkoph
phhil
ilin-22 ((PKPP
77-14
14
l n-2 (D
lein
(DSG
SG
SG-2
G 2), desmocollin-2 (DSC-2))7A few additiona
additional
nal causative mutations
na
mutationn have
2), desmoglein-2
(DSG-2),
also been identified
dent
de
entified in no
non-desmosomal
on-deesm
mossom
o all genes
gen
nes8,155. The
Th
he di
discovery
isccov
o eryy ooff DS
DS-gene
S-g
gen
ne mu
m
mutations
t tioons involved
ta
in
nv
a pa
ase
ath
thog
hog
o enes
esiis
is hhas
as ooffered
ffered
ed
d tthe
he ppotential
oten
ot
ten
nti
tial
all of
of a mole
m
ole
leccula
larr ge
la
gene
netic di
dia
agnoosi
sis
is off A
R
in the disease
pathogenesis
molecular
genetic
diagnosis
ARVC
in the clinical
c settingg16.
cal
For some genetic
etiic heart
h rt diseases
dis
such
s chh as LQT
LQT syndrome
s nd
ndrome
d
molecular
molec
oll llar genetic
etiic testing
t tii has
hha
been shown to allow prediction of arrhythmic risk and genotype-based optimization of therapy17.
At present, the prognostic impact of genotype in ARVC patients remains poorly understood and
the arrhythmic risk stratification relies on phenotypic predictors including the young age at the
time of diagnosis, previous cardiac arrest or syncope, VT, severe RV or left ventricular (LV)
dysfunction and inducibility at programmed ventricular stimulation18. Previous genotypephenotype correlation studies failed to identify specific genes and/or mutations distinctively
associated with an unfavourable arrhythmic outcome and so far there is little evidence to support
the use of genetic screening for prognostic assessment19. More recent studies, based on
comprehensive analysis of ARVC causative DS-genes, reported that compound heterozygosity
3
DOI: 10.1161/CIRCGENETICS.113.000288
(co-inheritance of different disease alleles of a single gene) and digenic heterozygosity (coinheritance of disease alleles for two different genes) is not uncommon and may contribute
significantly to varying ARVC penetrance and expression20-24. However, a systematic evaluation
of the genetic predictors of life-time arrhythmic outcome and SCD has not been previously
performed. Such information would be useful in making decision about treatment, particularly
for asymptomatic individuals7.
Thus, the present study was designed to evaluate the impact of genotype, with particular
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
reference to a complex genetic status with multiple DS-gene mutations,
s, to pr
ppredict
edict the pprobability
roba
ro
b
of experiencing a first major arrhythmic event including SCD, since birth
consecutive
irth in
in a consecut
tiv
ivee series
s
of ARVC DS-gene
D ge
DS-ge
gene
ne mutation
mut
u attio
ion carriers. The evaluation
on of a “gene-dose”
“gene-dosee” effect of
compound/digenic
status on
was
made
possible
digenic
ig
g
on the
th
he life-time
liife
fe--time
me arrhythmic
arrrhhytthmicc rrisk
iskk w
as mad
ade pos
ssib
iblle byy tthe
ib
he
comprehensive
s ge
sive
genetic
ene
neti
tic an
ti
analysis
alys
al
lys
ysiis
is of
of al
all
ll known
kn n ARVC-causing
ARVC
AR
VC-ca
VC
caus
usin
us
ingg DS-genes.
in
D -ggennes
DS
nes.
Methods
Study sample
The patients’ cohort of this single-institution study was drawn from the Inherited
Arrhythmogenic Cardiomyopathy Unit of the Department of Cardiac, Thoracic and Vascular
Sciences of the University of Padua (Padua, Italy), which is a national referral centre for the
diagnosis and the management of ARVC patients.
The initial cohort comprised 90 unrelated living probands with a clinical diagnosis of
ARVC based on the 2010 International Task Force (ITF) criteria25, who underwent
comprehensive molecular screening of DS-genes. One or more causative mutations of DS-genes
were found in 44 (49%) ARVC probands. Mutation screening of living family members (N=217)
of the 44 genotype-positive probands identified other 90 (41%) DS-gene mutation carriers.
4
DOI: 10.1161/CIRCGENETICS.113.000288
Overall, the population of the present study included 134 DS-gene mutation carriers (44
probands and 90 family members) from 44 consecutively genotyped ARVC families. Of these 44
families, 16 were reported in an earlier investigation with different study design and endpoint20.
The study was approved by the institutional review board, and all subjects gave their
informed consent.
Clinical evaluation
Clinical evaluation including detailed personal and family histories, physical examination, 12Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
lead-electrocardiogram (ECG) recording, and chest x-ray, signal-averaged
ag
ged ECG; 24-hou
24-hour
ur H
Holter
monitoring, exercise testing, 2-dimensional transthoracic echocardiography.
raphy
y. Sixty-three
Sixtty-th
Si
threee ppatients
th
at
e wen
erw
entt ad
addi
diti
di
t on
onal
a imaging studies such aass ccontrast-enhanced
ontrast-enhanceed cardiac magnetic
(46%) underwent
additional
resonance an
aand/or
ndd//or cardiac cath
catheterization
hetterizzationn iincluding
ncl
cludin
ing RV and
nd LV
LV cineangiography;
cinnean
ngioggraphhy; 38
38 (28%)
(28%
8%
8%)
%)
re st
tud
udie
die
iedd byy intracardiac
int
ntra
traca
card
rddiac el
electroph
phys
ph
hys
y iolo
iolo
l giic study
stud
st
udyy including
ud
incl
clud
cl
udin
ud
i g programmed
in
prog
ogra
og
ramm
ra
med
d ventricular
ven
entr
tr
patients were
studied
electrophysiologic
stimulation..
According
cording
di tto th
the
h 20
2010
10 IT
ITF
F criteria
it ia25, th
the
h diagnosis
di
sii off ddefinite
finite
i ARVC
ARV
RVC
C was
as ffulfilled
lfilled
lfil
lf
illledd in
i the
presence of two major criteria or one major criterion plus two major criteria or 4 minor criteria
from different categories. The ARVC diagnosis was considered borderline when 1 major and 1
minor or 3 minor criteria from different categories were fulfilled.
Mutation screening
A comprehensive open reading frame/splice site mutational analysis of all amino acid coding
exons and intron boundaries of the 5 ARVC-susceptibility DS-genes has been carried out as
previously described20.
According to the 2010 ITF criteria25, a pathogenetic mutation was defined as a DNA
alteration previously associated with ARVC that was unobserved or rare in a large non-ARVC,
5
DOI: 10.1161/CIRCGENETICS.113.000288
ethnically matched control population and was predicted to alter the structure or the function of
the encoded protein or cosegregated with disease phenotype in the family. All other sequence
variants were classified as polymorphisms26.
All DNA-identified variants were compared with a control group of more than 300
unrelated, healthy individuals (600 alleles) from the Caucasian population. Type of mutations
were divided into missense and non-missense (i.e. nonsense, frame shift, and splice mutations).
Multiple mutations were coded as a binary variable (either single or multiple mutations).
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
All subjects had given written informed consent after counselling
ng in accordance w
with
i the
it
ethical standards of the Declaration of Helsinki (2001) and with recommendations
mmenddattions given
given
n by
by the
a Et
al
thi
h ca
call Co
C
omm
mmittee.
mm
Institutional
Ethical
Committee.
Life-time arrhythmic
rrh
hyt
y hmic out
outcome
tcom
me andd endpoints
enddpo
p ints
V D
VC
S-ggen
e e mu
mut
tati
tati
tion
on carriers
carrie
ieers
rs are ex
expo
p seed to
po
to the
thee genetic
gen
enetticc ri
iskk ooff li
life
fe threeat
fe-t
aten
tenin
i g
in
Since ARVC
DS-gene
mutation
exposed
risk
life-threatening
y hmias andd SC
CD ffrom
rom bbirth,
irth
h, the
th
he ge
ggenotype-related
noty
type
ty
p -rellatedd ar
rhhyt
ythhmic risk assessme
e
ventricular arrhyt
arrhythmias
SCD
arrhythmic
assessment
was based on th
the
he ccumulative
m llati
atii e probabilit
probability
obbabil
biliit off experiencing
e periencing
ie in a fi
firstt arrh
arrhythmic
rhh th
thmic
hmiic event
e ent
nt or SCD
SCD during
d
life-time using date of birth as start point for the time to event analysis17.
The medical history of each subject was obtained by review of medical records, clinical
evaluation and patients’ interview during regular outpatient visits (at 6 to 12-months intervals).
For each subject, the follow-up period extended to the most recent evaluation or censoring event
up to June 2012.
The endpoint for the survival analysis was composite and included first major arrhythmic
events occurring during follow-up such as SCD, aborted SCD due to VF, sustained VT, and
appropriate intervention of implantable defibrillator (ICD). Sudden cardiac death was defined as
any natural death occurring instantaneously or within one hour from symptoms onset. Aborted
6
DOI: 10.1161/CIRCGENETICS.113.000288
SCD was defined as successful cardiopulmonary resuscitation/defibrillation for cardiac arrest
due to VF. Appropriate ICD intervention was defined as an ICD shock delivered in response to
VT or VF and documented by stored intracardiac ECG data, as previously reported6.
Statistical analysis
Results are summarized as mean or n (%) for continuous and categorical variables, respectively.
&DWHJRULFDOGLIIHUHQFHVEHWZHHQJURXSVZHUHHYDOXDWHGE\WKHȤWHVW of the Fisher exact test as
appropriate. Continuous variables were expressed as median (25%-75%iles) and compared with
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Wilcoxon rank-sum test or Kruskal-Wallis test. The cumulative probability
bility of experienc
experiencing
cin
ing a
first arrhythmic event during life-time was determined by using date off birth
birtth as st
start
tartt ppoint
oint
oi
nt ffor the
nt analysis.
anal
an
allys
ysis
iss. Subjects
Suubj
b ects were censored at the
th
he time
t me of their first
ti
firsst event or the time of their
time to event
last clinicall ffollow-up.
ollow-up. Sur
Survival
urrvivvall di
dist
distributions
s ribuuti
st
tionns du
during
uriing li
life-time
ife-ti
time
ti
m off the
me
th
he composite
comp
mpoosite arrhythmic
mp
arrhhythm
ar
mic end
e
eestimated
ated
at
ed using
ng Kaplan-Meier
Kap
apllan
lan-Me
Meie
Me
ier curves.
ie
curv
rves
rv
es.
es
point was estimat
e of covariates on ti
ect
ime to the
h comp
posiite arrhythmic
arrhyt
hy hm
h ic
i end
d-ppoiint was investiga
g tedd using
Effect
time
composite
end-point
investigated
a Cox proportional
ortional
tii all hazard
ha
h ard
d model
model.
dell H
Hazard
Ha ard
d ratio
tii (H
(HR)
R) and
ndd 995%
5% C
Confidential
fiddentiiall Intervals
fi
IInter
nte al
als
l (CI)
(CI) were
always presented.
Inflation in standard error due to familiar correlation of subjects was taken into account
using a Huber-White (sandwich) estimator implemented in the RMS Libraries of the R System27
as described by Harrell 28. Proportionality of hazards was assessed visually using Shoenfeld
residuals plots.
Due to the small sample size, multivariable model was built using a forward approach
and adopting AIC (Akaike Information Criterion) as selection criterion. A significance of 0.10
was required to variables for being candidates for the model. Model validation was performed
using bootstrap (1000 resamples) and Somer’s Dxy as measure of fitting adequacy. A value of
7
DOI: 10.1161/CIRCGENETICS.113.000288
P<0.05 was taken as a threshold for statistical significance.
All analyses have been performed using SPSS version 17 (SPSS Inc, Chicago, III) and
the R system.
Results
The study population included 134 DS-gene mutation carriers [68 males; median age 36 years
(22-52)], whose clinical characteristics at the time of the last evaluation are reported in Table 1.
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Four patients had been involved in competitive sports activity before diagnosis
of ARVC and/or
g
genotyping. Prophylactic antiarrhythmic drug/beta-blocker therapy forr prima
primary
mary
ma
ry pprevention
reve
re
vent
ve
ntio
nt
io
on of
life-threatening
ning arrhythmias
arrrhhyt
arrh
y hm
hmia
iaas was prescribed in 47 (35%)
(355%)
% patients; 25 (19%)
(19
1 %) patients were
implanted with
secondary
(N=22)
primary
(N=3)
w h an ICD, either
wi
eitther
ei
er for
for sec
se
econd
n arry (N
nd
N=
=2
22)) oorr pr
prim
im
mar
ary (N
N=3)
3 pprevention.
rev
ev
venti
tiion
on.
DS-gene mutations
m ta
muta
ati
t on
ns
Mutations identified
by comprehensive
i
co
ompre
mpre
mp
r heens
n iv
ve sequencing
sequ
se
quen
qu
en
nci
c ngg of
of DS-genes
DS-ggen
e es are
aree ssummarized
umma
um
mari
ma
r zed in Table 22. A
ri
total of 48 DS
DS-gene
mutations
absent
them,
D
DS-ge
g ne mu
gene
muta
tati
tion
ti
onss ab
abse
sent
nt iin
n at lleast
east
ea
stt 6600
00 reference
reffer
eren
ence
ce aalleles
llel
ll
eles
es we
were
re ffound.
ound
ou
ndd. Am
Amon
Among
ongg tth
h
16 were non-missense mutations.
A single pathogenetic mutation was identified in 113 patients (84%) with the following
gene distribution: DSP, n=44 (39%); PKP-2, n=38 (34%), DSG-2, n=30 (26%) and DSC-2, n=1
(1%). No one carried JUP mutations. Of the 113 patients, 62 carried missense mutations and 51
non-missense mutations.
A complex genetic phenotype with multiple DS-gene mutations was detected in 21
carriers (16%): compound heterozygosity in 7 and digenic heterozygosity in 14 (for details on
multiple DS-gene mutations see Table 3).
Disease penetrance
Of the 134 DS-gene mutations carriers, 78 (58%) fulfilled the 2010 Task Force Criteria for
8
DOI: 10.1161/CIRCGENETICS.113.000288
definite or borderline ARVC (Table 4). The disease penetrance was higher in patients with
multiple mutations than in those with single mutations (17/21 vs. 61/113, p=0.03); the difference
remained statistically significant after adjustment for age and gender (OR 3.1; 95%CI 1.29.1,p=0.03). Among patients with single mutation the disease penetrance did not differ according
to specific DS-genes or to the presence of missense vs. non-missense mutations.
Phenotypic expression
Among the 78 patients with a borderline or definite disease, males significantly more often
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
showed low QRS voltages in limb leads (42% vs. 26%, p=0.04) and LV
with
V dysfunction wit
th
ejection fraction <55% (28% vs. 8%, p=0.02). The presence of multiple
mutations
le DS-gene
DS
S-gene m
utat
t tio
ions was
associated with
with:
ECG abnormalities
waves (39% vs
vs.
w
h: 1)) more
m re frequent
mo
fr
abnormalitie
ies in the form of eepsilon
ie
ps
psilon
s 8%,
p=0.001), right
precordial
T-waves
extending
beyond
lead
(56%
ight
ig
ght precordia
al nnegative
ega
gaativee T
-wa
wavees ext
tenndiingg be
beyo
yond
yo
nd lea
ead V3 (56
566% vvs.
s. 26%
% p=
pp=0.02),
=0.
and late potentials
SAECG
p=0.04);
and
echocardiographic
t ial
tenti
alss on
o SAE
AECG
AE
CG (89%
CG
(89
8 % vs.
vss 64%,
% p=0
= .004)
=0
4);; an
nd 2) mo
more
ree ssevere
eveere ec
ev
echo
h caarddiogr
ho
iograp
aphh
alterations as
a shown byy a lower mean RV
RV fractional
fractiionall area change
g [[21
21 ((17-29)
17-29)
29) vs 29 ((24-31)
24-31))
p=0.001] and
higher
prevalence
ndd a hi
gh
h pre
alence
le
off multiple
m lltiple
tiiplle RV wall
all
ll motion
otiio abnormalities
ab
b
ali
litiie UHJLRQV72%

UHJLRQV
JLL V 77
vs 39%, p=0.01),
In the subgroup of 61 patients with single mutation who fulfilled the ITF criteria for
either borderline or definite ARVC, carriers of single non-missense mutations significantly more
often had right precordial T-wave inversion extending beyond lead V3 (45% vs. 13%, p=0.006)
and showed a trend towards a lower mean RV fractional area change [27 (20-30) vs. 30 (27-32),
p=0.06], compared with carriers of single missense mutations. No statistically significant
differences in the clinical phenotype were attributable to specific DS-genes, with the exception
of DSP-gene mutations carriers who distinctively had more frequent negative T-waves confined
to leads V4-V6 (DSP=9% vs. PKP-2 DSG-2, and DSC-2 =0%; p=0.02), low QRS voltages in
9
DOI: 10.1161/CIRCGENETICS.113.000288
limb leads (DSP=57% vs. PKP-2=18%, DSG-2=17% and DSC-2 =0%; p=0.02) and LV
dysfunction with ejection fraction <55% (DSP=38%, DSG-2=10%, PKP-2=6% and DSC-2 =0%;
p=0.01).
Life-time arrhythmic outcome
During a median observation period (from birth to last clinical follow-up) of 39 (22-52) years,
22 (16%) subjects from 20 different families reached the composite arrhythmic end-point: 1 died
suddenly, 6 experienced aborted SCD due to FV, 14 had sustained VT and 1 received an
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appropriate ICD discharge.
The age at the time of first arrhythmic event was 35 (27-40) years,
events
ars, with
wit
ithh no event
it
ntss
nt
occurring befo
bbefore
ore tthe
he aage
g ooff 15 (Figure 1-4).
ge
There
were
events
(27%)
and
events
among
the
family
re w
re
ere 12 even
en
nts aamong
mong
n thee 444
ng
4 pprobands
robbandss (27%
7%)) an
7%
nd 10 eve
veentts amon
ongg th
on
he 990
0 fa
a
members (11%).
subjects
1 %)
11%)
%). No sub
bje
j ct
cts
ts who
whh experienced
ex
xpe
perience
e
cedd ar
ce
aarrhythmic
rh
hyt
ythhm
hmic events
eve
ventss had
ve
had been
be ppreviously
reviou
re
ousl
sly
l in
iinvolved
vo
vo
in competitive
activity.
Subjects
followt sports
tive
spo
p rts activi
ity
ty. S
ubbjjeects with
h and
andd without
wiithhout major
majo
jor arrhythmic
arrhhyt
ythm
h ic events duringg fo
o
up did not differ
which
diff
di
ff with
iith
thh regard
rdd to
t prophylactic
proph
h llactic
actiic antiarrhythmic
antiarrh
ntiia h th
thmic
hmii drug/beta-blocker
ddrr g/beta
/beta bblocker
/b
lock
k therapy
th
therap
he
hic had
hi
been prescribed for primary prevention of life-threatening arrhythmias (8/22, 36% vs. 39/112,
35%; p=0.89).
Kaplan-Meier analysis of freedom from major arrhythmic events from date of birth
stratified by gender, genetic locus, type of mutation, and number of DS gene-mutations is
reported in Figures 1 to 4.
On multivariable Cox-regression analysis, multiple gene mutations and male gender
remained independent risk factors for life-time occurrence of major arrhythmic events, with
hazard ratios of 3.71 (95% CI: 1.54-8.92) and 2.76 (95% CI:1.19 to 6.41), respectively (Table 5),
showing a good model adequacy (Somer’s Dxy = 0.71).
10
DOI: 10.1161/CIRCGENETICS.113.000288
Male patients carrying multiple mutations were at the highest risk (63% life-time rate of
events), followed by females with multiple mutations (27%), males with single mutation (13%)
and females with single mutation (7%) (Figure 5). The median observation period was similar in
the 4 subgroups [44 years (32-51); 45 years (32-60); 35 years (26-44); 40 years (22-56)].
Family history of premature SCD
In the history of the 44 families, premature SCD (<35 years) had occurred in 12 subjects (10
males). Of 12 SCD victims, 9 (75%) belonged to families with a complex genotype (Table 6).
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Discussion
This is the first study
stud
st
udyy designed
ud
d siign
de
g ed to assess the value of genotype
g notype to pred
ge
predict
dic
ict the cumulative
probability ooff eexperiencing
major
event
life-time
DS-gene
xperienccin
ingg a ffirst
irrst
rst ma
m
jo
or ar
aarrhythmic
r ythm
rh
h ic ev
hm
vent
ntt dduring
uringg li
ur
life
fee-tiimee in DS
DS-g
gene
ne rrelated
e att
el
ARVC. Thee ma
majo
major
jo
or study
s ud
st
u y results
reesu
s lts were
were
re tthat
h t co
ha
comp
compound/digenic
mpou
mp
ound
ou
nd/d
nd
dig
gen
nic hheterozygosity
e erroz
et
ozyg
yg
gos
o itty wa
wass id
identified
den
enti
tiifieed in
tif
16% of our cohort of ARVC
DS-gene
mutation
This
genetic
VC D
S-ge
gene
nee m
uttat
atio
io
on carriers.
car
a ri
rier
errss.. Th
his
i ccomplex
ompl
om
ompl
plex
ex ge
ex
gene
neti
ne
t c status was a
ti
powerful and
risk
malignant
during
nd independent
nd
ind
ndep
epen
ende
dent
ntt ri
iskk ffactor
acto
ac
torr off m
alligna
alig
gnant
ntt aarrhythmic
rrhy
rr
hythm
hy
hmic
i eevents
ic
vent
ve
ntts an
andd su
sudd
sudden
dden
en ddeath
eath
ea
th dduri
ur
life-time. The study results support the use of comprehensive molecular screening of DS-genes
in ARVC not only for early diagnosis but also for arrhythmic risk stratification and optimization
of therapeutic interventions aimed to prevent SCD, particularly in individuals who are
asymptomatic.
Genotype-phenotype correlations
Molecular genetic diagnosis of ARVC by screening for mutations in DS-genes is successful in
up to 45% of patients16,19. Diagnostic genetic testing is applied to probands with a clinical
suspicion of ARVC with the aim of establishing a definitive diagnosis. Identification of a
pathogenetic DS-gene mutation in the proband enables cascade family screening which offers the
11
DOI: 10.1161/CIRCGENETICS.113.000288
potential of a preclinical diagnosis among relatives and allows to target resources to proven
ARVC mutation carriers 20-24,29.
The value of underlying molecular genetic etiology for risk stratification of ARVC
patients remains poorly understood and current management guidelines for patients with ARVC
give little consideration to the prognostic impact of molecular genotyping19. Most of previous
genotype-phenotype correlations studies were based on a few families, separately addressed
mutations in different DS- genes and compared clinical manifestations of ARVC mutation
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
carriers vs non carriers30,31. Although some differences have been reported
orted with regard tto
o a series
of clinical, ECG and morpho-functional RV abnormalities variables between
etween AR
ARVC
VC ge
gene
genenene
positive andd ggene-negative
enee-ne
en
e-ne
nega
gative
ga
ve patients, detection of ARVC
ARV DS-gene mutations
mut
uta
tations was not associ
associated
with an enhanced
hancced suscept
ha
susceptibility
tibbility
ty
y to li
llife-threatening
fe-th
thrreat
th
ateninng arrhythmias
arrrhhytthm
hmiias and
and di
did
id not
id
ot predi
predict
dict
di
c ar
arrhythmic
rrhy
ythm
outcome compared
o ar
ompar
ared
ed w
with
ithh an unk
unknown
nkkno
n wnn ggenotype.
enotyp
ypee. Th
yp
The
he few
fe av
avai
available
vai
aila
lablle st
la
stud
studies
tuddies
ie correlating
co
orr
rrelattin
i g the
th
he
phenotype to
t the underlying
underly
yingg genotype
g noty
ge
type
ty
p provided
proviided contradictory
contradi
d ctoryy fi
di
findings
ndi
dings
g withh regard
reg
gard to the
association between
bbet
et een specific
ifiic DS
if
D
DS-genes
S genes and/or
d/ mutation
m ta
tation
tii and
ndd clinical
linii l ffeat
features.
eat res A specific
ifiic tren
if
ttrend
towards more prevalent LV involvement was attributed to DSP mutations by Quarta et al.23 and
to DSG-2 mutations by Fressart et al.22, whereas no association was reported by others21. In our
study we found that DSP mutation carriers distinctively exhibited a more prevalent LV
involvement. However, in accordance with previous studies, neither non-missense mutations nor
mutations in a particular DS-gene were able to predict an unfavourable arrhythmic outcome of
ARVC patients.
Genotype complexity
Recent reports indicate that a sizeable proportion of ARVC patients carry more than one gene
mutation (i.e., compound and digenic heterozygosity) which may contribute to varying ARVC
12
DOI: 10.1161/CIRCGENETICS.113.000288
penetrance and expression 20-24.
Xu et al.24 reported that missense mutations of DS-genes, mostly the PKP-2, although
relatively common in ARVC probands, may be not enough for the disease phenotype to be overt
and require additional mutation(s). In this study subjects harbouring more than 1 variant in PKP2
or PKP2 plus other DS- gene variants had a more penetrant and expressed disease with an
earlier onset. Quarta el al.23 reported that ARVC probands were significantly more likely than
first-degree relatives to harbour >1 rare genetic variant; within families, diallelic individuals
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
more often developed disease expression and became the first family member to receivee a
diagnosis, while among relatives, the presence of >1 rare genetic variant
associated
with
ant was asso
ciiated
t dw
it a
significant (5-fold)
increase
expression.
(5-fo
fold
fo
ld
d) in
incr
c eaase in risk of disease expres
essi
es
s on. Likewise, in
n the study by Bauce et al.20,
multiple-mutation
utat
tation carriers
carrierrs showed
show
ow
wed
d a sign
significantly
gnifiicaantlly high
gn
higher
gherr pprevalence
reva
re
v leence of
va
of ECG
CG
G rep
repolarization
polariizattio
on
changes and
d LV
V iinvolvement,
nvol
nv
olveeme
ment
ntt, but
bu noo a worse
se arrhythmic
arr
rrhhyth
hyth
thmi
m c outcome
outc
ou
tcom
tc
omee du
om
dduring
uri
ring
ing a sshort-term
hort-te
ho
t rm ffollow-up.
te
ollo
ol
llo
Hence, the ppresent
resent studyy was spe
specifically
p ciifi
ficallly de
ddesigned
siign
g ed
d to assess as pprimary
riimary
y end
endpoint
dpo
p int the value of
genotype to
arrhythmic
o predict
edi
dict th
the
h ccumulative
m llati
atii e probability
probabilit
babi
bili
lit off eexperiencing
periencing
rii cii a ffirst
irst major
ajj arrh
h th
thmic
h ic eevent
e and
SCD since birth in DS-gene related ARVC. Compound/digenic heterozygosity was identified as
the most important genetic determinant of malignant arrhythmic outcome since birth.
We found that compound and digenic heterozygosity was present in 16% of cases. This
overall rate for multiple mutations was greater than in previous studies from other countries
where the reported prevalence of multiple mutations did not exceed 10% of cases21-24. In
agreement with previous studies, carriers of multiple mutations showed a more penetrant disease
and more severe phenotypic expression. However, our study results extended previous
observation by showing a “gene-dose” arrhythmic effect, given than multiple DS-gene mutations
were the most powerful genetic predictor of composite arrhythmic end point. Complex genotype
13
DOI: 10.1161/CIRCGENETICS.113.000288
identified ARVC patients at increased risk of life-time major arrhythmic events/SCD and
remained an independent determinant of malignant arrhythmic outcome during life-time at
multivariable analysis.
According to the study design, our study population was limited to living probands or
relatives who underwent a comprehensive screening of DS-genes implicated in the ARVC
etiology. Subjects who experienced SCD at young age (<35 years) before undergoing genetic
analysis were unavoidably not included in the analysis of genetic predisposition to life-time
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
arrhythmic events and SCD. However, it is noteworthy that the majority
young
ty
y (75%) of these yo
you
SCD victims belonged to families with multiple mutations, which further
her suggests
sugg
g estts an association
assso
soc
between complex
genotype
events.
mp
ple
lexx ge
geno
noty
no
ype and increased risk of fatal
fata
fa
tal arrhythmic eve
ta
ents.
The pprecise
mechanism
more
malignant
recise mechan
an
nism
m underlying
unde
d rlyiing a mo
de
oree ma
malign
nan
nt arrhythmic
arrhhythm
mic ccourse
oursee iinn ppatients
atieents
carrying multiple
DS-gene
Whether
worse
ventricular
electrical
u le D
ultiple
S-geene mutations
muttati
mu
tat onss is speculative.
specu
cula
cu
lati
la
t vee. Wh
ti
Whet
ethe
et
herr wo
he
wors
rss ve
rse
ntricu
nt
t cu
ula
lar el
lec
ecttric
tric
i all
instability iss secondary
y to a ge
ggenetically-determined
netiicall
lly
ll
y-de
d termin
i ed
d greater
greater extent off ventricular
ventriicular fibrofattyy
myocardial scar32 or iitt results
res llts
t directly
di
directl
ctll ffrom molec
molecular
le llar
a ddefects
effect off ddesmosomess and/or
d/ abnor
abnormal
bn
gap junction/sodium channel interactions3,33,34 remains to be elucidated by further studies.
Gender
Previous studies on ARVC index cases consistently demonstrated a male predominance,
suggesting that males develop a more severe disease phenotype which make them more likely to
come to medical attention21,30,35,36. Although in the present study multiple mutations had the
single most important role in predicting arrhythmic events, this factor was importantly
modulated by male gender. Arrhythmic prognosis was worse in male DS-gene mutation carriers
and males with multiple mutations exhibited the highest cumulative risk (63%) of experiencing a
malignant arrhythmic event. In addition, Kaplan-Meier analysis showed that DS-gene carriers
14
DOI: 10.1161/CIRCGENETICS.113.000288
remained arrhythmia-free for a long period of time, with events occurring after the pubertal
development. This finding is in agreement with a previous study showing that the majority of
familial cases are not diagnosed in infancy and both ARVC clinical onset and arrhythmic events
are characteristically delayed to adolescence and young adulthood, with a peak at the age interval
of 15 to 35 years35,37. All these findings suggest a direct influence of sex-hormones on the
disease pathobiology and development of the arrhythmogenic myocardial substrate. In this
regard, estrogens, such as 17-beta-extradiol, at physiologic concentration exert an inhibitory
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
effect on myocardial cell apoptosis38, which has been shown to play a role in myocyte
myo
y cyte ddeath
eath and
ea
subsequent fibrofatty myocardial replacement in ARVC39. This protective
tive eff
effect
ffectt off estrog
estrogens
ogen
og
expl
plai
pl
ainn wh
ai
hy fe
fem
males with DS-gene muta
tati
ta
tions tend to develop
deveelo
lop a less severe disease
dise
may partly explain
why
females
mutations
phenotype an
aand
nd fewer arrh
arrhythmic
hythm
mic complications
com
o pllic
om
i attio
ons com
compared
ompaareed with
w th
wi
hm
males.
ales.
Study limitations
a ns
ation
Sequence alterations
a
were ddivided
iviide
d d iinto
nto ppathogenic
athhoge
g niic mutations andd polymorphisms
p ly
po
lymorpphisms according
g to
generally accepted
ccepted
pted
d criteria
rite
i rii 25. Recent
Re nt st
studies
t di
dies on the
th
h presence off DS
DS-gene
gene variants
ariants
iant iin
n th
the
he gen
general
population, demonstrated that while non-missense DS-gene variants are high-probability ARVCcausing mutations, the pathogenetic potential of rare missense mutations should be interpreted
with greater caution and in the context of race and ethnicity, mutation location, and sequence
conservation 26,40. In vitro functional analysis or animal studies would have been required to
conclusively proof the causative nature of the missense variants, which we lack. However, it is
important to stress that these limitations are exactly the same present in all previous genotypephenotype studies in ARVC.
Although genetic and sex-linked hormonal factors may play a primary role, greater risk of
developing life-threatening ventricular arrhythmias may be related to environmental factors, such
15
DOI: 10.1161/CIRCGENETICS.113.000288
as prolonged and intense physical activity that may influence the disease phenotype and
outcome. It is believed that the incorporation of genetically defective proteins into cardiac
desmosomes may provoke detachment of myocytes at the intercalated discs41, particularly under
mechanical stress conditions, such that occurring during regular and vigorous sports
training/competition8. Therefore, one may expect that the more intensively a given subject is
involved in physical activity in the teen years, the more severe the ventricular fibrofatty scar
lesion and the more malignant the arrhythmic disease manifestations. However, in our study the
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
poorer arrhythmic outcome in multiple-mutation than in single-mutation
on carriers cannott be
b
related to sports performance, because none of the former group was involved
nvollvedd iin
n compe
competitive
peeti
tittiv
ti
sports.
mp
pliccations andd conc
ncclusi
siion
Clinical implications
conclusion
t the
he rresults
he
esultss of
es
of th
tthee pr
presen
nt sstudy,
tudy, compound/digenic
com
ompo
poun
po
und/
d/di
d/
diige
dige
geni
nic
ni
i heterozygosity
het
he
etero
terozygoosity
sity off ARVCsi
ARVC
AR
ARVC
VCAccording to
present
S g ne mutations predicts
S-ge
p edicts
pr
d
hyt
ythmiic outcome because
because of a “doub
b
causing DS-gene
a more severe arrh
arrhythmic
“double
t Th
Thi
This
is highlights
highl
hliighht the
th
h importance
im rt
off screening
in the
th
h entire
tii panel
ell off DS
genes eeven
en after
dose” effect.
DS-genes
a single gene mutation has been identified. Moreover, the occurrence of double and compound
mutations and the increased chances of inheriting the gene defect have significant implications
for genetic counselling. If a subject is a double heterozygote with a mutation on two different
DS-genes inherited independently, the risk that a child will receive at least one gene mutation is
75%, while the chance of inheriting both gene mutations is 25%. In subjects with a compound
heterozygosis, there is a 50% risk of passing on the multiple gene defect if the two mutations are
both on the same allele (cis), and a 100% risk of transmitting one of the two mutations if they are
on different alleles (trans). Cardiologists and clinical/medical geneticists dealing with ARVC
families should be skilled in offering an appropriate genetic counselling, aimed to elucidate and
16
DOI: 10.1161/CIRCGENETICS.113.000288
discuss the patterns of inheritance and clinical consequences of multiple mutations which may
assist couples in making a more informed decision about prenatal genetic testing and
management of the pregnancy.
In addition, our study provides insights on pre-symptomatic arrhythmic risk stratification
for SCD in ARVC. Clinical manifestations of ARVC usually develop during adolescence or
young adulthood and are preceded by a long preclinical phase7,8,35. Cardiac arrest may occur as
the first manifestation of disease in previously asymptomatic adolescents and young adults1,2.
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
The most important aim of genotyping families with ARVC is to diagnose
nose ggenetically
enetically aff
affected
ffeec
ff
relatives before a malignant clinical phenotype occur. According to our
results,
of
ur result
lts, ddemonstration
lt
emonst
strra
st
ra
rat
multiple DS-gene
S ge
S-ge
gene
ne mu
muta
mutations
tation
ta
ons on genotyping may bee a m
on
major
ajor factor pred
predicting
ed
dicting life-threatenin
life-threatening
n
arrhythmias.
Preclinical
young
individual
multiple
DS-gene
mutations
s. P
s.
reclinical identification
identiifi
ficati
tiion of
of yo
oung in
ndivi
viduual ccarrying
a ryin
ar
ing mu
ultip
ip
ple D
S-gen
ene mu
en
m
utaa
may lead too establish
estab
abli
ab
lish
li
s a focused
sh
foc
ocus
used
ed
d prevention
preven
en
nti
tion strategy
str
traateg
tr
e y wi
eg
with
ith
t a ccloser
lo
oser cl
cli
clinical
linic
inic
i all follow-up
fol
ollo
ol
low-upp an
low
andd a m
more
aggressive treatment.
t
It is reasonably
reasonab
bly
l to assume,, on the
h bbasis
asis off our fi
ffindings,
nddings
g , that lifestyle
lifesty
yle
modifications, such
h as restriction
t i tii ffrom competitive
ti
titi
itii sport,
t and
d prophylactic
h l tii th
therapy
h
with
iith
th
hb
betablockers are indicated in carriers of multiple DS-gene mutations, particularly male individuals
who have the highest arrhythmic risk. By contrast, the decision to institute prophylactic therapy
in single mutation carriers, particularly females, at lower risk of events is not justified and should
be individualized. Whether a lower threshold for ICD therapy should be used among
asymptomatic carries of multiple DS-gene mutations remains to be established by larger cohort
studies.
Acknowledgments: The authors acknowledge Drs. Alice Niero and Kim Nguyen for their
contributions in collecting clinical information.
Founding Sources: This study was supported by the Ministry of Health, Rome, Italy;
17
DOI: 10.1161/CIRCGENETICS.113.000288
Fondazione Cariparo, Padova and Rovigo, Italy; Italian Telethon Foundation, Rome GGP09293;
and Registry of Cardio-Cerebro-Vascular Pathology, Veneto Region, Venice, Italy.
Conflict of Interest Disclosures: None.
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18
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desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular
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4.
21
DOI: 10.1161/CIRCGENETICS.113.000288
Table 1 – Clinical characteristics of the study population at the time of the last evaluation
Overall
N=134
Median age, years (range)
36 (22-52)
Males, n (%)
68 (51)
Symptoms
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Palpitations, n (%)
37 (28)
Chest pain,, n (%)
11 (8)
Syncope, n (%)
(
(5
7 (5)
ECG findings
Low QRS-voltage in limb leads , n (%)
30 (22)
(2
Epsilon waves
w ve
wave
vess , n (%)
(%
%)
12 (9)
(99
Negative T-wave
T-w
wave in le
leads
eads V
V1-V2/V3
1-V
-V
-V2/
V2/
2/V3
V oonly
V3
nlyy , n (%)
nl
(%
%)
15 (11)
(1
Negative T-wave
T-w
-wav
a e be
beyond
eyo
y ndd leadd V33 , n ((%)
%))
28 (21)
(22
ds V4-V6
V4-V
V4
V6 on
only
ly
y , n (%)
(%))
Negative T-wave in lead
leads
Late-potentials
n
ntials
on SAECG , n (%
((%))
8 (6)
(66
55 (41)
(4
Morpho-functional
unctional
u
ncti
nc
tion
ti
onal
al abnormalities
abn
bnor
orma
mali
liiti
ties
es
RV dilation , n (%)
75 (56)
RV dysfunction, n (%)
47 (35)
RV regional WMA, n (%)
52 (39)
LV dilation, n (%)
26 (19)
LV dysfunction, n (%)
18 (13)
5 (4)
LV regional WMA , n (%)
Therapy
Beta-blockers, n (%)
24 (18)
Anti-arrhythmic, n (%)
39 (29)
ICD, n (%)
25 (19)
ECG=electrocardiogram; ICD=implantable cardiac defibrillator; LV=left ventricle; RV=right
ventricle; SAECG=signal-averaged electrocardiogram; WMA=wall motion abnormalities
22
DOI: 10.1161/CIRCGENETICS.113.000288
Table 2 – Mutation screening results
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Locus
Exon
NT
AA
Functional meaning
N. of patients
PKP2
1
1
1
1
2
2
3
3
3
7
10
10
12
12
148_151delACAG
c.175C>T
c.184C>A
c.184_185delCA
c.227A>G
c.336G>T
c.627C>G
c.631C>T
c.663C>A
c.1643delG
c.2009delC;
c.2119C>T
c.2333T>C
c.2447_2448delCC
T50SfsX61
Q59X
Q62K
Q62DfsX23
N76S
K112N
S209R
Q211X
Y221X
G548VfsX15
K672RfsX12
Q707X
I778T
T816RfsX10
Frameshift
Nonsense
Missense
Frameshift
Missense
Missense
Missense
Nonsense
Nonsense
Frameshift
Frameshift
Nonsense
Missense
sense
Frameshift
eshi
es
hift
hi
ft
9+2*
1
2*
2
2
2
1*
1*
1*
4
7
7+7*
1+1*
1+1
3+1*
3+
1*
DSP
1
Intron2
Intron3
tron3
7
7
9
111
11
23
23
23
2233
23
23
23
24
24
c.88 G>A
c.273+5G>A
c.423-1G>A
c 423-1G>A
cc.817C>T
.817C>T
cc.897C>G
c.
89
97C
7C>G
>G
G
c.1124A>T
c.112
24A
A>T
T
c.1372A>T
c.13772A
A>T
T
c.1408A>G
c.14
14008A>
14
A G
c.3337C>T
c.33
c.
3337C>T
T
c.3764G>A
c.37
3764
644G>
G>A
A
c.3774C>A
c.4803G>A
c.48
c.
4803
48
033G>
03G>
G>A
A
c.4961T>C
c 49
4961
4961T>C
61T>
T>C
C
c. 4973C>T
c.3203_3204delAG
c.7622G>A
c.7039A>G
V30M
Missense
sense
Splicing
cing
Splicing
Nonsense
Nons
nssense
Missense
Miss
Mi
ssen
ss
e se
Missense
Missseense
s
se
Missense
Missseense
se
Missense
M ssen
Mi
ensee
Nonsense
Nons
sen
ense
Missense
Miss
Mi
s en
ss
e se
Missense
Missense
Misssen
Mi
Miss
ense
se
Missense
Miss
Mi
ssen
ense
se
Missense
Frameshift
Missense
Missense
7+1*
7+1*
3
3
4
10
1
1**
3
2
4
2*
2
4
6
6*
1*
2*
2+1*
1*
4
4
5
6
7
8
8
8
9
9
Intron12
13
14
15
15
15
c.260A>G
c.298G>C
c.437G>A
c.689A>G
c.797A>G
c.880A>G
c.890A>G
c.991G>A
c.1174G>A
c.1250_1253dupATGA
c.1880-2G>A
c.1912G>A
c.2033G>C
c.2491C>T
c.2990delG
c.2773C>T
G638R
G678A
L831F
G997VfsX20
P925S
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Frameshift
Splicing
Missense
Missense
Missense
Frameshift
Missense
1
2
1*
1+1*
4
3
1*
1+2*
5
2+1*
2*
1*
4
5+2*
2+1*
2*
8
c.1034T>C
I345T
Missense
1
DSG2
DSC2
Q273X
Q273
73
3X
S299R
S
S2
99
9R
N375I
N
3755I
N458Y
N
458
8Y
K470E
K470
K4
70E
70
E
R1113X
R111
13X
3
R1255K
R
R1
2 5K
25
K
D1258E
M1601I
M160
M1
601I
60
1II
L1654P
L165
L1
654P
4P
S1658F
p.E1068VfsX19
R2541K
I2347V
Y87C
G100R
R146H
E230G
N266S
K294E
D297G
E331K
V392I
D419X
*Indicates the number of patients with the mutation in compound or double heterozygotes
.
23
DOI: 10.1161/CIRCGENETICS.113.000288
Table 3 – Complex genotypes with multiple DS-gene mutations
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Age at last FU
Gender
(yrs)
Gene/exon mutation1
Gene/exon mutation2
Gene/exon mutation3 Task Force Criteria
Arrhythmic events
(age)
54
M
PKP2/ex3 S209R
PKP2/ex12 T816RfsX10
DSG2/ex5 R146H
Definite
48
M
PKP2/ex3 Q211X
PKP2/ex12 I778T
DSP/ex23 R1255K
Definite
77
49
F
F
PKP2/ex3 Y211X
PKP2/ex1 Q62K
DSP/ex23 R1255K
PKP2/ex10 Q707X
DSP/ex23 L1654P
Borderline
Definite
56
M
PKP2/ex1 Q62K
PKP2/ex10 Q707X
Definite
59
F
DSP/ex23 L1654P
PKP2/ex10 Q707X
Definite
Aborted SCD
(42)
Aborted SCD
(39)
Aborted SCD
(36)
-
21
F
DSP/ex23 L1654P
PKP2/ex10 Q707X
P
Phenotype
negative
-
67
40
M
M
DSP/ex23 L
L1654P
L1
16554P
DSP/ex23 L1
L1654P
L
1654P
PKP2/ex10
PK
KP2
P2/eex1
x 0 Q707X
Q 07
Q7
07X
PKP2/ex10
P
PK
P22/ex1
x 0 Q707X
Q707
07X
Definite
Definite
-
18
F
DSP/ex23 L1654P
L 65
L165
654P
4P
PKP2/ex10
PKP2
PK
P /ex1
P2
x10
x1
0 Q707X
Q707
Q7
0 X
07
Phenotype
negative
P
-
48
F
DSG2/ex6 E230G
E
DSG2/ex8
DS
SG2
G2/e
/ x88 D297G
D297
D2
97G
97
G
Definite
50
F
DSP/ex9 N375I
DSG2/ex13 G638A
Definite
42
F
DSP/ex23 S1658F
DSP/ex24 I2347V
Definite
Sustained VT
(40)
Sustained VT
(45)
-
23
F
DSP/ex1 V30M
DSP/ex24 R2541K
Definite
-
26
M
DSG2/ex9 D419X
DSG2/ex15 G997VfsX40
Definite
-
63
F
DSG2/ex8 E331K
DSG2/int12 c.1881-2G>A
Definite
62
F
DSG2/ex8 E331K
DSG2/int12 c.1881-2G>A
Phenotype negative
61
M
PKP2/ex1 T50SfsX61
DSG2/ex9 V392I
Definite
60
F
PKP2/ex1 T50SfsX61
DSG2/ex9 V392I
Definite
Sustained VT
(56)
Sustained VT
(32)
-
23
F
DSP/ex23 p.Glu1068ValfsX19
DSG2/ex15 p.Pro925Ser
Phenotype negative
-
32
M
DSP/ex23 p.Glu1068ValfsX19
DSG2/ex15 p.Pro925Ser
Definite
Sustained VT
(17)
CH=compound heterozygosis; DH=digenic heterozygosis; F=female; M=male; FU=follow up; SD=sudden death; VT=ventricular tachycardia.
24
DOI: 10.1161/CIRCGENETICS.113.000288
Table 4 – Disease penetrance in the population of DS-gene carriers (2010 ITF Criteria).
Overall
N=134
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Morpho-functional criteria
Major
43 (32)
Right ventricular regional wall motion abnormalities + LAX RVOT >32 mm and/or SAX
RVOT 36 mm > and/or FAC <33%
Minor
8 (6)
Right ventricular regional wall motion abnormalities + LAX RVOT 29-32mm and/or SAX
RVOT 32-36 mm and/or FAC 33-40%
Electrocardiographic Criteria
Major
42 (31)
TWI in V1-V3 in the absence of complete right bundle branch block
12 (9)
Epsilon waves in the right precordial leads
Minor
13 (9)
jeects wi
w
th
TWI in V1-V2 only or right precordial TWI extending beyond lead V3 in subjects
with
complete bundle branch block
68 (51)
68
Post-potentials on SAECG or terminal QRS activation time > 50 msec
Arrhythmic criteria
Major
111 (8)
a ned or susta
ain
tained VT with a left
ta
lefft bundle branchh block/left
block/left
f axi
is deviation
Non-sustained
sustained
axis
morphology
ogy
ogy
Minor
Non-sustained
bundle
a need or
aine
o sustained
sus
usta
sta
tained
ed
d VT
T with
with a left
lef
eftt bu
bund
n le
nd
l branch
bra
ranc
n h block/right
bloock/
k/ri
righ
ri
g t axis
gh
axis
is ddeviation
eviatiion or
unknown morphology
24-hour
Holter
monitoring
morpho
h logy and/or
and
d/o
or >500 premature
premature ectopic bbeats
eats on 24
4-hhour H
ollter moni
itoring
41 (31)
Diagnosis:
d
Established
ccausing
ausing mutation
muta
ta
tati
ati
tion
on ((major
majo
ma
majo
jor criteria)
crrit
crit
iter
eria
ia)) + at lleast
east
ea
st oone
st
ne oother
ther
th
er m
ajor
aj
orr criteria
criite
teri
ria or
ria
or ttwo
wo m
inor
in
or
Disease ca
major
minor
599 (44)
criteria
Borderline
Disease causing mutation (major criteria) + one minor criteria
19 (14)
FAC=fractional area change; LAX=long axis view; RVOT=right ventricular outflow tract; SAECG=signal
averaged electrocardiogram; SAX=short axis view; TWI=T-wave inversion; VT=ventricular tachycardia.
Table 5. Genetic determinants of arrhythmic events since birth.
Univariate analysis
HR
95% CI
P
2.24
0.92-5.46
0.07
3.01
1.42-6.37
0.004
1.53
0.53-4.42
0.4321
Gender (male)
Multiple mutations
Non-missense mutations*
DS genes*,#
PKP2
1
DSP
1.41
0.28-3.05
0.89
DSG
1.53
0.38-6.15
0.38
* among patients with single mutation.
# The single carrier of DSC-2 gene mutation was excluded from the analysis.
25
Multivariable analysis
HR
95% CI
P
2.76
1.19- 6.41
0.02
3.71
1.54-8.92
0.003
DOI: 10.1161/CIRCGENETICS.113.000288
Table 6. Non genotyped relatives with premature (<35 years of age) sudden cardiac death
Age
(years)
34
15
15
Sex
M
M
M
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Mutation(s) in
Family
PKP2
DSP
DSP
Exon
10
7
23
NT
2009delC
897C>G
3774C>A
AA
N670TfsX14
S299R
D1258E
34
M
PKP2
PKP2
DSG2
12
3
5
2447_2448delCC
625C>G
437G>A
T816RfsX10
S209R
R146H
30
M
PKP2
PKP2
DSP
3
12
23
631C>T
2333T>C
3764G>A
Q211X
I778T
R1255K
22
24
M
M
PKP2
PKP2
10
1
2119C>T
184C>A
Q707X
Q62K
Q62K
30
M
DSP
DSP
1
24
88G>A
7622G>A
M
V30M
R254
R2
541K
54
1K
R2541K
26
M
21
F
DSP
DSG
DS
G
DSG
PKP2
P22
PKP2
23
9
1
4961T>C
1174G>A
11
174
74G>
G>A
A
145-148del
145-1448d
14
8dell CGA
CGA
L1654P
V3921
V392
V3
9211
92
T50SfsX10
T5
50SfsX
X100
15
M
DSP
D P
DS
DSP
D P
DS
233
24
c.4973
c 49
c.
4 73 C>T
C>T
>
c.7039A>G
c.70
7 399A>
A G
S1658F
S165
658F
8
I2347F
24
F
DSG
D G
DS
DSG
DSG
DS
G
6
6
689A>G
689A
A>G
G
890A>G
890A
89
0A>G
0A
>G
G
E230G
D297G
D297G
D2
F=female; M=male
M=
=ma
male
le
Figure Legends:
Figure 1: Kaplan-Meier analysis of survival from the composite arrhythmic end-point in the
study population according to sex.
Figure 2: Kaplan-Meier analysis of survival from the composite arrhythmic end-point in the
study population according to the presence of multiple mutations
26
DOI: 10.1161/CIRCGENETICS.113.000288
Figure 3: Kaplan-Meier analysis of survival from the composite arrhythmic end-point in the
subgroup with single mutation according to the defective DS-gene. The single carrier of DSC-2
gene mutation was excluded from the analysis.
Figure 4: Kaplan-Meier analysis of survival from the composite arrhythmic end-point in the
subgroup with single mutation according to the functional meaning of the mutation.
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Figure 5: Kaplan-Meier analysis of survival from the composite arrhythmic
ythmic end-point in
n the
th
entire study population according to gender and the presence of multiple
mutations.
ple mut
tattions.
SM=single mutation;
mu
uta
tati
tiion
on;; MM=multiple
MM=
MM
=multiple mutations
27
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Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
Downloaded from http://circgenetics.ahajournals.org/ by guest on June 17, 2017
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Compound and Digenic Heterozygosity Predicts Life-Time Arrhythmic Outcome and Sudden
Cardiac Death in Desmosomal Gene-Related Arrhythmogenic Right Ventricular
Cardiomyopathy
Ilaria Rigato, Barbara Bauce, Alessandra Rampazzo, Alessandro Zorzi, Kalliopi Pilichou, Elisa
Mazzotti, Federico Migliore, Martina Perazzolo Marra, Alessandra Lorenzon, Marzia De Bortoli,
Martina Calore, Andrea Nava, Luciano Daliento, Dario Gregori, Sabino Iliceto, Gaetano Thiene,
Cristina Basso and Domenico Corrado
Circ Cardiovasc Genet. published online September 26, 2013;
Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas,
TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 1942-325X. Online ISSN: 1942-3268
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circgenetics.ahajournals.org/content/early/2013/09/26/CIRCGENETICS.113.000288
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
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