APPENDIX
Methods
Patient recruitment
Inclusion criteria were: 1) DCM characterized by a left ventricle ejection fraction (LVEF) <50% and an
indexed left ventricular end diastolic diameter LVEDDI >33 mm/m2 (men) or >32 mm/m2
(women)(10); 2) endomyocardial biopsy performed; 3) genetic evaluation; 4) age ≥18 years; 5)
written informed consent.
Exclusion criteria included presence of:
1) previous history of myocardial infarction and/or significant coronary artery disease (stenosis
>50%) using coronary angiography;
2) primary valvular disease (mitral regurgitation grade≥3, aortic regurgitation grade≥2, or aortic
stenosis < 1cm2 );
3) hypertensive heart disease
4) congenital heart disease;
5) (suspected) acute myocarditis defined by either an episode of viral prodromes (febrile
infection of the bronchial tree, the gut, or the urinary tract) within the last 6 months and at
least one of the following features not related to myocardial ischemia: increase in serum
concentrations of myocardial necrosis markers, pericardial effusion, (non)sustained
ventricular tachycardia or ventricular fibrillation of unknown origin and impaired global or
regional left ventricular systolic function;
6)
(likely) diagnosis of arrhythmogenic right ventricular dysplasia
Patients with possible reversible DCM (tachycardiomyopathy, alcohol-induced, chemotherapyinduced) were optimally treated for at least 6 months before they were included in the registry.
1
Patients with previously unknown systemic diseases which were diagnosed during the clinical
diagnostic work-up were included in the registry. Distribution of systemic diseases known to be
associated with DCM included rheumatoid arthritis (n=6), sarcoidosis (n=4), vasculitis (n=2), systemic
lupus (n=1), polymyositis (n=1), Crohn’s disease (n=1), morbus Bechterew (n=1), coeliac disease
(n=1).
Transthoracic echocardiography
Echocardiographic measurements were performed in the standard parasternal, apical and
subxiphoidal views according to the recommendations of the American Society of Echocardiography
(Sonos 5500 and iE33; Philips Medical Systems, Best, The Netherlands)(10). Two independent
echocardiographers blinded to patient details performed the measurements, including left
ventricular (LV) end-diastolic (EDD) and end-systolic diameter (LVESD), end-diastolic thickness of the
interventricular septum (IVS) and LV posterior wall (LVPW). LV mass was calculated as previously
defined. LVEF was measured with Simpson’s biplane method from apical view. LV reversed
remodeling (LVRR) was defined as an absolute increase in LVEF of ≥10% or a LVEF of ≥50% in
combination with a decrease in LVEDDI of ≥10% or LVEDDI ≤33 mm/m2(11).
Endomyocardial biopsy
Right ventricular EMBs were obtained using a transcatheter bioptome (Cordis, Miami, FL, USA). A
total of six endomyocardial biopsies were taken from the right ventricle. Two to three specimens
were used for immunohistological analysis and three for the detection of viral genomes.
Histopathological examinations were done on 4 um-thick tissue section from formalin-fixed, paraffinembedded EMBs stained using hematoxylin, eosin, Sirius red, CD3+, CD45+ and CD68+ antibodies
according to the manufacturer’s protocol (DAKO, Glostrup, Denmark) and further quantified as
described previously (13). Patients with active, fulminant or giant cell myocarditis were excluded
(n=22). Increased cardiac inflammation is defined as ≥14 infiltrating cells per mm2, according to the
2
task force of the World Heart Federation’s Council on Cardiomyopathies (14). Collagen volume
fraction (CVF) was quantified as percentage Sirius red stained area per total myocardial tissue area,
excluding perivascular and endocardial fibrosis (13).
Three frozen myocardial specimens were used for polymerase chain reaction (PCR) and reverse
transcriptase PCR analysis to detect the presence of cardiotropic viruses. DNA of parvovirus B19
(PVB19), Human herpes virus-6 (HHV-6), adenovirus, and Epstein-Barr virus (EBV) was extracted from
three pooled endomyocardial biopsies using a QIAmp DNA blood mini kit (Qiagen, Venlo, The
Netherlands). Extractions were performed according to the manufacturer’s instructions. DNA
concentrations were determined using a nanodrop instrument (Thermo Fischer Scientific,
Wilmington, DE, USA). RNA for enterovirus detections was isolated using TRIZOL reagents
(Invitrogen, Paisley, Scotland, UK). Before extraction, all samples were spiked with murine
cytomegalovirus (CMV) DNA or RNA, which was used as an extraction and amplification control.
Reverse transcription was performed using Taqman reverse transcriptase reagents (Applied
Biosystems, Foster City, CA, USA). Primers and probe sequences were obtained from the literature,
as previously described (15). The PCR mix consisted of 20 µl isolated DNA, final concentrations of 600
nM of each primer and 200 nM of the probe and 1x absolute QPCR mix (Abgene, Epsom, UK). All
probes were labelled with FAMTM as a reporter dye and TAMRATM as a quencher dye. All real-time
PCR reactions were performed using an ABI prism 7000 (Applied Biosystems). The PCR assay used has
a linear quantitative range from 1.0x102-1.0x108 copies with a detection probability above 95%.
Below this range semiquantitative detection is performed by extrapolation of the standard curve. We
evaluated the variation of each DNA concentrations in the standard curve over a period of 3 months.
Our results showed that standard deviation was ≤0.15 log10 for all concentrations >500 copies/ml.
Only for the two lowest concentrations (500 and 200 copies/ml) standard deviations up to a
maximum of 0.38 log10 were measured. The quality of the assays was assured by positive and
negative controls as well as a test on amplification inhibition in each sample by an external
amplification control. For quantification of viral loads, standard curves were included in each run.
3
Significant viral load was defined as ≥500 copies per microgram DNA, as previously described (16).
4
MOGES
Organ involvement
Organ involvement is routinely assessed by the clinical geneticist with additional standardized
questionnaires. extra-cardiac organ involvement was assessed according to the proposed organs in
the MOGES classification, i.e. muscle-skeletal, nervous, cutaneous, eye, auditory, kidney,
gastrointestinal, skeletal. Data considering extra-cardiac organ involvement such as dysphagia,
severe obstipation indicating additional diagnostics and chronic medication, congenital deafness,
objective muscular weakness or dystrophy with or without increased creatinine kinase levels,
vascular purpura, alopecia, glomerulonephritis, epilepsia, congenital blindness, uveitis, episcleritis)
were collected from the patient records and a standardized questionnaire, and reviewed by two
investigators. Disagreements were settled by consensus. Extra-cardiac organ involvement was
considered positive if the condition was proven by a specialist and has a known or suspected relation
to DCM. Extra-cardiac organ involvement most likely secondary due to heart failure or treatment was
not considered as organ involvement for the MOGES classification.
Genetic or familial DCM
All index patients underwent genetic counseling and testing. A minimum of three generations family
history of cardiomyopathy and/or sudden cardiac death was drafted by a clinical geneticist.
Systematic non-invasive cardiac evaluation including electrocardiogram and echocardiography was
performed in all first-degree relatives of probands who consented, as indicated.(17) Familial
inheritance was based on the presence of two or more affected individuals in a single family or in the
presence of a first-degree relative with well documented unexplained sudden cardiac death (18) at
<60 years of age. The selection of tested genes was based on the expertise of the clinical geneticist.
In specific cases of cardiomyopathies, non-cardiac features or mixed-phenotypes were initially
considered based on clinical assessment, leading to the evaluation of additional genes. During the
inclusion period of 10 years we screened genes consecutively, based upon the knowledge at that
5
time. This strategy changed over time, due to increasing knowledge and improving techniques, new
genes were identified and tested accordingly. All genes were analyzed using Sanger sequencing
(details available upon request). Variants were classified in 5 different classes: Pathogenic (Path),
Likely Pathogenic, variant of clinical unknown significance (VUS), likely benign or benign. Both
pathogenic and likely pathogenic mutations were classified as pathogenic mutations. All others were
considered as non-pathogenic.
Genetic/familial DCM was classified as presence of familial inheritance pattern and/or presence of a
pathogenic mutation.
Rhythm disturbances or toxic exposure
DCM is categorized as due to rhythm disturbances in case of initial tachycardia-induced
cardiomyopathy (atrial fibrillation or atrial tachycardia with a mean ventricular response of >100 bpm
recorded on 24-hour Holter), however did not improve after 6 months optimal treatment and were
referred for further evaluation of the DCM. Secondly, premature ventricular complex (PVC)-induced
cardiomyopathy (≥20% PVCs of total QRS-complexes) was also characterized as a rhythm disturbance
associated with development of DCM.(7-9)
DCM is categorized as toxic if the cardiomyopathy is related to heavy alcohol use (>8 oz/day of
ethanol for 6 months) or abuse of cocaine, ecstasy or heroin; or if the cardiomyopathy is related to
cancer therapy (doxorubicin, daunorubicin, trastuzumab, epirubicin, mitoxantrone, rituximab,
idarubicin, Taxane, fluorouracil and/or radiation in the chest area).(5-9)
References for supplementary table 2
The following references indicate the articles validating the (likely) pathogenic nature of the
respective mutations shown in supplementary table 2.
6
31.
Karkkainen S, Helio T, Jaaskelainen P et al. Two novel mutations in the beta-myosin heavy
chain gene associated with dilated cardiomyopathy. Eur J Heart Fail 2004;6:861-8.
32.
Dausse E, Komajda M, Fetler L et al. Familial hypertrophic cardiomyopathy. Microsatellite
haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain
gene. J Clin Invest 1993;92:2807-13.
33.
Scharner J, Brown CA, Bower M et al. Novel LMNA mutations in patients with Emery-Dreifuss
muscular dystrophy and functional characterization of four LMNA mutations. Hum Mutat
2011;32:152-67.
34.
Benedetti S, Menditto I, Degano M et al. Phenotypic clustering of lamin A/C mutations in
neuromuscular patients. Neurology 2007;69:1285-92.
35.
Girolami F, Ho CY, Semsarian C et al. Clinical features and outcome of hypertrophic
cardiomyopathy associated with triple sarcomere protein gene mutations. J Am Coll Cardiol
2010;55:1444-53.
36.
Rodriguez-Garcia MI, Monserrat L, Ortiz M et al. Screening mutations in myosin binding
protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy. BMC Med Genet
2010;11:67.
37.
DeWitt MM, MacLeod HM, Soliven B et al. Phospholamban R14 deletion results in late-onset,
mild, hereditary dilated cardiomyopathy. J Am Coll Cardiol 2006;48:1396-8.
38.
Kapplinger JD, Tester DJ, Alders M et al. An international compendium of mutations in the
SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic
testing. Heart Rhythm 2010;7:33-46.
39.
Hegde MR, Chin EL, Mulle JG et al. Microarray-based mutation detection in the dystrophin
gene. Hum Mutat 2008;29:1091-9.
7
Supplemental Table 1. Baseline characteristics of DCM patients with and without complete
diagnostic work-up for DCM
Baseline variables
Complete work-up
Incomplete work-up
P value
N=213
N=181
Age of onset (years)
51±13
50±13
0.65
Gender (M/F)
128/85
41/29
0.89
HR (bpm)
74±15
79±19
0.14
SBP (mmHg)
129±20
127±21
0.32
DBP (mmHg)
79±12
78±14
0.28
Body height (cm)
175±9,6
174±9
0.80
Body weight (kg)
82±18
82±19
0.83
BSA
1,6±0,27
1,7±0,25
0.72
Sympt duration to
4 [1-19]
5 [1-21]
0.50
LBBB
55 (26)
38 (21)
0.28
AV block
10 (5)
7 (4)
0.64
AF
36 (17)
35 (20)
0.48
LVEF
29±11
29±11
0.69
LVEDD
62±8
61±9
0.48
LVEDDI
40±8
40±8
0.65
History of HT
83 (40)
71 (39)
0.97
Hyperlipidemia
36 (17)
29 (15)
0.46
OSAS
21 (10)
15 (8)
0.58
Diabetes Mellitus
23 (11)
21 (12)
0.32
Hyperthyroidism
3 (1)
6 (3)
0.32
Hypothyroidism
6 (3)
5 (3)
1.00
Systemic disease
17 (8)
presentation (months)
ECG
Echocardiographic
Co-morbidities
EMB performed
N=213
N=107
Cardiac inflammation
87 (41)
46 (45)
0.74
CD3+ (cells/mm2)
4.7 [2.4- 8.3]
5.2 [2.6-9.1]
0.65
CD45+ (cells/mm2)
8.5 [5.5-13]
9.4 [5.4-13]
0.98
CD68+ (cells/mm2)
3.0 [1.2-6.2]
3.3 [1.4-7.0]
0.57
5.7 [3-10]
6.6 [3.8-12]
0.22
Fibrosis (%)
8
162 (76)
91 (85)
0.31
PVB19
152 (75)
86 (80)
0.27
HHV6
48 (23)
21 (20)
0.42
EBV
11 (5)
5 (5)
0.80
Adenovirus
4 (2)
1 (1)
0.66
Enterovirus
0 (0)
2 (2)
0.13
Duration (months)
47 [30-67]
48 [30-68]
0.
All-cause death
15 (7)
18 (10)
0.30
Htx
1 (1)
2 (1)
0.60
Life-threat. arrhythmia
12 (6)
11 (6)
0.85
CEP
27 (13)
24 (13)
0.86
Lost-to-follow-up
0 (0)
3 (2)
0.10
Viral presence
Follow-up
Abbreviations, M indicates male; F, female; bpm, beats per minute; SBP, systolic blood pressure; DBP, diastolic
blood pressure; BSA, body surface area; OHCA, out-of-hospital cardiac arrest; sx, symptoms; LBBB, left bundle
branch block; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic diameter; HT,
hypertension; OSAS, obstructive sleep apnea syndrome; EMB, endomyocardial biopsy; PVB19, Parvovirus B19;
HHV6, human herpes virus-6; EBV, Ebstein-Barr virus; Htx, heart transplantation; Life Threat. Arrhythmia, lifethreatening arrhythmia; CEP, combined end-point
9
Supplemental Table 2. Overview of 18 identified mutations in 16 patients of 213 index patients with dilated cardiomyopathy
Gene
Nucleotide change
Amino acid change
References in online
appendix
(31)
(19)
This study
(32)A
This study
MAF
Add evidence in own MUMC lab
p.(Arg1500Trp)
p.(Asp1096Tyr)
p.(Glu981Lys)
p.(Arg403Trp)
p.(p.Phe735Valfs*3)
No. of index
patients
1
1
1
1
1
MYH7
c.4498C>T
c.3286G>T
c.2941G>A
c.1207C>T
c.2201dupA
Not
Yes (MAF=0.0002)
Not
Not
Not
Coiled-coil disturbance in silico
Coiled-coil disturbance in silico
Coiled-coil disturbance in silico
n.a.
n.a.
LMNA
c.810G>A
p.(Lys270Lys)
1
(33)
Not
c.992G>A and c.1879C>T
p.(Arg331Gln) and
p.(Arg627Cys)
1
(34)
This study (Arg627Cys)
Not
aberrant RNA splicing, nuclear
malformations in patient fibroblast
Nuclear malformations
MYPBC3
c.2905C>T
c.3065G>C (+ PLN:
c.40_42delAGA)
p.(Gln969Ter)
p(.Arg1022Pro) (+PLN:
p.(Arg14del))
1
1
(35)B
(36)B
Not
Not
n.a.
n.a.
PLN
c.40_42delAGA
p.(Arg14del)
2
(37)
Not
n.a.
SCN5A
c.2482C>T
c1858C>T
p.(Leu828Phe)
p.(Arg620Cys)
1
1
unpublished C
(38)
Not
Not
Functional characterized
n.a.
BAG3
c.910C>T
p.(Gln304Ter)
1
This study
Not
n.a.
MYPN
c.806C>T
p.(Pro269Leu)
1
This study
Not
Ig-domain affected in silico
DMD
Inframe deletion of exon
45 - 55
p.Glu2147_Gln2739del
1
(39)
Not
Inframe deletion of exon 45-55
MIT
m.15546A>G
p.(His267Arg)
1
This study
Not (in mtDB or
Cytochrome B, heteroplasmic in
10
mtSNP)
skeletal muscle, urine, blood
Abbreviations:
MAF: Allele frequencies present in Exome sequencing project (EVS) variants (MAF), or present in Human mitochondrial genome database (mtDB or mtSNP)
A
: DCM phenotype without signs of (end-stage) HCM, and presence of HHV6 inflammatory cardiomyopathy according to EMB.
B
: both DCM phenotype without signs of (end-stage) HCM
C
: functionally characterized, data not shown (available upon request, abstract presented at Heart Rhythm 2012)
11
Supplemental Table 3. Echocardiographic measurements of patients with or without left
ventricular reversed remodeling after a mean of 12 months.
Echo FU
LVRR- (A)
LVRR+ (B)
P value
N=198
N =128
N = 70
A vs B
LVEF
29±11
32±10
23±9
<0.001
LVEDD
62±8
60±8
66±8
<0.001
LVESD
53±10
49±10
58±9
<0.001
LVEDDI
40±7,5
40±7
42±8
0.08
IVST
8.7±1.4
8.5±1.4
9±1.4
<0.05
PWT
8.8±1.4
8.6±1.4
9.1±1.3
0.009
LV-mass
234±74
216±71
267±69
<0.001
LV-mass index
152±45
143±42
169±44
<0.001
E/A
1.2±0.7
1.1±0.7
1.4±0.8
0.08
LVEF
40±11
37±10
46±9
<0.001
LVEDD
57±8
59±8
54±6
<0.001
LVESD
46±9
48±9
42±7
<0.001
LVEDDI
38±7.0
39±7
34±6
<0.001
IVST
8.7±1.3
8.5±1.2
9.3±1.3
<0.001
PWT
8.7±1.3
8.5±1.2
9.1±1.3
<0.001
LV-mass
201±63
203±69
196±51
0.47
LV-mass index
101±31
104±33
97±21
0.10
E/A
0.9±0.4
1.0±0.4
1.0±0.3
0.57
Baseline echo
Follow-up echo (12 months)
Abbreviations: Echo FU, echo follow-up; LVRR-, no presence of left-ventricular reversed remodeling; LVRR+,
presence of left-ventricular reversed remodeling; LVEF, left ventricular ejection fraction; LVEDD, left
ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; LVEDDI, indexed left
ventricular end-diastolic diameter; IVST, interventricular septum thickness; PWT, posterior wall thickness; LV,
left ventricular
12
Supplemental Figure 1.
13
Supplemental Figure 2.
14
Supplemental Figure 3.
15