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Clinical genetics and outcome of left ventricular
non-compaction cardiomyopathy
Farbod Sedaghat-Hamedani
1,2†
, Jan Haas
1,2†
, Feng Zhu
1,3†
, Christian Geier
4,5
,
Elham Kayvanpour
1,2
, Martin Liss
5,6
, Alan Lai
1,2
, Karen Frese
1,2
,
Regina Pribe-Wolferts
1
, Ali Amr
1,2
, Daniel Tian Li
1,2
, Omid Shirvani Samani
1,2
,
Avisha Carstensen
1
, Diana Martins Bordalo
1,2
, Marion Mu¨ ller
1,2
, Christine Fischer
8
,
Jing Shao
3
, Jing Wang
9
, Ming Nie
3
, Li Yuan
10
, Sabine Haßfeld
11
, Christine Schwartz
4
,
Min Zhou
12
, Zihua Zhou
3
, Yanwen Shu
3
, Min Wang
3
, Kai Huang
3
, Qiutang Zeng
3
,
Longxian Cheng
3
, Tobias Fehlmann
7
, Philipp Ehlermann
1
, Andreas Keller
7
,
Christoph Dieterich
2,13
, Katrin Streckfuß-Bo¨ meke
14,15
, Yuhua Liao
3
,
Michael Gotthardt
5,6
, Hugo A. Katus
1,2
*, and Benjamin Meder
1,2
*
1
Department of Medicine III, Institute for Cardiomyopathies Heidelberg (ICH), University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany;
2
DZHK
(German Centre for Cardiovascular Research), Heidelberg, Germany;
3
Department of Cardiology, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology, 430022 Wuhan, China;
4
Experimental and Clinical Research Center (ECRC), A Joint Cooperation of Charite´ Medical Faculty and Max
Delbru¨ck Center for Molecular Medicine (MDC), Augustenburger Platz 1, 13353 Berlin, Germany;
5
DZHK (German Centre for Cardiovascular Research), Berlin, Germany;
6
Neuromuscular and Cardiovascular Cell Biology, Max Delbru¨ ck Center for Molecular Medicine, Robert-Ro¨ ssle-Str. 10, 13092 Berlin, Germany;
7
Department of Bioinformatics,
University of Saarland, Building E2.1, 66123 Saarbru¨ cken, Germany;
8
Department of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg,
Germany;
9
Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan, China;
10
Department of
Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan, China;
11
Department of Cardiology, Virchow Klinikum,
Charite´ University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany;
12
Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan, China;
13
Department of Medicine III, Klaus Tschira Institute for Computational Cardiology, University of Heidelberg, Im
Neuenheimer Feld 410, 69120 Heidelberg, Germany;
14
Department of Cardiology and Pneumology, Georg-August-University Go¨ ttingen, Robert-Koch-Str. 40, 37075 Go¨ ttingen,
Germany; and
15
DZHK (German Centre for Cardiovascular Research), Go¨ ttingen, Germany
Received 12 October 2016; revised 29 May 2017; editorial decision 2 September 2017; accepted 20 September 2017; online publish-ahead-of-print 6 October 2017
Aims In this study, we aimed to clinically and genetically characterize LVNC patients and investigate the prevalence of
variants in known and novel LVNC disease genes.
........................................................................ ............. ............. ............. .................. ............. ............. .................. ......................
Introduction Left ventricular non-compaction cardiomyopathy (LVNC) is an increasingly recognized cause of heart failure, ar-
rhythmia, thromboembolism, and sudden cardiac death. We sought here to dissect its genetic causes, phenotypic
presentation and outcome.
........................................................................ ............. ............. ............. .................. ............. ............. .................. ......................
Methods
and results
In our registry with follow-up of in the median 61 months, we analysed 95 LVNC patients (68 unrelated index pa-
tients and 27 affected relatives; definite familial LVNC = 23.5%) by cardiac phenotyping, molecular biomarkers and
exome sequencing. Cardiovascular events were significantly more frequent in LVNC patients compared with an
age-matched group of patients with non-ischaemic dilated cardiomyopathy (hazard ratio = 2.481, P= 0.002).
Stringent genetic classification according to ACMG guidelines revealed that TTN, LMNA, and MYBPC3 are the most
prevalent disease genes (13 patients are carrying a pathogenic truncating TTN variant, odds ratio = 40.7, Confidence
interval = 21.6–76.6, P< 0.0001, percent spliced in 76–100%). We also identified novel candidate genes for LVNC.
For RBM20, we were able to perform detailed familial, molecular and functional studies. We show that the novel
* Corresponding author. Tel: þ49 6221 564835, Fax: þ49 6221 564645, Email: Benjamin.Meder@med.uni-heidelberg.de
†
The first three authors contributed equally to the study.
Published on behalf of the European Society of Cardiology. All rights reserved. V
CThe Author 2017. For permissions, please email: journals.permissions@oup.com.
European Heart Journal (2017) 38, 3449–3460 BASIC SCIENCE
doi:10.1093/eurheartj/ehx545 Heart failure/cardiomyopathy
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variant p.R634L in the RS domain of RBM20 co-segregates with LVNC, leading to titin mis-splicing as revealed by
RNA sequencing of heart tissue in mutation carriers, protein analysis, and functional splice-reporter assays.
........................................................................ ............. ............. ............. .................. ............. ............. .................. ......................
Conclusion Our data demonstrate that the clinical course of symptomatic LVNC can be severe. The identified pathogenic vari-
ants and distribution of disease genes—a titin-related pathomechanism is found in every fourth patient—should be
considered in genetic counselling of patients. Pathogenic variants in the nuclear proteins Lamin A/C and RBM20
were associated with worse outcome.
䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏
Keywords RBM20 •Non-compaction cardiomyopathy •Titin •Next-generation whole-Exome sequencing
Translational perspective
In the current study, we show that symptomatic left ventricular
non-compaction (LVNC) patients have unfavourable outcomes in
comparison to patients with dilated cardiomyopathy (DCM). We
provide a comprehensive landscape of genetic variation in LVNC
by applying whole exome sequencing and stringent ACMG crite-
ria for classification and nomenclature of genetic testing results.
By molecular investigations in cardiac tissue and in in vitro sys-
tems, we provide evidence for the causal role of RBM20 and titin
variants for LVNC pathophysiology. Carriers of pathogenic TTN,
LMNA,andRBM20 variants did show unfavourable outcomes.
Introduction
Left ventricular non-compaction cardiomyopathy (LVNC, non-
compaction cardiomyopathy = NCCM) is characterized by the pres-
ence of excessive left ventricular trabeculae, deep intratrabecular
recesses, and a thin compacted myocardial layer.
1
The phenotype of
left ventricular non-compaction or hypertrabeculation is an increas-
ingly recognized finding.
2
It also became clear that the detection of
this morphological trait is not sufficient to define a pathological condi-
tion, as recently found in a population-based imaging study.
1,3
Hence,
estimating the prevalence of cases with a non-compaction phenotype
and that of cases that are also prone to develop the typical triad con-
sisting of heart failure, arrhythmia, and embolism is difficult, present-
ing a unique challenge for larger, prospective trials to assess its
pathogenesis, genetic aetiology, management, and outcomes.
4
The pathogenesis of LVNC is not fully resolved. The inherited
phenotype can arise due to a gene mutation that disrupts the physio-
logical compaction of the developing embryonic myocardium, a pro-
cess that normally progresses from the base to the apex of the heart.
Other cases are observed sporadically or may even be acquired as
observed in highly-trained athletes or in pregnant women.
2,5
Also, in
families with other types of cardiomyopathy (HCM, DCM), a non-
compaction phenotype may be found, which raises the question if
LVNC is a distinct cardiomyopathy or a sub-trait. While the
American Heart Association (AHA) classifies LVNC as an independ-
ent genetic cardiomyopathy, the European Society of Cardiology
(ESC) defined it as an unclassified entity
6
.
From differently sized cohorts of paediatric or adult cases, it can
be estimated that 18–44% of LVNC cases are genetic,
7,8
with
autosomal dominant transmission being the by far most common
mode of inheritance, followed by X-linked and maternal modes.
9
The
available, mostly small-sized cohort studies showed that various un-
related genes are potential disease causes. The genes that have been
reported mainly encode for sarcomeric, Z-disc and nuclear-envelope
proteins including ACTC1,MYH7,MYBPC3,TNNT2,TPM1,TTN,LDB3,
LMNA,andDTNA.
10–14
Others encode mitochondrial proteins such
as TAZ,
12
regulators of the NOTCH pathway,
15
or even ion channels
such as HCN4 as reported recently by our groups.
16
In the present analysis of the ‘Genetics in Non-Compaction
Registry’, we aimed to clinically characterize a larger cohort of symp-
tomatic LVNC cases, assess their longitudinal follow-up in compari-
son to DCM and investigate the prevalence of known and novel
LVNC disease genes. For the first time, we link genetic variants in
RBM20 with LVNC and precisely delineate the molecular down-
stream effects by RNA sequencing, titin protein analysis, and func-
tional assays. We provide the first comprehensive landscape of
genetic variation in LVNC by applying stringent ACMG criteria for
classification and nomenclature, supporting genetic testing and coun-
selling of patients.
Materials and methods
Patient recruitment and clinical evaluation
Consecutive symptomatic index patients with LVNC presenting at partic-
ipating tertiary centres were enrolled if they had given written informed
consent for clinical investigations and genetic testing. The echocardio-
graphic diagnosis for LVNC was based on the criteria of Sto¨llberger
et al.
17
and Jenni et al.
18
A subgroup of index patients additionally received
MRI and fulfilled the criteria by Petersen et al.
19
For more details, please
see Supplementary data online, methods.
Biomaterial processing, genotyping, and
genetic mapping
A peripheral blood sample was collected from each participant.
Sequencing was performed in-house on an Illumina HiSeq 2000.
Demultiplexing of the raw sequencing reads and generation of the fastq
files was done using CASAVA v.1.82.
20
Variant calling and quality filtering
of variants were performed with Genome-Analysis-Toolkit.
21
For anno-
tation of genes and functional variant effects, we primarily used
ANNOVAR.
22–24
For known LVNC disease genes (Figure 2), we applied
ACMG criteria.
25
For more details and for global splicing analysis,
Western Blot,
26
RNA analysis,
27–30
and splice reporter assay
26
please see
Supplementary data online, methods.
3450 F. Sedaghat-Hamedani et al.
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Results
Ventricular arrhythmias are frequent
manifestations of left ventricular
non-compaction cardiomyopathy
In total, n=95probandswithLVNC(68index patients and 27 affected
relatives) were investigated on a phenotypic and genetic level (51 from
Univ. Heidelberg, 34 from Univ. Wuhan, and 10 from Univ. Berlin).
The baseline clinical characteristics of the index patients at time of
blood sampling are listed in Supplementary material online, Table S1.
In all, 23.5% of the patients had a definite familial disease defined by at
least one additionally affected first-degree relative in the family, who
was available for clinical studies at one of the centres (16 pedigrees)
and showed the full phenotype of LVNC. Follow-up was in the me-
dian 61 months. During follow-up of index cases, seven patients
(10.3%) received heart transplantation due to progressive disease.
Clinically relevant arrhythmias such as atrial fibrillation (AF) (29.4%)
and ventricular tachycardia (VT/VF) (35.3%) were frequently docu-
mented findings. As much as 18.2% of implantable cardioverter defib-
rillator (ICD) carriers had an appropriate ICD shock. All outcome-
related events during follow-up are listed in Table 1.
Distribution of non-compacted myocardial segments was as previ-
ously shown typical for LVNC, with apical/lateral segments being
mainly affected, which was not observed in a cohort with (non-
ischaemic) DCM patients (Figure 1A). Left-ventricular ejection frac-
tion was in the mean moderately decreased (LVEF 38% ± 15.3),
attributed to the fact that only symptomatic cases of LVNC were
studied (Figure 1B), while accidental findings of LVNC or probands
from population-based studies were not included. The number of
composite events including cardiovascular (CV) death, sudden car-
diac death (SCD), aborted SCD [appropriate ICD shock, reported
sustained ventricular tachycardia, or cardiopulmonary resuscitation
(CPR)], or HTx was significantly higher in LVNC index cases com-
pared with a cohort of age-matched DCM patients (HR = 2.481,
P=0.002)(Figure 1C). CV death was 10.8% in LVNC (7 from 65) vs.
4.9% in DCM patients (12 from 247); VT was 30.8% in LVNC (20
from 65) vs. 5.7% in DCM patients (14 from 247); HTx rate was 10.8
in LVNC (7 from 65) vs. 2.4% in DCM patients (6 from 247) and CPR
rate was 4.6% in LVNC (3 from 65) vs. 3.6 in DCM patients (9 from
247). The LVEF was higher in the LVNC group compared with the
DCM cohort, showing that the arrhythmogenic substrate in LVNC is
most likely not solely based on reduced ejection fraction.
Prevalence of pathogenic variants in
known left ventricular non-compaction
cardiomyopathy-associated gene loci
To genetically characterize the enrolled patients with LVNC, we per-
formed next-generation exome sequencing in altogether 106 sub-
jects (68 unrelated LVNC probands, 27 affected, and 11 healthy
relatives). When only considering the established disease genes for
LVNC, we find in 38.2% of index patients a rare variant that is classi-
fied as ‘pathogenic’ according to the stringent ‘American College of
Medical Genetics and Genomics’ (ACMG) guidelines for the inter-
pretation of sequence variants (Figure 2,seeSupplementary material
online, Table S4).
The most frequently affected gene was the sarcomeric elastic-fibre
gene TTN, which was very recently described to be associated with
LVNC.
14
From 13 patients that had a pathogenic TTN variant, 13
were truncating variants due to a frameshift insertion/deletion or
Stop-gain, which is a new finding for LVNC and 1 patient had add-
itionally a mis-sense variant. The percent spliced in (PSI) values for
the variant-harbouring exons was 100% in 11 cases of truncating vari-
ants, for 1 it was 76% and 1 was within exon 48 were no reliable PSI
annotation is given [https://cardiodb.org (August 2017)]. The mis-
sense variant p.G4714D was also in an exon with PSI of 100%. The
ODDs ratio for enrichment of truncating TTN variants compared
with EXaC was 40.7 (95% confidence interval: 21.6–76.6; P< 0.0001)
and for none of the pathogenic variant the associated confidence
interval included the 1 (P< 0.0001) (see Supplementary material on
line, Table S4).
The truncating TTN variants were mainly located in the A-band
(Figure 3A), which is a region that harbours most pathogenic titin vari-
ations. From three Stop-gain variant-carriers, we were able to perform
mRNA-sequencing from myocardial biopsies, showing that nonsense-
mediated RNA-decay (NMD) is the likely pathomechanism leading to
loss-of-function of the titin-allele (bar graphs on top of Stop-gain vari-
ants).
31
In four pedigrees, we found segregation of one mis-sense and
three frameshift TTN variants with the LVNC phenotype (Figure 3B).
The second most commonly affected disease gene was LMNA (5%).
For MYBPC3, which is carrying a pathogenic variant in 4% of the index
patients, we report co-segregation of a Splice-variant in a large family
with nine affected individuals all having cardiac non-compaction cardio-
myopathy (see Supplementary material online, Figure S2). For valid-
ation, we performed Sanger sequencing and confirmed this variant as
well as randomly picked further variants, which have been classified as
pathogenic in TTN,LMNA,MYBPC3,orMYH7 (see Supplementary ma
terial online, Table S2 and Figures S3 and S4).
.................................................................................................
Table 1 Follow-up of 68 unrelated left ventricular
non-compaction cardiomyopathy index patients
(median follow-up: 61 months)
Clinical characteristics
Neuromuscular disorders (%) 2 (2.9)
AF (%) 20 (29.4)
VT and/or non-sustained (%) 24 (35.3)
All-cause mortality (%) 9 (13.2)
CV death (%) 8 (11.8)
Successful CPR (%) 3 (4.4)
HTx (%) 7 (10.3)
VAD implantation (%) 2 (2.9)
ICD/CRT-D implantation (%) 22 (32.3)
Appropriate ICD shock (%)
a
4 (18.2)
Stroke/TIA (%) 6 (8.8)
Systemic embolism (%) 7 (10.3)
Syncope (%) 12 (17.6)
AF, atrial fibrillation; CPR, cardiopulmonary resuscitation; CV, cardiovascular;
ICD, implantable cardioverter-defibrillator; CRT, cardiac resynchronization ther-
apy; TIA, transient ischaemic attack; VAD, ventricular assist device; VT, ventricu-
lar tachycardia.
a
Percentage of ICD-carriers.
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Clinical genetics and outome of LVNC 3451
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months follow-up
Composite end-point
DCM
LVNC
P=0.002
DCM CohortLVNC Cohort
LV-EF (%) LV-EF (%)
Distribution of
non-compaction/hypertrabeculation
A
B
C
0%-10%
10%-20%
20%-30%
30%-40%
40%-50%
50%-60%
60%-70%
70%-80%
>80%
Scale
inferior
anterior
lateral
septal
anterior
lateral
septal
inferior
DCM (n) 247 202 146 91 40 13 5 2
LVNC (n)654538272312 9 4
% of patients
% of patients
Number at risk:
10
20
30
10
20
30
Figure 1 Clinical evaluation and event-free survival of LVNC patients compared with DCM cohort. (A) Distribution of the non-compacted myo-
cardium according to 17-segment model in the LVNC index patients compared with DCM. Bulls-eye plot showing the proportion of non-compac-
tion/hypertrabeculation in a 17-segment model of the left ventricle (LV) in LVNC (left) and DCM (right). The percentage of patients with NC/C > 2
is colour coded (middle). (B) Distribution of LVEF in LVNC index patients compared with DCM cohort. (C) Kaplan–Meier survival analysis of the
composite end-point heart failure associated events in 65 patients with LVNC compared with 247 age-matched DCM patients. The rate of HF associ-
ated eventsincluding CV death, SCD, aborted SCD, or HTx is significantly higher in LVNC patients (P=0.002).
3452 F. Sedaghat-Hamedani et al.
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Using this very stringent classification, more than 38.2% can be ex-
plained by a pathogenic variant in a known LVNC disease locus.
However, this underlines that in several probands with either spor-
adic or familial disease, a currently unknown gene could be causally
involved. As such, we find in candidate genes derived from LVNC ani-
mal models, potentially causal variants in the TTN-interaction partner
Obscurin (OBSCN),
32
which is an established disease gene for DCM
and HCM in humans
33,34
(see Supplementary material online, Figure
S5A). In our cohort, Obscurin variants were located near the
Immunoglobin I-set or protein kinase region of the protein. Other
candidate genes include NCOR2 and XIRP2, both resulting in LVNC in
mice models when depleted (see Supplementary material online,
Figure S5B and C). In carriers of variants in these three candidate
genes, no family members were available to show segregation.
Hence, the pathogenic role remains speculative.
RBM20 as a novel disease gene for left
ventricular non-compaction
cardiomyopathy
Using our whole-exome sequencing strategy, we stratified identified
sequence variants for potential pathogenicity using in-silico prediction
and further genetic and clinical work-up. In the RNA binding motif pro-
tein 20 gene (RBM20), an important splice regulator of TTN,wefound
four non-synonymous (p.I413V, p.R634L, p.P798L, and p.P1105L)
rare genetic variants (see Supplementary material online, Table S3),
from which three have a CADD Phred score (p.R634L, p.P798L, and
p.P1105L) that is in agreement with a protein damaging effect. For
the novel variant p.R634L, which is located in a mutational hot spot
of the protein (RS domain), we are able to provide high evidence for
pathogenicity on different levels.
Within pedigree 16 (Figure 4A,seeSupplementary material online,
Table S5), the index patient (III.2) was diagnosed with LVNC at age of
17. He underwent cardiac transplantation due to progressive disease
at the age of 21. The patient’s mother (II.3) also presented with se-
verely reduced LVEF (20%) and with left ventricular non-compaction/
hypertrabeculation at age 39 (Figure 4B). Non-sustained ventricular
tachycardia (nsVT) was recorded in her Holter-ECG. Accordingly,
she received an ICD for primary prevention. Endomyocardial biopsies
of the index patient and his mother showed severe myocardial fibrosis
without signs of myocarditis (Figure 4B). The index patient’s brother
(III.1) died at the age of 16 as a consequence of tetralogy of Fallot
(TOF). His grandmother (I.2) suffered SCD at age 59. His uncle (II.2)
was also known to have non-ischaemic heart failure. No cardiac image
sequences were available to exclude non-compaction or
hypertrabeculation.
The exonic rare variant (RBM20: p.R634L, c.G1901T) identified by
NGS was found in both available patients and was validated by
Sanger sequencing (Figure 4C). This novel variant is located in exon 9
and the amino acid change affects the arginine/serine-rich (RS) do-
main of RBM20, which is highly conserved across species (Figure 4C).
‘PROVEAN’, ‘SIFT’, and ‘Poly-Phen2’ predicted the functional effect
of the R634L variant to be ‘deleterious’ (PROVEAN score = -2.67
and SIFT score = 0) and ‘probably damaging’ (Poly-Phen 2
Score = 0.999). Also, the recently established ‘Combined Annotation
Dependent Depletion’ (CADD) score,
35
which combines informa-
tion from in total 63 annotation sources including functional data
from ENCODE, calculated the variants effect to be among the most
harmful 1% variants in the whole genome (CADD-Phred = 26.7).
Importantly, the RBM20 variant carrier did not show a pathogenic
variant in any other cardiomyopathy disease gene.
1
To further elucidate the potential effect of the variant, we per-
formed expression analysis of the mutant and wild-type allele. By
mRNA sequencing of myocardial tissue of both patients, we show that
the wild-type and mutant allele are expressed at comparable levels,
suggesting that RNA-decay is not the pathomechanism involved (Figure
4Bbottom). Western blot analysis supported this finding (Figure 4B
bottom). Hence, to establish the detected variant as pathogenic in this
family, we performed further molecular and functional investigations.
The RBM20 variant p.R634L leads to
severe titin mis-splicing and expression
of a giant titin protein
To determine a potential downstream causal effect of the non-
synonymous RBM20 variant p.R634L, we performed mRNA deep
sequencing in left-ventricular myocardial tissue of the index patient (ex-
planted heart after heart transplantation) and the endomyocardial bi-
opsy of his mother (Figure 5A). The two LVNC cases (blue line) and
unaffected controls (red line) did not differ in the splicing patterns of im-
portant structural and sarcomeric cardiac genes (see Supplementary
Patients with pathogenic variants in
known LVNC disease genes
TTN
19%
LMNA
5%
MYBPC3
4%
DSP
4%
MYH7
3%
CASQ2
2%
Figure 2 Prevalence of pathogenic variants in known LVNC dis-
ease genes. Shown is the fraction of patients affected by a patho-
genic variant in one of the known LVNC disease genes. For all
shown variants, stringent ACMG guideline criteria for the interpret-
ation of sequence variants have been applied. ‘Likely pathogenic’
variants or ‘variants of unknown significance’ are not included. TTN
variants are mainly truncating variants of the A-band.
Clinical genetics and outome of LVNC 3453
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material online, Figure S6). However, TTN transcripts were significantly
altered in their splicing pattern, as shown by the DPSI (green line). By
percent-spliced-in (PSI) analysis, the region of mis-splicing could be
mapped to the I-band region of titin (PEVK and immunoglobulin-rich re-
gion), confirming the hypothesis that the p.R634L variant alters titin
splicing predictably by reduced exon skipping.
To further prove the functional relevance of the rare RBM20 variant,
we next performed targeted in-vitro transcript-splicing assays (Figure 5B).
To do so, we cloned wild-type and the three predicted damaging vari-
ants (p.R634L, p.P798L, and p.P1105L) constructs and expressed them
together with a titin subfragment, which was tagged by sequences cod-
ingforRanillaandFirefly-Luciferase,respectively.AsshowninFigure 5C,
the p.R634L mutant constructs (Aminoacid numbering based on rat
RBM20 sequence: R637L) lead to a strong increase of the ratio
Luciferase/Ranilla Luminescence, similar to the positive control
(P641L), indicating highly defective splicing activity with reduced exon-
skipping, which is predicted to result in a longer titin isoform as seen in
the RNA-seq experiments and which was consecutively validated by
splice-analysis of the mini-gene constructs (Figure 5D).
To substantiate this finding, we performed agarose protein gel
electrophoresis of isolated protein from the myocardial specimens of
the two available LVNC patients. While titin isolated from wild-type
tissue contains mainly the shorter N2B form and to a lesser extent
the longer N2BA isoforms, both affected patients showed a marked
shift towards a very high-molecular-weight titin protein. This so-
called giant N2BA isoform is not present in controls or RBM20-muta-
tion negative patients with DCM (Figure 6) and is only expressed due
to defective RBM20.
Discussion
Cardiomyopathies are complex diseases that require detailed clinical
investigations to delineate the individual subtype and disease aeti-
ology. While there is consensus about the diagnostic criteria for
dilated,
36
hypertrophic
37
and arrhythmogenic
38
cardiomyopathies,
the classification and phenotypic definition of LVNC are not ad-
dressed in guidelines of international cardiac societies. This uncer-
tainty in the definition of LVNC renders estimations of its prevalence
very difficult. Furthermore, the reported clinical outcomes vary
largely describing LVNC as a trait of normal morphological variation
3
up to an infaust disease leading to stroke, end-stage heart failure or
A
B
Figure 3 Titin variants are a major cause of LVNC. (A) Protein domain structure of titin [according to http://cardiodb.org/titin/ (August 2017)]
showing the distribution of non-synonymous, frameshift and stop variants. As shown, most frameshift or stop variants are located in the A-band. For
three Stop-gain variants, myocardial transcriptome sequencing of biopsies revealed lower expression of the mutant (yellow) compared with the
wild-type (black) allele, indicating non-sense mediated RNA decay (B) Pedigrees with co-segregation of LVNC with TTN variants. three out of the
four segregating variants are small deletions predictably leading to a frame-shift.
3454 F. Sedaghat-Hamedani et al.
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A
BC
Figure 4 LVNC pedigree showing co-segregation of a mis-sense variant in RBM20.(A) The index patient of pedigree 16 was diagnosed with
17 years and underwent heart transplantation only 3 years later due to progressive heart failure. (B) The left and right panels show echocardiography
images with pronounced non-compaction in the apical, anterolateral, and inferolateral regions of the index patient and his mother. Histopathological
examination of left ventricular endomyocardial biopsy of both LVNC cases demonstrated extensive cardiac fibrosis. Expression analysis indicates
equal expression of the wild-type (black) and mutant (red) alleles and RBM20 protein amounts. (C) Verification by Sanger Sequencing of the mis-
sense variant RBM20: p.R634L, which is predicted as deleterious by computational algorithms. The high cross-species conservation of the residues is
shown below. To further reveal the pathogenic role of RBM20variants, molecular and functional characterization was performed. *Non-compacted
myocardium. TOF, tetralogy of Fallot; N.A., not accessible for analysis.
Clinical genetics and outome of LVNC 3455
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A
B
E
F
CD
Figure 5 Functional evidence for a pathogenic role of RBM20: p.R634L. (A) Transcriptome sequencing from heart tissue of the two affected
RBM20 LVNC patients (blue line) compared with normal controls (n=3; red line). As shown by PSI analysis, a strong exon inclusion rate is found in
the I-band region of titin (DPSI, green line), which is predicted to result in an abnormally long titin protein. (B) Splicing activity of RBM20 was deter-
mined by a dual luciferase splicing assay (DLR). In case of a functional (wild-type) RBM20, Luciferase activity is diminished, whereas defective (LOF,
loss of function) RBM20 will lead to a high Luciferase activity. (C) PEVK exon 4-13 splice reporter in HEK293.EBNA cells without RBM20 (CTRL),
wild-type RBM20 (WT) and RBM20 mutations (R637L and P641L, corresponding to human R634L and P638L) (n=8). The high Luciferase activity in-
dicates a detrimental effect of the variants on RBM20. One-way analysis of variance (ANOVA) test P<0.001 (***), P<0.01 (**), P<0.05 (*),
P=1.1102e
-16
(D) Splicing activity of rat RBM20 as determined by RT-qPCR on genomic minigene of human TTN exon 241-243 in HEK293 (n=3).
One-way ANOVA test P<0.001 (***), P<0.01 (**), P<0.05 (*), P= 2.0505e
-09
.(E) Exon structure and functional domains of rat RBM20.(F)
Sequence conservation in the RS region of RBM20. Dots indicate patient mutations affecting amino acids R637L (red) and P641L (blue). AA, amino
acid; CTRL,control; f.c., fold change; FLuc, firefly luciferase; MHC, myosin heavy chain; RLuc, renilla luciferase; RRM, RNA recognition motif; RS,arge-
nin/serin rich region; WT, wildtype; ZnF, zinc finger; PSI, percent spliced-in.
3456 F. Sedaghat-Hamedani et al.
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sudden cardiac death.
7
Altogether, these facts led to inconsistent rec-
ommendations for diagnosis and therapy, for instance regarding
prophylactic ICD implantation or oral anticoagulation.
In our cohort, we find 10.3% of patients with thromboembolism
during the whole observational period. Whether these incidents
were caused by thrombus formation in the deep recessi of the left
ventricle as observed in one of our cases that underwent heart trans-
plantation or by undetected AF cannot be finally concluded. In 33%
of patients with TIA/stroke, we did not detect AF even after repeat
Holter monitoring and 4.5% (2 from 44) of patients free of AF (base-
line and follow-up) did develop systemic embolism.
The patients were furthermore frequently affected by potentially
life threatening ventricular arrhythmias. VT/VF was documented at
least once in as many as 35.3% of cases till the end of the follow-up
time, which is in agreement with several previous reports.
39
Acom-
parison between the LVNC cohort and an age-matched DCM cohort
showed significantly higher rates of HF associated events in LVNC.
This finding is not explained by LVEF alone, which was higher in the
LVNC group. It can only be speculated that the different myocardial
properties (non-compaction, fibrosis) play a role as substrate for ar-
rhythmias.
39
In cMRI scans of the here enrolled patients, as many as
66.7%, had LGE as indicator of myocardial fibrosis.
The data presented on clinical outcomes should encourage phys-
icians, independent of the genotype, toroutinely follow-up symptom-
atic LVNC cases—especially in case of concomitant reduction in
LVEF—by repeat Holter-ECG to detect atrial and ventricular ar-
rhythmias.
2
Besides the clinical risk stratification, genetic information
may aid the process of diagnosis, predictive testing of relatives and
prognostication in LVNC cases. The present study provides for the
first-time estimations of the contribution of the known disease genes
in a larger cohort using uniform next-generation sequencing.
Following stringent criteria defined by the ‘American College of
Medical Genetics and Genomics’ (ACMG), we show that 38.2% of
patients have a pathogenic variant in one of six known disease genes.
For titin, we identified a role of truncating variants that were previ-
ously not known to cause non-compaction cardiomyopathy. Taking
this class of genetic variation into account, TTN variants are the most
frequent cause for LVNC, showing an odds ratio that is expected for
Mendelian disease and suitable for genetic counselling. By transcrip-
tome sequencing of the TTN mutation carriers, we show that the mu-
tant alleles undergo complete or partial nonsense-mediated RNA
decay. In case of the two patients with the pathogenic RBM20 variant
p.R634L, we demonstrate the consistent change of titin protein ex-
pression due to transcriptomic alteration of its messenger RNA, ren-
dering loss-of-function and dominant negative changes in titin a
pathomechanisms in LVNC. Although the small numbers do not
allow a final conclusion, carriers of pathogenic TTN, LMNA,and
RBM20 variants did show unfavourable outcomes, which is in-line
with observations made in DCM.
LVNC shares many symptoms and clinical findings with other car-
diomyopathies. Towbin et al.
39
characterized the phenotypic expres-
sivity of LVNC and identified distinct subtypes including benign
LVNC with preserved systolic and diastolic function, dilated LVNC,
hypertrophic LVNC, restrictive LVNC, and LVNC with arrhythmia.
The reasons for this remarkable variability in clinical findings are not
understood, but may be due to the underlying pathogenic variant or
the genetic background. In the present study, we could investigate 95
patients mainly with dilated LVNC phenotypes, which is to our
knowledge the largest series available. The reason why the patho-
genic alleles for RBM20 and TTN did result in a LVNC phenotype and
not classical DCM is not finally answered. Titin mutations were not
only found to be a frequent cause of DCM
40
but are also related to
Peripartum Cardiomyopathy (PPCM), indicating that yet unknown
additional factors are involved. Titin is a high-molecular weight pro-
tein in striated muscles, which spans half of the sarcomere from the
Z-disc to the M-band. Titin is required for proper sarcomere assem-
bly, force transmission and confers to sarcomere elasticity.
41
The
TTN gene contains 363 exons and is expressed as different isoforms
generated by alternative splicing. It is important to know that titin
undergoes significant changes in its isoform expression during em-
bryogenesis and at the time of birth. We are only at the beginning of
understanding the machinery that dynamically adapts the exons of
titin during these important stages of haemodynamic alterations, but
it is known that a very tight regulation of exon-numbers is required.
The human heart normally expresses different major titin isoforms,
two during embryonic development and three after birth. In the fetal
phase, heart tissue expresses the N2BA-N1 isoform (3.7 MDa),
which is replaced by the neonatal isoform (N2BA-N2, 3.6 MDa) and
later the adult isoforms, N2BA-A1 and -A2 (3.2–3.4 MDa) and N2B
(3.0 MDa).
26,42
The N2BA isoform contains more extensible elem-
ents in the I-band and is less rigid compared with the N2B isoform,
but also is structurally weaker.
43
We observed in our data that the
Figure 6 Effect of p.R634L mutated RBM20 on titin isoform ex-
pression. Titin isoform expression in healthy human heart tissue
(WT), patient II.3 (mother), patient III.2 (index), in a DCM patient
with S635A (positive CTRL) and in patient with DCM and without
RBM20 mutation. Both affected patients show a marked shift to-
wards a high-molecular weight titin protein (giant N2BA isoform).
Clinical genetics and outome of LVNC 3457
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splicing patterns in distinct regions of titin are significantly different in
LVNC patients compared with DCM patients with RBM20 variants. It
may be a matter of TTN gene dose and protein function
26
that is pre-
sent during development or by additional genetic background vari-
ation that disturb embryogenesis or adaptive hypertrophic growth
during the stress of cardiac failure, ultimately leading to LVNC.
44
RBM20 is a key cardiac splice regulator that controls the process-
ing of several important transcripts. Hence, mutations in RBM20 can
result in mis-splicing of several targets, often leading to progressive
DCM with conduction diseases as well as atrial or malignant ventricu-
lar arrhythmia.
26,45
Until now, there were to best of our knowledge
no described cases of LVNC due to RBM20 mutations. In the current
study, we could identify a novel non-synonymous variant in Exon 9 of
RBM20 that results in significantly altered splicing-regulator activity.
Other coding variants that were identified in this cohort (RBM20
p.P798L, and p.P1105L) did not show altered titin splicing, at least not
in the assays applied here (see Supplementary material online,
Figure S7). The majority of known RBM20 mutations are localized in
exon 9, which functionally disrupts normal RNA splicing of target
genes.
26
However, the exact mechanisms leading to DCM as well as
LVNC in RBM20 mutation carriers are still not completely under-
stood. Li et al.
46
proposed that RBM20 regulates titin alternative splic-
ing by exon skipping or exon shuffling. RBM20 binds to specific
regions of titin pre-mRNA and inhibits introns from being spliced out.
Following this step, the 5’ and 3’ splice-sites of the exons flanking the
RBM20-repressed region splice with each other to complete the al-
ternative splicing process. Brauch et al.
45
were the first to report
RBM20 mutations in DCM patients, most having high morbidity and
mortality. In RBM20 deficient animal models an increase of subendo-
cardial fibrosis has been reported, accompanied by electrical abnor-
malities such as conduction disease or ventricular arrhythmia.
26
In
the family reported here, we found severe endomyocardial fibrosis
and progressive LVNC with dilated cardiomyopathy, heart failure
and ventricular arrhythmia. We showed that RBM20 p.R634L variant
leads to the production of abnormally giant TTN isoform (G-N2BA).
It is known that this isoform causes an increase in TTN elasticity that
has implications for diastolic function and also the Frank-Starling
mechanism. Furthermore, the flaccid titin filament causes a compen-
satory increase in collagen biosynthesis and leads to fibrosis and ar-
rhythmia aswe could observe in thesetwo patients.
26
Besides the findings discussed above, we further detected variants
in genes that have previously been reported to result in a LVNC
phenotype in animal models (see Supplementary material online,
Figure S5). The nuclear receptor corepressor 2 (NCOR2) is facilitating
the recruitment of histone deacetylases (HDACs) to DNA pro-
moters. In a NCOR2 loss of function mouse model, the compact zone
formation of the myocardium was severely affected.
47
We could re-
port here four non-synonymous SNVs human patients, rendering
NCOR2 a highly likely new disease gene for LVNC. The same is true
for variants found in the Xin actin-binding repeat containing 2
(XIRP2),
48
but further functional investigations and family studies are
required to provide the same level of evidence as we have shown in
this study for RBM20 and titin.
The present study highlights the role of mis-sense and non-sense
variants in the titin gene and titin-interacting partner proteins
(Obscurin, RBM20). We show that 19% of all LVNC cases are caused
by TTN variants (mainly truncating) that only partly underlie NMD. In
the family with an aggressive LVNC course, we show by multiple lines
of evidence the pathogenic role of RBM20 p.R634L. The pathogen-
icity involves most obviously severe titin mis-splicing, resulting in a
mechanically altered elastic filament protein. Importantly, the splicing
patterns observed in LVNC patients, is in distinct regions of TTN dif-
ferent compared with DCM patients with RBM20 variants. Although
speculative, these minute changes in TTNisoforms caused by RBM20
or TTN variants may explain why some patients develop DCM while
others are presenting with a LVNC phenotype (see Supplementary
material online, Figure S8). It is important to further the understanding
how different alleles in the same gene can result in significantly differ-
ing cardiac phenotypes, which is the case for several other disease
genes such as MYH7, MYBPC3, LMNA,orSCN5A to name only few ex-
amples. As such, we investigated all families for overlapping pheno-
types with HCM. In one family we for instance detected the known
HCM causing variant MYH7 p.R719W. The index case was available
for phenotypic analysis and presented an abnormal septal wall thick-
ness of 12 mm. Although not fulfilling the diagnostic criteria of HCM,
this may indicate that there is not only a genotypic but also pheno-
typic overlap.
Potential limitations
There is an on-going and controversial debate whether LVNC is a
morphological trait of normal variation, adaptive mechanism to
physiological growth stimuli, subtrait of a major cardiomyopathy such
as DCM or distinct cardiomyopathy. When applying morphological
criteria only, the presence of non-compaction in population-based
samples is far higher than expected for a detrimental disease.
3
On the
other hand, there are several retrospective studies showing progres-
sive disease and bad prognosis in non-compaction patients. The diffi-
cult, but utterly important discrimination of both extremes is not
addressed in current guidelines, leaving cardiologists alone in estab-
lishing the correct diagnosis. In our patient series, we included pa-
tients that were referred to our tertiary centres and were mostly
symptomatic at the time of diagnosis or had pathological findings sug-
gestive of a disease (biomarker increase, ECG alterations). Hence,
there is a potential referral bias that might explain the bad prognosis
of our patients. To control for this effect, we included age-matched
DCM patients from our centres. Importantly, the prognosis of DCM
patients was favourable in comparison to LVNC although LVEF was
better in our LVNC cohort.
Whole-exome sequencing could be performed in 27 affected rela-
tives of 68 index patients. Not all relatives could be genotyped in fa-
milial cases due to lack of biomaterial or informed consent. Due to
the missing co-segregation data in some families, the rate of ‘variants
with unknown significance’ is considerably high. Furthermore, it is
currently unclear how particular RBM20, TTN,orotherpathogenic
variants lead to LVNC and not to other cardiomyopathy phenotypes
and how the mutation frequencies would translate to another LVNC
cohort with milder phenotypes. It is speculated that alternative titin
isoforms arise from truncating mutations that may, besides a loss-of-
function mechanism of the main isoform, interfere in a dominant
negativemode. Additionally, the genetic background of patients could
modify the phenotype expression. Long-read transcriptome
sequencing and ribosomal profiling are thought to likely answer these
questions in the nearer future, since animal models for non-
compaction are sparse and partly provide non-reproducible
3458 F. Sedaghat-Hamedani et al.
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phenotypes as shown for knock-in mice of the human LVNC TNNT2
(p.E96K) mutation,
49
the natural occurring p.R820W mutation in
MYBPC3 that causes a HCM phenotype in rag-doll cats, but a LVNC
in humans
50
or knock-out of DTNA that causes muscle dystrophy and
DCM instead of LVNC.
12
In summary, we were able to characterize 95 patients with LVNC
both by clinical phenotyping and whole-exome sequencing. We
show that LVNC is associated with unfavourable outcome in com-
parison to DCM. Furthermore, we report the prevalence of patho-
genic variants in known LVNC disease genes, show that truncating
TTN variants are the leading cause for LVNC and introduce variants
in RBM20 as a novel pathomechanism for LVNC.
Supplementary material
Supplementary material is available at European Heart Journal online.
Funding
German Center for Cardiovascular Research (DZHK), Berlin;
‘Bundesministerium fu¨r Bildung und Forschung’ (CaRNAtion); The
European Union (FP7 BestAgeing); and ‘Deutsche Gesellschaft fu¨r
Kardiologie’ (DGK).
Conflict of interest: none declared.
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