University of Groningen Matters of the heart: genetic and ... · Peripartum cardiomyopathy (PPCM) was observed in two DCM families carrying RBM20 mutations (p.R636H and p.S637N)

University of Groningen

Matters of the heart: genetic and molecular characterisation of cardiomyopathiesPosafalvi, Anna

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2015

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Posafalvi, A. (2015). Matters of the heart: genetic and molecular characterisation of cardiomyopathies.University of Groningen.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 28-03-2021

https://research.rug.nl/en/publications/matters-of-the-heart-genetic-and-molecular-characterisation-of-cardiomyopathies(089769ad-4b9c-4294-b03f-e949596ddad7).html

CHAPTER 2CANDIDATE GENE

SCREENING

Chapter 2.1

Mutational characterisation of RBM20 in dilated cardiomyopathy

and other cardiomyopathy subtypes

Anna Posafalvi, Ludolf G Boven, Cindy Weidijk, Paul A van der Zwaag, Jan G Post, Karin Y van Spaendonck-Zwarts, Imke Christiaans,

Maarten P van den Berg, Robert MW Hofstra, Gerard J te Meerman, Richard J Sinke, J Peter van Tintelen, Jan DH Jongbloed

Manuscript in preparation

ABSTRACT

Introduction: Dilated cardiomyopathy (DCM) is an insidious disease of the myocardium, leading to impaired heart function. RBM20, a recently discovered gene associated with DCM encodes the RNA-binding motif protein 20. It is involved in the tissue-specific splicing of titin and several other proteins with an essential function in the heart, and is known to have a five amino acid mutation hotspot in exon 9.

Objectives: Our aim was to perform mutation screening of RBM20 in a Dutch cardiomyopathy patient cohort, and to design a spicing assay for evaluation of the pathogenicity of the identified variants.

Methods and results: We performed mutation screening of RBM20 by Sanger (DCM patients only; n=436) and gene-panel based (targeted) next generation sequencing (all cardiomyopathy subtypes included; n=1311 patients). This resulted in the identification of 18 novel and 5 known, rare missense variants in 35 probands. In total 10 likely pathogenic or pathogenic missense mutations were identified: 7 missense variants in the RS-rich domain, 1 novel missense variant of the RNA-recognition motif and 2 outside of these important domains. Peripartum cardiomyopathy (PPCM) was observed in two DCM families carrying RBM20 mutations (p.R636H and p.S637N). A differential splicing assay of the known RBM20-target LDB3 was developed to evaluate the predicted pathogenicity of 11 missense variants, but the results were inconclusive.

Conclusion: Our study identified a significant number of novel and potentially pathogenic RBM20 mutations, particularly in the RS domain. In addition, known hotspot mutations were identified. Analysis of all cardiomyopathy subtypes by next generation sequencing revealed that likely pathogenic or pathogenic mutations were found almost exclusively in DCM patients. The RBM20 mutations that we found in patients with PPCM were not unexpected as previous studies had shown TTN mutations are a frequent cause of PPCM. They lead to its shifted isoform composition, and TTN is the best characterized splicing target of RBM20.

Keywords: dilated cardiomyopathy, RBM20, genetic screening, differential splicing assay

55RBM20 IN DILATED CARDIOMYOPATHY

CHA

PTER

2.1

INTRODUCTION

The RNA-binding motif protein 20 gene, RBM20, is a known disease gene in dilated cardiomyopathy (DCM) (Brauch et al, Li et al 2010, Millat et al, Refaat et al, Guo et al 2012, Wells et al). It was initially reported as missense mutations clustering in a hot-spot between amino acids 634-638, encoding an RS-rich domain of unknown function. It has also been observed that RBM20 carriers sometimes exhibit an unusually severe phenotype, or early onset of the disease (Brauch et al).

Recent studies showed that RBM20 is involved in the heart-specific splicing of the mRNA molecules of several target genes. Some of these targets, such as the sarcomeric giant titin gene (TTN), as well as the cytoskeletal LIM domain binding 3 gene (LDB3), the calcium/calmodulin-dependent protein kinase II delta gene (CAMK2D), and the gene encoding one of the voltage-dependent calcium channel proteins involved in membrane polarisation (CACNA1C) (Guo et al 2012) have been linked to cardiomyopathy but also to other cardiac phenotypes. Li et al (2013) demonstrated complex exon skipping and shuffling patterns of titin in Rbm20-/- rats were demonstrated. These animals were shown to suffer from ultrastructural changes in the heart, including Z line streaming, abnormal myofibril width and orientation, and aggregation of mitochondria (Guo et al 2013). A more recent study crosslinking RBM20 molecules to their RNA targets, followed by immunoprecipitation and RNA sequencing (CLIP-Seq) suggests a role for RBM20 in differential splicing of PDLIM3, LMO7, RTN4 and RYR2 as well, while confirming its previously observed role in splicing of CAMK2D, LDB3 and TTN (Maatz et al). Finally, the phenotypic influence of RBM20 knock down was studied in a mouse stem cell model, which led to the identification of intracellular enrichment of actin stress fibres and thin, elongated sarcomeric ultrastructures during cardiac differentiation, an observation that agrees with the abnormal sarcomere geometry in the thin, weakened myocardium of DCM patients (Beraldi et al). This study also showed disturbed Ca2+ handling in shRBM20 cardiomyocytes and abnormal splicing of TTN and CAMK2D.

To date, the extent to which RBM20 contributes to DCM in the Netherlands has not been studied, nor has the putative role of the gene in other cardiomyopathy subtypes. The aim of this study was to determine if RBM20 mutations are responsible for DCM and other types of cardiomyopathy in the Dutch population and to gain functional evidence for the pathogenicity of some of the identified variants.

56 SANGER SEQUENCING

METHODS

Mutational screening

Patients: Written informed consent was obtained from index patients and their relatives involved in the first phase of the study with approval of the medical ethics committees of the participating hospitals. Patients included in this phase fulfilled the formal diagnostic criteria for DCM (Mestroni et al). Cardiomyopathy patients in the second phase of this study were evaluated in a standard diagnostic setting. The clinical diagnosis of these patients was inferred from the referral diagnosis to our laboratory, and included a variety of cardiomyopathy subtypes.

DNA sequencing: Peripheral blood was collected from cardiomyopathy patients and their relatives when applicable. DNA was isolated according to the standard protocols. In the first phase of this study, Sanger sequencing of whole or part of the RBM20 gene was performed in 436 DCM patients. For this purpose, 19 amplicons covering all exons and the flanking intronic regions of the RBM20 gene were amplified by AmpliTaq Gold (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using a standardized PCR protocol (for primer sequences see also table S1). After purification, the PCR products were sequenced on an ABI3730xI sequencer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Resulting sequences were visualized for analysis by Mutation Surveyor software (SoftGenetics LLC, State College, PA, USA). In the second phase of this study, patients’ DNA (n=1311) was analyzed using our gene-panel-based targeted next generation sequencing (NGS) method as described previously (Sikkema-Raddatz et al, chapters 4.1 and 4.2), and including RBM20.

Variant classification: The identified genetic variants were classified as ‘benign’ (B), ‘likely benign’ (LB), ‘variant of unknown significance’ (VOUS), ‘likely pathogenic’ (LP), and ‘pathogenic’ (P) as described in chapter 4.1. In short, our classification was based on the changes in the physicochemical nature of the affected amino acid residues, the evolutionary conservation of the respective residue and the region harbouring the variant, the predicted pathogenicity using various software (such as AGVGD, PolyPhen, SIFT, and MutationTaster), and data from literature and online databases when available. Variant frequency information from additional databases (dbSNP, ExAC (including 1000 Genomes and ESP6500), and GoNL) was taken into consideration.

Marker analysis: Several genetic markers (and their respective primers) were selected in the region both 5cM up- and down-stream of the R634W


CHA

PTER

2.1

mutation from the deCODE high-resolution genetic map, as shown in table S2a. Lengths of PCR products were determined on the ABI 3730xl DNA Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and the results analyzed using the GeneMapper v 4.1 software (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).

Functional analysis

Plasmids: Flag-tagged, kanamycine-resistant pCMV6-Entry vectors con-taining the human RBM20 cDNA sequences (wild type, and two mutants: R634P and Y681C) were obtained from OriGene Technologies Inc (Rockville, MD, USA). Site-directed mutagenesis was applied to introduce additional variants as follows: (1) primer pairs were designed that could anneal back to back to the plasmid and for this purpose were phosphorylated at the 5’ end, then the desired mutation was introduced in the middle of the forward or reverse primer, with about 10-15 perfectly matched nucleotides upstream and downstream of the mutated nucleotide; (2) the mutation was introduced with a PCR reaction, using 2.5ng wild-type vector, the mutation specific primers and the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, Waltham, MA, USA); (3) PCR products were visualized on 1% agarose gel and, when produced in sufficient amounts, circularized by Quick T4 DNA ligase (New England Biolabs, Ipswich, MA, USA); (4) plasmids were transformed into competent DH5α E. coli cells, colonies selected for kanamycine resistance were mini-cultured and isolated using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Waltham, MA, USA) kit; (5) plasmids were Sanger-sequenced and the presence of the introduced mutations confirmed. Tables S3 and S4 show the primers used for site-directed mutagenesis and prior and subsequent plasmid sequencing.

Transfection, cell lines: HEK293 cells were cultured in a 6-well culture plate in DMEM containing 10% foetal bovine serum and 1% penicillin-streptomycin. Transfection was performed using 150 µl serum-free DMEM including 1 µg plasmid DNA and 5µl polyethilenimine per sample. Cells were cultured at 37oC. RNA was isolated with the standard Trizol method 48 hours after transfection, and subsequently reverse transcribed into cDNA by using oligo(dT)18 primers and the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction.

Differential splicing assay: First, PCR and gel electrophoresis of cDNA samples was performed to enable several quality checks, including


analysis of the expression of house-keeping genes and RBM20 mRNA, and sequencing RBM20 to confirm the presence of introduced mutations (PCR and sequencing reactions were performed as described above; the primers used for the analysis of RBM20 expression are shown in table S5). As several putative target mRNA molecules were available for analyses of potential effects of RBM20 mutations on differential splicing, results of these molecules in non-transfected (endogenous), wild type and hotspot mutation (R634P) transfected HEK293 cells were evaluated (primers designed for amplification of target mRNA molecules are shown in table S6). Based on this evaluation, we selected LDB3 for further follow up. As a start, we extracted separated PCR products of various lengths from agarose gel and sequenced them. Next, PCR conditions for amplification of LDB3 were optimised to enable termination of amplification before reaching saturating conditions (36 cycles; 62oC annealing), while allowing parallel quantification of several PCR products of different fragment sizes. Then, 16 cDNA samples originating from transfected HEK293 cells (2 of non-transfected HEK293 cells, 2 of cells transfected with plasmid containing wild type RBM20, 11 of cells transfected with plasmids carrying a single nucleotide variant each, and 1 non-template control) were placed on a 96 well PCR plate in triplo (PCR experimental replicates). To avoid possible plate-position-related effects, samples were distributed according to a computer generated random position. All LDB3 mRNA products were PCR-amplified. Resulting PCR products were then purified using the High Pure PCR Product Purification Kit (Roche Applied Science, Penzberg, Germany). Subsequently, aliquots of these samples were transferred to a 384-well plate and loaded on the Caliper GX capillary electrophoresis system (Perkin Elmer, Waltham, MA, USA), which separates PCR products by size and quantifies and visualizes the separated products. Each sample was quantified three times (capillary electrophoresis experimental replicates).

Data analysis: We first performed peak detection of the three dominant product sizes that correspond to the previously sequenced fragments (the peaks were detected at an approximate fragment length corresponding to the 245, 472 and 613 bp long PCR products). The position of the peak was determined and an area under the curve (AUC) was determined by adding the intensity values +/- 6 bases from the peak position. Thus 9 data points were available for each peak of each wild type/mutant/control transfected sample (3x PCR and 3x capillary electrophoresis experimental replicates). Normalization was performed by setting the value of the AUC to 100 and assigning the proportional value to the


CHA

PTER

2.1

other peaks. We used the group of certainly benign (wild-type) and the group of certainly pathogenic (R634P, R634W, R636H) transfected cells as controls during the data analysis, and compared the splicing pattern of cells transfected with the remaining variants of uncertain significance to these two control groups. Analysis of variance was used to evaluate differences between cells.

RESULTS

Mutational screening

To study the contribution of RBM20 mutations to the development of cardiomyopathy, specifically DCM, in the Dutch population, we analyzed this gene by Sanger sequencing and targeted NGS (as part of a panel of 55 cardiomyopathy genes).

Initially, we studied RBM20 in 62 DCM patients by Sanger-sequencing all exons and the flanking intronic sequence. In an additional cohort of 374 patients, we performed sequencing of amplicons in which potentially interesting genetic variants were previously identified (in the literature or in our 62 patients). Exon 9 was chosen since it encodes the RS-rich (Arginine and Serine-rich) domain and carries the known mutation hot-spot of five neighbouring amino acids between positions 634-638 (Brauch et al). Exons 6 and 7 were chosen because they encode the RNA-recognition motif (RRM) domain and a putative nuclear localization signal between c.1770-1842 (Filippello et al). The RRM domain, in particular, is highly homologous to RRMs of other spliceosomal and SR-proteins, is expected to play an essential role in the protein function and reported by Li et al (2010) to carry one missense variant. Finally, exon 11 was chosen because it harbours the missense variant D888N reported by Refaat et al as causative mutation. However, when our targeted NGS method was implemented into our routine diagnostics (Sikkema-Raddatz; chapter 4.1), we also evaluated the contribution of RBM20 mutations to both DCM and other cardiomyopathy subtypes for 1311 patients. In this study, we focus only on those patients who had interesting RBM20 variants.

In 35 patients, we identified 23 missense variants (18 novel) that were either absent or present at very low frequency (MAF<0.0005) in the ExAC (http://exac.broadinstitute.org) and/or the GoNL (http://www.nlgenome.nl) databases. Table 1 contains clinical information is given on RBM20 patients and their family members who were identified within the initial phase (Sanger


sequencing of DCM patients), while table 2 summarizes information on all the potentially interesting variants identified in this study, including their classification. As expected from their low population frequency, all variants were classified, at minimum, as VOUS (table 2). Together, 13 variants were classified as VOUS, 7 as LP and 3 as P (hot-spot mutations). Six variants were identified in two or more patients, including the pathogenic R634W mutation identified in 3 different patients and the likely pathogenic mutations Y681C and Y1193C identified in 5 and 3 patients, respectively.

The variants found in the known mutation hot-spot (R634P, R634W, and R636H) of the RS-rich domain (residues R632-S654) were classified as “pathogenic” not only as a consequence of their high evolutionary conservation, complete absence in control populations, and pathogenic predictions by multiple in silico programmes, but also due to several previous reports on hotspot mutations leading to an unusually severe phenotype and cosegregation with disease in several families, including the R634P mutation in our studies (table 1). In addition to the mutations mentioned above, we identified four other novel, likely pathogenic mutations in residues of the RS domain: R623Q, R632K, P633L, and S637N.

We also identified likely pathogenic mutations outside the RS domain: V535L, Y681C, and Y1193C. The novel missense variant V535L resides in the RRM, a region in which only one genetic variant affecting the same amino acid position (V535I) was previously reported. We considered this variant as

“likely pathogenic”, because it is located in an area of very high evolutionary conservation, is not found in any population database, and is predicted to be pathogenic by three of the four prediction programmes we used (PolyPhen, SIFT, MutationTaster). The Y681C mutation is located just outside the RS-rich domain, affects a very conserved residue residing in a significantly conserved region and is predominantly predicted to be a harmful variant. It has been identified in one individual in the GoNL population and twice (MAF=0.0001028) in the ExAC population. Finally, Y1193C resides at the end of a Zinc-finger(like) domain (residues 1158-1193), affects a conserved residue and is surrounded by conserved residues, is predicted to be pathogenic by three out of four prediction programs we used, and has never been identified in the control populations of GoNL and ExAC.

In addition to the likely pathogenic and pathogenic mutations described above, 13 missense variants were detected outside the RBM20 domains or regions of known importance and were classified as VOUS (table 2). It is


CHA

PTER

2.1

Table 1. Clinical information of RBM20 families identified during phase I; Sanger sequencing. The mutations as well as the result of segregation analysis, when applicable, are indicated.

Family Year of birth gender RBM20

mutationAge at

diagnosisclinical status clinical details

family 1 1935 M unknown 66 affected DCM

1961 M p.L100F 40 affected DCM

family 2 1942 F p.R634P 33 affected LV-dysfunction; LVEF 11% (63yr); NSVT; ICD (64yr)

1946 M no sample 58 affected DCM; died at age 62yr

1938 M no sample na affected? SCD 41yr

1967 M p.R634P 39 affected DCM; NSVT; PVCs; ICD (39yr)

1969 M p.R634P 31 affected? NSVT; LVEF 53%; PVCs

1910 F no sample na affected? SCD 46yr

family 3 1934 M no sample 50 affected DCM; HTX (53yr); hypertrophic cardiomyocytes on histology

1929 M none 72 unclear severe atherosclerosis; LVEF 35% (72yr); VT, MI (72yr), ICD (72yr); AF (79yr)

1959 F p.S637N 35 affected PPCM (35yr); VT; HTX (37yr)

1992 F p.S637N 16 affected DCM (16yr); NSVT; PVCs; ICD and MI (17yr); HTX (19yr)

family 4 1945 M p.Y681C 56 affected DCM; NSVT; PVCs; AF (59yr)

1974 M non-carrier 21 affected DCM; AF (20); ICD (30)

1942 M no sample 44 affected DCM; AF; died at age 45yr; interstitial fibrosis on histology

1907 M no sample healthy Died at age 59yr

family 5 1947 p.V535L 57 affected LV dilatation; AVB; sporadic case

family 6 1938 p.R634W 55 affected LV dysfunction; LVEF 35% (67yr); died at age 70yr

no sample affected LV dilatation; died at age 67yr

no sample affected LV dilatation; died at age 40yr

unknown affected DCM

family 7 1978 p.R634W and p.G672S 20 affected DCM

family 8 1944 F p.R636H 47 affected DCM leading to HTX

1968 F p.R636H 23 affected Died at age 23yr (PPCM); cardiomegaly (post mortem)

Abbreviations: AF – atrial fibrillation, AVB – atrioventricular block, DCM - dilated cardiomyopathy, HTX – heart transplantation, ICD – implantable cardioverter-defibrillator, LV – left ventricle, LVEF – left ventricular ejection fraction, MI - myocardial infarction, NSVT – non-sustained ventricular tachycardia, PPCM – peripartum cardiomyopathy, PVC

- premature ventricular contraction, SCD – sudden cardiac death, VT – ventricular tachycardia

important to note that a considerable subset of these 13 variants were identified in non-DCM patients, mainly those with hypertrophic cardiomyopathy (HCM). This is in contrast to our likely pathogenic and pathogenic mutations, which were almost exclusively identified in DCM patients, with the only exception being the Y681C and Y1193C mutations identified in both DCM and HCM patients. In addition, two mutations, the likely pathogenic S637N and the pathogenic R636H, were identified in DCM families in which peripartum


Tabl

e 2.

RBM

20 v

aria

nts

iden

tifie

d by

San

ger

sequ

enci

ng (

S) a

nd t

arge

ted

sequ

enci

ng (

T) T

he t

able

incl

udes

info

rmat

ion

on

each

mut

atio

n, t

he m

etho

d by

whi

ch t

he m

utat

ion

was

iden

tified

, the

num

ber

of p

atie

nts

carr

ying

the

mut

atio

n an

d th

eir

part

icul

ar

card

iom

yopa

thy

subt

ype,

put

ativ

e ca

rrie

rshi

p of

oth

er li

kely

pat

hoge

nic

or p

atho

geni

c m

utat

ions

, the

freq

uenc

y in

the

ExAC

pop

ulat

ion

and

the

resp

ectiv

e cl

assi

ficat

ion

of th

e m

utat

ions

. Evo

lutio

nary

con

serv

atio

n, p

redi

cted

pat

hoge

nici

ty (b

y AG

VGD

, SIF

T, P

olyP

hen2

and

M

utat

ionT

aste

r sof

twar

es) a

nd a

llele

freq

uenc

ies

(in d

bSN

P, Ex

AC, a

nd G

oNL)

wer

e ta

ken

into

con

side

ratio

n fo

r cla

ssifi

catio

n.

Prot

ein

chan

gecD

NA

coo

rdG

enom

ic c

oord

(37)

met

hod

# of

pat

ient

s (C

M

subt

ype)

othe

r mut

atio

nsM

AF

ExA

C db

ase

clas

sific

atio

nH

GM

D

L100

Fc.

298C

>T10

:112

5406

65S/

T2

(DCM

)LP

SCN

5A &

MYH

6 (in

1 p

at)

0VO

US

G

284R

c.85

0G>A

10:1

1254

1217

T2

(DCM

; HCM

)

0.00

0459

9VO

US

G

309E

c.92

6G>A

10:1

1254

1293

T1

(HCM

)

0VO

US

V4

87M

c.14

59G

>A10

:112

5445

79T

1 (H

CM)

0.

0000

5045

VOU

S

V535

Lc.

1603

G>C

10:1

1255

7341

S1

(DCM

)

0LP

V5

45I

c.16

33G

>A10

:112

5573

71T

1 (D

CM)

0.

0000

4516

VOU

S

R623

Qc.

1868

G>A

10:1

1257

0208

T1

(uns

p)LP

MYH

70

LP

R632

Kc.

1895

G>A

10:1

1257

2050

T1

(uns

p)

0LP

P6

33L

c.18

98C>

T10

:112

5720

53T

1 (u

nsp)

0.

0000

6948

LP

R634

Pc1

901G

>C10

:112

5720

56S/

T1

(DCM

)

0P

yes;

oth

er a

a ch

ange

R634

Wc1

900C

>T10

:112

5720

55S/

T3

(DCM

)

0P

yes

R636

Hc1

907G

>A10

:112

5720

62S

1 (P

PCM

/DCM

)

0P

yes

S637

Nc1

910G

>A10

:112

5720

65T

1 (P

PCM

/DCM

)

0LP

yes;

oth

er a

a ch

ange

G67

2Sc2

014G

>A10

:112

5721

69S/

T3

(2 D

CM; 1

HCM

)

0VO

US

R6

73Q

c.20

18G

>A10

:112

5721

73T

1 (D

CM)

0.

0002

682

VOU

Sye

s; o

ther

aa

chan

geS6

75T

c.20

23T>

A10

:112

5721

78T

1 (D

CM)

0

VOU

S

Y681

Cc2

042A

>G10

:112

5721

97S/

T5

(4 D

CM; 1

HCM

)P

PLN

(in

1 pa

t)0.

0001

028

LP

G75

8Sc.

2272

G>A

10:1

1257

2427

T1

(HCM

)LP

MYH

70

VOU

S

D78

6Gc.

2357

A>G

10:1

1257

2512

T1

(DCM

)

0.00

0046

47VO

US

I9

21F

c.27

61A

>T10

:112

5811

38T

2 (1

DCM

; 1 H

CM)

LP N

EXN

& T

NN

T2 (i

n 1

pat)

0VO

US

V1

102A

c.33

05T>

C10

:112

5816

82T

1 (H

CM)

0

VOU

S

Y119

3Cc.

3578

A>G

10:1

1259

5630

T2

(1 D

CM; 1

HCM

)

0LP

A

1208

Vc.

3623

C>T

10:1

1259

5675

T1

(DCM

)

0VO

US

A

bbre

viat

ions

: CM

– c

ardi

omyo

path

y, D

CM –

dila

ted

card

iom

yopa

thy,

HCM

– h

yper

trop

hic

card

iom

yopa

thy,

HG

MD

– H

uman

Gen

e M

utat

ion

Dat

abas

e, L

P: l

ikel

y pa

thog

enic

, MA

F –

min

or a

llele

freq

uenc

y, P

– p

atho

geni

c, P

PCM

– p

erip

artu

m c

ardi

omyo

path

y, V

OU

S –

varia

nt o

f unk

now

n si

gnifi

canc

e. G

ene

sym

bols

: MYH

6 –

Myo

sin,

he

avy

chai

n 6,

car

diac

mus

cle,

alp

ha, M

YH7

– M

yosi

n, h

eavy

cha

in 7

, car

diac

mus

cle,

bet

a, N

EXN

– N

exili

n (F

act

in b

indi

ng p

rote

in),

PLN

– P

hosp

hola

mba

n, S

CN5A

– so

dium

ch

anne

l, vo

ltage

gat

ed, t

ype

V al

pha

subu

nit a

nd T

NN

T2 –

Tro

poni

n T

type

2 (c

ardi

ac).


CHA

PTER

2.1

cardiomyopathy (PPCM) was also diagnosed. We also identified the missense variant D888N, which was formerly considered to be pathogenic (Refaat et al). However, we anticipate that D888N is a rare polymorphism, as it was only identified in 6 of our 374 Sanger-sequenced DCM patients (0.16%), in 13 of our 1200 NGS-analyzed patients (0.11%), and in comparably low frequencies in control populations: 0.18% in Dutch controls (GoNL; 9 in 996 alleles, which means 9/498 healthy individuals) and 0.28% in the ExAC database, D888N is therefore not included in summary table 2.

Cosegregation and haplotype analysis

In cases where DNA of affected family members was available, we studied the carriership status of those individuals (for details, see table 1). We were able to show co-segregation of mutations R634P and R636H with the disease phenotype in the respective families. We did not find co-segregation of Y681C in the one small family that was available for testing.

We also identified unrelated index patients carrying the same variants: three patients carrying the pathogenic mutation R634W and five patients having the likely pathogenic mutation Y681C. Although no family relation between these patients was known, we hypothesized that these mutations were inherited from common ancestors (founders). Therefore, we performed haplotype analysis using markers within the approximately ±5cM region surrounding the respective mutations. These studies revealed that the patients carrying the R634W mutation share a relatively large haplotype (see table S2b; results shown for two of the three patients), suggesting that the mutation originated from a common founder. In contrast, no shared haplotype could be identified for the Y681C mutation (data not shown). Together with lack of cosegregation of this variant in the small family studied, the fact that it was also found in 1/996 alleles in the GoNL database, and that one of the Y681C patients is also carrying a certainly pathogenic PLN deletion, these results suggest that Y681C is less likely to be pathogenic. However, more data is needed to verify this conclusion.

Functional evaluation

In order to evaluate our classification of variants L100F, V535L, R634P, R634W, R636H, G672S and Y681C using a functional approach, we designed a differential splicing assay. In addition, we included the likely benign variants W768S, W768L and the D888N variant, which was formerly reported


as pathogenic but that we now designate as benign. For this purpose, we transfected HEK293 cells with wild type and “mutated” human RBM20 cDNA expressing vectors. The RNA isolated from these cells was reverse transcribed, and we then studied potential differences in the composition of isoforms of putative splicing targets of RBM20 (Guo et al 2012, Maatz et al) or other spliceosomal proteins. For this purpose, primers were designed for the CAMK2D, CAMK2G, LDB3, SH3KBP1, SORBS1(1), SORBS1(2), TNNT2, TPM1 and TRDN targets, and differences in splicing patterns in presence of wild type, R634P, or endogenous RBM20 production were analyzed. Out of the nine potential targets, only LDB3 showed clear effects in this assay. We therefore decided to continue evaluating differential splicing of the known cardiomyopathy gene (Vatta et al) and RBM20 target (Guo et al 2012, Maatz et al) LDB3. We first sequenced the three different length PCR products of LDB3 detected on agarose gel, using the primers corresponding to 3’ sequences of exon 3 and 5’ sequences of exon 7, respectively. The two longer products we identified were shown to correspond to transcripts NM_001080114.1 and NM_001080116.1 (product of 472 nucleotides, including exon 3, 5, 6 and 7), and transcripts NM_0070782 and NM_001080115.1 (product of 614 nucleotides, including exon 3, 4 and 7). However, the shorter 245 nucleotide product did not correspond to the remaining isoforms NM_001171610.1 and NM_001171611.1 and only contained sequences of exon 3 and 7 (see also figure 1).

Next, we analyzed the resulting LDB3 products in cDNA derived from HEK293 cells expressing the different RBM20 mutations/variants. A pre- liminary screening (a PCR under saturating conditions followed by gel electrophoresis) did not show an obvious presence/absence of certain LDB3 products when comparing wild type cells with cells expressing one of the hotspot mutations. However, as we observed subtle differences in LDB3 product intensities between the different samples (data not shown), subsequent analyses were aimed at quantifying the respective products under non-saturating conditions. Unfortunately, this did not lead to the identification of significant differences in product intensities between cells carrying the vector expressing wild type or certainly pathogenic mutations of RBM20 (figure 2). As shown in figure 2, we did observe some differences in the product intensity patterns between non-transfected, wild type transfected and mutation (R634P(1), R634P(2), R634W and R636H) transfected cells, although this was less apparent for the R636H transfected cells. The most prominent effect we saw was relatively high amounts of the 613bp product in non-


CHA

PTER

2.1

Figure 1. LDB3 splicing products identified in transfected HEK293 cells. PCR amplified and sequenced LDB3 transcripts expressed by the HEK293 cells are shown. Two fragments correspond to known LDB3 transcripts/isoforms (472bp and 613bp), while the exon composition of the shortest one (245bp) does not correspond to known transcripts. The known transcripts of LDB3 have the following NCBI IDs: transcript/isoform 1 – NM_007078.2, 2 – NM_001080114.1, 3 – NM_001080115.1, 4 – NM_001080116.1

transfected cells, while we observed high proportions (90-100%) of the 245 bp product in wild type transfected cells and slightly smaller proportions (<80%) of this short product in mutation transfected cells (with the exception of R636H) (figure 2). In most of the other cases (G672S, W768S, W768L and D888N transfected cells) the product intensity patterns resembled that of wild type transfected cells. The only exception to this was V535L transfected cells, which looked more like those of the certainly pathogenic mutation expressing cells, suggesting that this variant may be pathogenic as well.

During our analysis of variants we grouped together the certainly pathogenic, wild type, non-transfected and unknown variants. A significant effect of the base position was present, but there was no significant effect of the baseposition x mutation type interaction. As an illustration, the results for the interaction are shown in figure 3. In summary, we were not able to show the presence of different splicing products or a shift in isoform composition among the samples using our in vitro splicing assay.


Figure 2. Individual LDB3 splicing plots of transfected and non-transfected HEK293 cells. On each plot the X axis shows the basepositions (corresponding to the 245, 473 and 613 bp long fragments), while the associated area under the curve (AUC) values are indicated on the Y axis. Samples marked with * are biological replicates.

DISCUSSION

In this study, we aimed to identify and functionally evaluate mutations in the RBM20 gene in DCM patients, as well as in patients suffering from other subtypes of cardiomyopathy. This led to the identification of 23 different rare variants that might be involved in disease development. Importantly, the 10 mutations in 17 patients that were classified as likely pathogenic or pathogenic were almost exclusively found in DCM patients, underscoring the idea that RBM20 mutations are associated with the development of this cardiomyopathy


CHA

PTER

2.1

Figure 3. Grouped LDB3 splicing plots of transfected HEK293 cells.

subtype. Notably, a few non-hotspot variants of unknown significance or likely pathogenicity were found in HCM, or in both HCM and DCM patients. Moreover, the identification of two likely pathogenic or pathogenic mutations in PPCM cases suggests that RBM20 mutations could be specifically involved in the development of this particular manifestation of familial DCM as previously reported for the TTN gene (van Spaendonck-Zwarts et al).

Of the 23 variants we identified, 3 were classified as pathogenic mutations, 7 as likely pathogenic mutations and 13 as variants with unknown clinical significance. Based on its frequency in controls, we classified variant D888N, which was formerly reported to be pathogenic, as a rare polymorphism. To gain more insight into the putative pathogenicity of the promising variants we found, we performed additional tests such as segregation and haplotype analyses. When DNA sample from family members was available, we performed segregation analysis for the mutations found in the index patients. We found the mutations R634P and R636H co-segregated with the disease phenotype, while the Y681C variant did not co-segregate in the one family we could further investigate. One of the most interesting findings was the identification of the S637N mutation in a family with PPCM and DCM. However, the segregation analysis in this family could not prove that this novel RBM20 hot spot mutation (found in the PPCM patient and her DCM-affected daughter, but not in the grandfather who had possible DCM; Spaendonck-Zwarts et al) played and exclusive disease-causing role, leading to our classification of it as a VOUS. It is possible, that the RBM20 mutation together with another not-


yet-identified genetic mutation could explain the disease in this family, with the RBM20 mutation contributing to the PPCM phenotype. In a few other cases, the RBM20 variant was shown to be inherited in combination with other likely pathogenic mutations, e.g. in MYH7 or SCN5A (table 2). Such digenic and oligogenic inheritance is increasingly observed in various types of cardiomyopathy (Bauce et al, Xu et al 2010, Nakajima et al, Roncarati et al, Bao et al, Rigato et al, Pugh et al, Haas et al, chapter 2.2 and chapter 4.1). Likewise, the RBM20 mutation carriers published by Li et al (2010) carry other variants in LDB3 and LMNA as well.

For unrelated patients who carried the same mutation, we performed marker analysis in order to check if the mutation is part of the same relatively large haplotype. This resulted in linking three index patients carrying the same R634W mutation, which suggests that they have inherited it from a common ancestor.

Finally, in order to be able to further evaluate the pathogenicity level of the mutations found in our patients, we performed functional experiments. Several recent papers indicated that RBM20 has an important role in splicing dynamics of RNA molecules encoding cardiomyopathy-related proteins, mainly by studying these processes under in vivo conditions both in the presence of wild type or mutated RBM20 (Guo et al 2012, Guo et al 2013, Maatz et al). However, there were no myocardial biopsies available from the patients with RBM20 mutations in our study that could be used to evaluate the effect of these mutations on differential splicing in vivo. Therefore, it was our aim to design an in vitro assay in which this could be easily tested but not requiring an invasive procedure. Unfortunately, although our first experiments on transcripts of the validated RBM20-target LDB3 in HEK293 cells indicated minor differences in isoform intensities between samples, no significant differences were apparent upon quantification of the data and the results were not conclusive. One explanation for this could be that the cell line used in this assay has an embryonic kidney origin rather than being derived from embryonic or adult cardiomyocytes. If this were the case, splicing products resulting from compensating mechanisms and/or splicing procedures diverging from regular cardiac splicing may have concealed the true RBM20-related splicing effects. For example, one of the LDB3 transcripts we identified both in non-transfected and in wild type and mutated RBM20-transfected HEK293 cells does not correspond to the known isoforms, and it lacks the alternative Zasp-like motifs (exons 4, 5 or 6) which play a role in


CHA

PTER

2.1

actin-binding (Huang et al, Klaavuniemi et al). Hence, it might not be the ideal system for modelling cardiomyopathy. On the other hand, it is likely that RBM20 overexpression conditions in our assay influence the outcome more than the effects of the RBM20 mutations, as Maatz et al have recently shown dramatic differences in the splicing of TTN, RYR2, CAMK2D and LDB3 between heart failure patients with relatively high and low expression levels of endogenous RBM20. Moreover, the same study also showed that patients exhibiting high expression of wild type RBM20 had similar LDB3 splicing patterns to a DCM patient who carried the RBM20 mutation S635A. The fact that differences could be observed between LDB3 splicing patterns of the non-transfected (endogenous RBM20 expressing) and the transfected (wild type or mutant RBM20 overexpressing) HEK293 cells during our functional in vitro assay suggests that RBM20 overexpression, regardless of its wild type or mutant sequence, affects differential LDB3 splicing to an extent that conceals mutation effects, and thus hampers the evaluation of pathogenicity levels of the different RBM20 variants.

An intriguing finding is that in two RBM20-mutation-carrying families in our study, those carrying the S637N and the R636H mutations, family members were diagnosed with PPCM. Previously, no association had been reported between PPCM and RBM20 mutations. However, we recently showed that PPCM/DCM can be frequently caused by truncating TTN mutations (van Spaendonck-Zwarts et al). In fact, our functional studies on explanted tissue of a TTN truncating mutation carrier demonstrated that the passive force generation of the single cardiomyocytes is drastically decreased, but also showed that the TTN isoform composition (which provides the molecular basis for this passive tension) is shifted towards more long, compliant N2BA isoform production in place of short N2B isoform production (van Spaendonck-Zwarts et al). The splicing of TTN is known to be regulated by RBM20-mediated exon skipping, a process which is disrupted by mutations of RBM20 (Guo et al 2012). Moreover, Rbm20-/- rats were found to exclusively express the N2BA isoform at all ages, while this isoform was shown to only account for about 15% of expressed titin 20 days after birth in healthy animals (Guo et al 2013). Together, these findings suggest that titin isoform shift may be a common molecular pathway in PPCM/DCM patients with either RBM20 or TTN mutations.

RBM20 mutation carriers have been reported to have unusually severe symptoms with very early onset (Brauch et al, Li et al 2010, Wells et al). We have only observed this in two cases within the two families with PPCM/DCM:


a patient carrying R636H passed away at the age of 23 while another with the S637N mutation had to go through heart transplantation at the age of 19. It is likely that the more severe nature of the disease in the earliest reported RBM20 families was the result of patient selection bias as more recent papers reported contradictory results, although including patients carrying RBM20

“mutations” of which the pathogenic nature is questionable (like the S455L and D888N variants) may have affected the outcomes of these studies (Haas et al; Refaat et al).

Aberrant splicing, which is the key pathomechanism leading to cardiomyopathy and DCM in RBM20-mutation-carrying patients, has been associated with various diseases including, increasingly, cardiac diseases. For example, it was recently shown that heart failure (due to aortic stenosis, ischemic and DCM) is associated with altered splicing of sarcomeric genes, such as TNNT2, TNNI3, MYH7 or FLNC (Kong et al). Indeed, it is not only RBM20 that is known to contribute to the splicing machinery and splicing events in the physiological processes of the heart, several other splicing factors have also been connected to DCM in various animal models; SC35, ASF/SF2, and FXR1 are involved in the processing of calcium homeostasis or desmosomal proteins (Ding et al, Xu et al 2005, Whitman et al). There are also structural homologues of RBM20 that are known to play a role in the heart. One of these homologues is MATR3, which is involved in adult onset myopathy (Senderek et al), and which has been shown to interact with wild type RBM20, but to lose this ability in the presence of the S635A mutant (Maatz et al). RBM4 and PTB are antagonists of each other competing for the recognition element of TPM1 mRNA and regulating its exon selection (Lin & Tarn), while RBM24 is involved in cardiac differentiation and its targets are important for sarcomere assembly (Poon et al). According to the co-translational assembly model, the muscular RNA-binding proteins play an extremely important role in filament assembly, because they bind and stabilize their targets in the nucleus, and mediate the transports of those to close proximity to the sarcomere for immediate use (Zarnescu & Gregorio).

It would therefore be worthwhile to study whether some of these spliceosomal proteins/genes could also contribute to human cardiac diseases, e.g. cardiomyopathy or heart failure in general. Remarkable in this respect is that, apart from RBM20, no clearly Mendelian inherited mutations in other spliceosomal proteins causing DCM have yet been reported. It may be that these mutations are rare and private familial mutations that still have to be


CHA

PTER

2.1

discovered. On the other hand, as the splicing machinery is generally quite complex, involving a number of analogous proteins, it may be able to accept mutations in individual components of the machine without very drastic effect on its function. If this is the case, it will be even more intriguing to find out why RBM20 mutations specifically result in the development of DCM. Since the expression of RBM20 is not strictly limited to the heart (Filippello et al), and the RBM20 RNA-recognition element containing an UCUU core sequence (Maatz et al) is quite short and not very specific, neither of these can sufficiently explain the purely cardiac phenotype of the patients. Further research on how the interconnections of RBM20 with other spliceosomal proteins ultimately mediate the splicing process in the heart in a tissue- and life-stage specific manner is needed.

ACKNOWLEDGEMENTS

The authors would like to thank Jackie Senior and Kate Mc Intyre for editing the manuscript. Anna Pósafalvi was supported by grants from the Jan Kornelis de Cock Foundation.

REFERENCESBao JR, Wang JZ, Yao Y et al. Screening of pathogen-

ic genes in Chinese patients with arrhythmo-genic right ventricular cardiomyopathy. Chin Med J (Engl) 2013;126:4238-41

Bauce B, Nava A, Beffagna G et al. Multiple mu-tations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia. Heart Rhythm 2010;7:22-9

Beraldi R, Li X, Martinez Fernandez A et al. Rbm20-deficient cardiogenesis reveals early disruption of RNA processing and sarcomere remodeling establishing a developmental eti-ology for dilated cardiomyopathy. Hum Mol Genet 2014;23:3779-91

Brauch KM, Karst ML, Herron KJ et al. Mutations in ribonucleic acid binding protein gene cause familial dilated cardiomyopathy. J Am Coll Car-diol 2009;54:930-41

Ding JH, Xu X, Yang D et al. Dilated cardiomyopa-thy caused by tissue-specific ablation of SC35 in the heart. EMBO J 2004;23:885-96

Filippello A, Lorenzi P, Bergamo E et al. Identi-fication of nuclear retention domains in the RBM20 protein. FEBS Lett 2013;587:2989-95

Guo W, Pleitner JM, Saupe KW et al. Pathophys-iological defects and transcriptional profil-ing in the RBM20-/- rat model. PLoS One 2013;8:e84281

Guo W, Schafer S, Greaser ML et al. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med 2012;18:766-73

Haas J, Frese KS, Peil B et al. Atlas of the clinical genetics of human dilated cardiomyopathy. Eur Heart J 2014; pii: ehu301 [epub ahead of print]

Huang C, Zhou Q, Liang P et al. Characterization and in vivo functional analysis of splice variants of cypher. J Biol Chem 2003;278:7360-5

Klaavuniemi T, Kelloniemi A, Ylannet J: The ZASP-like motif in actinin-associated LIM protein is required for interaction with the α-actinin rod and for targeting to the muscle Z-line. J Biol Chem 2004;279:26402-10

Kong SW, Hu YW, Ho JW et al. Heart failure-asso-ciated changes in RNA splicing of sarcomere genes. Circ Cardiovasc Genet 2010;3:138-46

Li D, Morales A, Gonzalez-Quintina J et al. Identifi-cation of novel mutations in RBM20 in patients with dilated cardiomyopathy. Clin Trans Sci 2010;3:90-97


Li S, Guo W, Dewey CN et al. RBM20 regulates titin alternative splicing as a splicing repressor. Nu-cleic Acids Res 2013;41:2659-72

Lin JC & Tarn WY: Exon selection in alpha-tro-pomyosin mRNA is regulated by the antago-nistic action of RBM4 and PTB. Mol Cell Biol 2005;25:10111-21

Maatz H, Jens M, Liss M et al. RNA-binding protein RBM20 represses splicing to orchestrate cardi-ac pre-mRNA processing. J Clin Invest 2014 Jun 24. pii: 74523

Mestroni L, Rocco C, Gregori D et al. Familial di-lated cardiomyopathy: evidence for genetic and phenotypic heterogeneity. Heart Mus-cle Disease Study Group. J Am Coll Cardiol 1999;34:181-90

Millat G, Bouvagnet P, Chevalier P et al. Clinical and mutational spectrum in a cohort of 105 un-related patients with dilated cardiomyopathy. Eur J Med Genet 2011;54:e570-5

Nakajima T, Kaneko Y, Irie T et al. Compound and digenic heterozygosity in desmosome genes as a cause of arrhythmogenic right ventricular cardiomyopathy in Japanese patients. Circ J 2012;76:737-43

Poon KL, TanKT, Wei YY et al. RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility. Cardiovasc Res 2012;94:418-27

Pugh TJ, Kelly MA, Gowrisankar S et al. The land-scape of genetic variation in dilated cardiomy-opathy as surveyed by clinical DNA sequencing. Genet Med 2014;16(8):601-8

Refaat MM, Lubitz SA, Makino S et al. Genetic variation in the alternative splicing regulator RBM20 is associated with dilated cardiomyopa-thy. Heart Rhythm 2012;9:390-6

Rigato I, Bauce B, Rampazzo A et al. Compound and digenic heterozygosity predicts life-time arrhythmic outcome and sudden cardiac death in desmosomal gene-related arrhythmogenic right ventricular cardiomyopathy. Circ Cardio-vasc Genet 2013;6:533-42

Roncarati P, Viviani Anselmi C, Krawitz P et al. Doubly heterozygous LMNA and TTN muta-tions revealed by exome sequencing in a severe form of dilated cardiomyopathy. Eur J Hum Genet 2013;21(10):1105-11

Senderek J, Garvey SM, Krieger M et al. Autoso-mal-dominant distal myopathy associated with a recurrent missense mutation in the gene en-coding the nuclear matrix protein, matrin 3. Am J Hum Genet 2009;84:511-8

van Spaendonck-Zwarts KY, Posafalvi A, van den Berg MP et al. Titin gene mutations are com-

mon in families with both peripartum cardio-myopathy and dilated cardiomyopathy. Eur Heart J 2014;35:2165-73

Vatta M, Mohapatra B, Jimenez S et al. Mutations in Cypher/ZASP in patients with dilated cardio-myopathy and left ventricular non-compaction. J Am Coll Cardiol 2003;42:2014-27

Wells QS, Becker JR, Su YR et al. Whole exome se-quencing identifies a causal RBM20 mutation in a large pedigree with familial dilated cardiomy-opathy. Circ Cardiovasc Genetics 2013;6:317-26

Whitman SA, Cover C, Yu L et al. Desmoplakin and Talin2 are novel mRNA targets of Fragile X-related protein-1 in cardiac muscle. Circ Res 2011;109:262-71

Xu X, Yang D, Ding JH et al. ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell 2005;120:59-72

Xu T, Yang Z, Vatta M et al. Compound and digenic heterozygosity contributes to arrhythmogenic right ventricular cardiomyopathy. J Am Coll Cardiol 2010;55:587–97

Zarnescu DC & Gregorio CC: Fragile hearts: New insights into translational control in cardiac muscle. Trends Cardiovasc Med 2013;23:275-81


CHA

PTER

2.1

Supplementary table 1. RBM20 sequencing primers. PT-tails were attached to all primers that facilitated sequencing using PT-primers and resulted in better quality sequencing data.

amplicon direction primer sequence (5'-> 3')exon 1 F GCCACCGGGAAGGACAAGGG

R TCAGCAGGGACGGGAAATGAAGCexon 2/1 F GACCAGTGTGGGAAGGTCTTG

R CATTAGAGGGAAACCGGGTACTGexon 2/2 F AATCAACTGAGGCATCCGTCTG

R AAGGCAGCTTGACCATCCTGexon 2/3 F TATGGCCCTGAAACAGATGG

R GATAGCAACGCCTGGTCCTCexon 2/4 F CCCGAGGAACCAACCTCAGAC

R CTTGCCCATTGGCAGCGTGAGexon 3 F CAGCCTCTGGGCGCTCTGTG

R ACTTTGCCTGTTCTGGTCTCTGexon 4 F GGGCTGCTAGGAAGGTTTGG

R TGCTTTCTACATCCGTGAGAAGGexon 5 F CAGCATGTCCAGAGGTACAATC

R GCATTCCAGCCTGGCTGTCTCexon 6 F CCTGGGTGATGGGAGTGAGAC

R GTGGTCTGTGGCATATACACTGexon 7 F GACGTGGAATCATGCCTTGTG

R GCAATGGTTTGCCTCGAGATCCexon 8 F TGGTGGACCAGGCAATGAATG

R GAACAGGGCACAGCATGACTCexon 9/1 F TGCACAGTATATCTAAGACAGAGAC

R AGACCCAGATCTCGGGTACTTCexon 9/2 F CCGGCAACTGGACAAGGCTG

R CTCCTTCAGGGCCTGCCTCGexon 10 F GCTGGGACCTGCATTCAATATC

R GGGTCTCAGCCATATTCCATCCexon 11/1 F ATGGCCAAGTCTTGTGCCTTCC

R CCGCTCAGCATCCAGATTTAGGexon 11/2 F AGCCTCAAGTCACCCAGAGAAC

R GGTGAGCAGGAGTCCAATCAACexon 12 F GCTCCTAATGACAGTGCTTTGG

R CAAGCTCTTGAGGTTGCTATGGexon 13 F TGGAGCTCGTGGCTCCCATTTC

R AAACAGCCTGGTGTGCTTGGexon 14 F GCACAGATGCCAGGAGAGGGATG

R TGGGTGACTTGCTCCTGGAGAC


Supplementary table 2a. Marker analysis primers. The markers used to screen for a potentially shared haplotype inherited from a common ancestor were selected from the deCODE high resolution genetic map. The names and primers can be found in the first two columns. The RBM20 gene can be found at 112.5 M (NC_000010.10) and markers in a region of 5 cM at each side were selected (see Location and Distance coloumns).

Marker Primer sequences 5'- 3' Location (NC_000010.10)

Distance to RBM20 gene (cM)

D10S530 TCTAGCAGTAAGAGTTGTGTCTCC 107.5M / 123.53 cM 4.4TTGACAAGGCCATCAAAAC

D10S1778 CTTGGTTATGATCTCACATGGTCT 108.1M / 124.24 cM 3.7CTGCTCTGGATTGAATGTTT

D10S521 CTCCAGAGAAAACAGACCAA 109.3M / 125.85 cM 2.1CCTACCATCAATCAACTGAG

D10S597 GAATGAAGACATCCAGAGG 111.2M / 126.7 cM 1.3GCAAGTATCAGAAACCCAA

D10S543 AAAGATGTTCAGGTAGATAACACAC 111.8M / 127.43 cM 0.6ATCCCTCAGCCCCACT

MUTATION (R634W)D10S1760 GCGAGACTCCATCTCCATAG 113.7M / 128.63 cM 0.6

CCATATAGTGGGTGGCTTAAAD10S1429 GCTCGTAATAGCTTTGTCCA 114.1M / 129.4 cM 1.4

ATGAAACCATATATGTGACTTTTTGD10S168 CATGGCACTAATAGAGTTAAC 114.7M / 130.05 cM 2

TTCACTTGGGATGGAGGCAD10S554 GGAGGACTCATGTCAGACTT 116.0M / 132.47 cM 4.4

CCTACCTTTAATTCAGCCCTD10S468 CAGGCATGTCCATGTAGGTA 117.2M / 134.83 cM 6.7

TCTGTAAATAACTCATTTGTCCG

Supplementary table 2b. Results of marker analysis for the R634W mutation carriers

Markers

Expected product range (length in bp) Patient 1 Patient 2 Control 1 Control 2

allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 allele 1 allele 2D10S530 182-208 196 202 196 198 198 200 190 192D10S1778 216-226 220 220 218 220D10S521 155-189 173 177 173 181 177 181 173 177D10S597 206-222 214 216 214 215 214 218 215D10S543 129-149 129 138 138 137 140 138 MUTATION (R634W) D10S1760 112-155 107 113 107 113 109 113 109 137D10S168 159-165 160 160 162 162D10S554 150-162 148 150 150 150D10S468 82-96 87 83 87 89 81 87


CHA

PTER

2.1

Supplementary table 3. RBM20 vector sequencing primers

amplicon primer sequence (5'-> 3')VP1.5_F GGACTTTCCAAAATGTCG02A_R_PT2 CATTAGAGGGAAACCGGGTACTG02B_F_PT1 AATCAACTGAGGCATCCGTCTG02C_R_PT2 AGATAGCAACGCCTGGTCCTC02D_F_PT1 CCCGAGGAACCAACCTCAGACexon5_R CCACAGAAGCCAAAGGAAATGexon3_F CTGGGAGCTGCATGTGAAAG09A_R_PT2 AGACCCAGATCTCGGGTACTTC09B_F_PT1 CCGGCAACTGGACAAGGCTG11A_R_PT2 CCGCTCAGCATCCAGATTTAGG11B_F_PT1 AGCCTCAAGTCACCCAGAGAACXL39_R ATTAGGACAAGGCTGGTGGG

Supplementary table 4. Site-directed mutagenesis primers. The 5’ primer ends were phosphorylated.

Variant Primer sequences 5'- 3' TM Calculator Annealing

L100F_F CAGCTCACCTTCCACCGGC 70,84 71

L100F_R GGCCTGCAGTTGAGCCAGC 71,09 71

V535L_F GGCTGCCCTTTGGAAAGGT 67,73 68

V535L_R CCAGGTTAATGAGGTCATTCTCAGTG 67,73 68

R634W_F AGAAAGGCCGTGGTCTCGTAG 66,85 67

R634W_R GGGCCATATCTGTCTGCTTCC 67,13 67

R636H_F CGCGGTCTCATAGTCCGGT 67,79 68

R636H_R GCCTTTCTGGGCCATATCTGTC 67,99 68

G672S_F TCCTGGGAGCACTCTCCCTATGC 71,74 71,5

G672S_R GTCCCGGCTATTGCCCCAGT 71,45 71,5

W768S_F CAAAGCCAAGTCGGACAAGTATCTG 68,72 69

W768S_R GGCTCTTTCCGGTAGTAGCCGT 68,54 69

W768L_F CAAAGCCAAGTTGGACAAGTATCTGA 68,14 68

W768L_R GGCTCTTTCCGGTAGTAGCCGT 68,54 68

S637N_F CGGTCTCGTAATCCGGTGAGC 70,16 70

S637N_R CGGCCTTTCTGGGCCATATC 69,82 70

D888N_F AAAGTGAGGCAGAGGGGGAG 67,08 67

D888N_R CACTCTCCCAATTTTGTTCCTTCTT 66,44 67


Supplementary table 5. RBM20 cDNA primers

amplicon direction primer sequence (5'-> 3')exon 3 F CTGGGAGCTGCATGTGAAAGexon 5 R CCCACAGAAGCCAAAGGAAATGexon 9 F GGAGAAGTACCCGAGATCTGexon 9 R TGGCCTCGTCTTTCCTCCTGexon 10 F AACAGGAGGGCATGGAAGAAAGexon 11 R CATTCCCTACGGCCTTGACTC

Supplementary table 6. Target cDNA primers. The following primer pairs were used for differential splicing analysis of the target molecules of RBM20. Abbreviations: CAMK2D

– calcium/calmodulin-dependent protein kinase II delta; CAMK2G - calcium/calmodulin-dependent protein kinase II gamma; LDB3 – LIM domain binding 3; SH3KBP1 – SH3-domain kinase binding protein 1; SORBS1 – sorbin and SH3 domain containing 1; TNNT2

– troponin T type 2 (cardiac); TPM1 – tropomyosin 1 (alpha); TRDN – triadin.

target gene direction primer sequence (5'-> 3')CAMK2D F AAGGGTGCCATCTTGACAAC

R TCAAAGTCCCCATTGTTGATCAMK2G F CAACGATCCACGGTGGCATCC

R GTGTAGGCCTCAAAGTCCCCALDB3 F TCAAAGCGTCCCATTCCCATC

R TGAATTCTGTCCCCGTCATCTGSH3KBP1 F GTAGAGGAAGGATGGTGGGAA

R CTACTTCAATTGACCTTGGTCSORBS1 /1 F AGAGCACTCAGGACTTAAGC

R AGATGCAGGAAACTGGTAGGSORBS1 /2 F CGCTCTTCCTCACTGAAGTC

R GAGTAGGGCTGATGGCTGAGTNNT2 F CATAGAAGAGGTGGTGGAAGAG

R TCCTTCTCCCGCTCATTCCTPM1 F TGGACGCCATCAAGAAGAAG

R TCATATTTGCGGTCGGCATCTRDN F CAAAGACACTGGCGAAAG

R GCTTGTTCTGTCGGTAAGG

Chapter 2.2

Missense variants in the rod domain of plectinincrease susceptibility to

arrhythmogenic right ventricular cardiomyopathy

Anna Posafalvi, Petros Syrris, Vincent Plagnol, Ludolf G Boven, Marieke C Bolling, Judith A Groeneweg, MP van den Berg,

Marcel F Jonkman, Arthur AM Wilde, Richard NW Hauer, Richard J Sinke, William McKenna, J Peter van Tintelen, Jan DH Jongbloed

Manuscript in preparation

ABSTRACT

Aims: Although mutations causing arrhythmogenic right ventricular cardiomyopathy (ARVC) have been identified in several genes, they do not explain the disease in all patients. Since the majority of these genes encode desmosomal components, we hypothesized that proteins known to be physically linked to the desmosome might contain ARVC-causing genetic variations. Thus, we screened patients for mutations in the PLEC gene encoding the cytolinker protein plectin that anchors intermediate filaments to the desmosomes.

Methods & Results: We sequenced 107 ARVC patients from the Netherlands and 358 patients from the UK with either Sanger or high-throughput sequencing for the coding regions of PLEC, and identified 96 novel or low frequency (<2%) variants scattered across the gene. A comparison with the genetic variation of PLEC seen in the GoNL control population revealed an area of the rod domain harbouring multiple missense variants that are only present in ARVC patients. Careful classification of the variants based on their conservation, frequency, and predicted pathogenicity level, combined with the lack of genetic variability of this region in healthy controls, suggests that these variants, which are located in the domain responsible for homodimerization, may affect normal plectin function.

Conclusions: Although PLEC has been hypothesised as a promising candidate gene for ARVC, our current studies do not support mutations in this gene as the primary cause of ARVC. Our data do, however, suggest that PLEC missense mutations, in particular those in regions of the protein lacking variation in healthy controls, could play a risk factor role in an oligogenic inheritance model of ARVC.

Keywords: ARVC, oligogenic inheritance, plectin, rod domain

81PLEC IN ARRHY THMOGENIC CARDIOMYOPATHY

CHA

PTER

2

INTRODUCTION

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive heart disease characterized histologically by fibrofatty infiltration of the ventricular myocardium. Clinically, ARVC patients may suffer from ventricular arrhythmias, syncope, and sudden cardiac death as early as young adulthood (Sen-Chowdhry et al), with the majority of cases diagnosed before the age of 40 (Teekakirikul et al).

Familial ARVC is a genetically heterogeneous disease and is most commonly transmitted as an autosomal dominant trait with variable expression and incomplete penetrance (te Rijdt et al). However, oligogenic inheritance is increasingly observed (Bauce et al, Xu et al, Nakajima et al, Bao et al), and carriership of multiple rare variants was recently suggested to be related to a more severe disease outcome (Marcus et al 2013, Rigato et al). Currently, ARVC is considered a disease of the desmosome, an important cell-cell adhesion structure. Genes encoding desmosomal proteins such as plakophillin-2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC2), junctional plakoglobin (JUP), and desmoplakin (DSP) have been reported to carry ARVC-related mutations (te Rijdt et al). Most mutations causing familial ARVC have been found in the PKP2 gene (found in up to 70% of the patients) (van Tintelen et al). Mutations have also been identified in the DSC2 and DSG2 genes although in lower proportions (in up to 15% of patients). Mutations in the JUP and DSP genes are less frequent in ARVC patients, and are more often associated with the cardiocutaneous syndromes Naxos disease (McKoy et al) and Carvajal syndrome (Norgett et al), respectively. Finally, desmin (DES), the intermediate filament protein that builds up the cytoskeleton of cardiomyocytes and is anchored to the membrane via desmosomes has been implicated in ARVC as well (te Rijdt et al).

Recently, more ARVC-associated genes have been discovered: the cell-cell adhesion molecule α-3 catenin (CTNNA3); the nuclear envelope protein lamin A/C (LMNA); titin (TTN), which is involved in sarcomere assembly and passive elasticity; regulators of intracellular Ca-levels, namely ryanodine receptor 2 (RYR2) and phospholamban (PLN); transforming growth-factor β-3 (TGFB3) and transmembrane protein 43 (TMEM43), of which the function is unknown (te Rijdt et al). These new genes still only contribute to a small proportion of this predominantly desmosomal disease, and roughly half of ARVC cases remain unexplained (Jacob et al, Marcus et al 2013). It was therefore the purpose of this study to explore whether the putative candidate


gene PLEC, which encodes a protein (plectin) physically connecting the cardiac desmosome, is also involved in ARVC.

Plectin is a large cytolinker protein and belongs to the plakin family of proteins. Among other functions, plectin is believed to connect the desmosomes to the cytoskeleton by binding the DSP protein of the desmosomes and the desmin/keratin filaments in the myocardium/skin epithelium, respectively. In cardiac tissue, plectin is mainly localized at the intercalated disk and the sarcomeric Z-line, whereas in skin it is located in desmosomes and hemi-desmosomes. This means that plectin potentially has a general and fundamental function in junctional complexes (Wiche et al, Zernig et al). When fully knocked out in mice, the lack of plectin causes severe skin blistering, a phenotype similar to the symptoms of epidermolysis bullosa patients. Although this blistering condition in mice seemed lethal in itself a few days after birth, the animals also exhibited more phenotypic changes, such as generalized myopathies of the skeletal muscle, while ultrastructural differences (e.g. aberrant myofibril bundles and focal loss of Z-lines) were observed in the heart (Andrä et al).

Over the past two decades, PLEC has been shown to harbour mutations in patients suffering from various forms of inherited epidermolysis bullosa. Clinical phenotypes associated with mostly homozygous nonsense/frameshift mutations in the gene encompass autosomal recessive epidermolysis bullosa simplex (EBS) with muscular dystrophy (MD) (MIM#226670), EBS-MD with myasthenic symptoms (Winter & Wiche; not in OMIM), EBS-Ogna (autosomal dominant, heterozygous; MIM#131950), EBS with pyloric atresia (MIM#612138), and limb-girdle muscular dystrophy (MIM#613723) (reviewed by Winter & Wiche, Sonnenberg & Liem). Heterozygous carriership of missense mutations was recently reported in mild cases of EBS without cardio-muscular manifestation (Bolling et al 2013). Moving beyond these skin and muscle abnormalities, and based on the symptoms of the plectin KO mouse, PLEC was also suggested to be involved in further disease processes such as neurodegeneration or cardiomyopathy due to increased fragility of the desmosomes (Andrä et al). Nonetheless, only two incidental cases of cardiac involvement have been reported: a plectin mutation-carrying EBS-MD patient with ventricular hypertrophy (Schröder et al) and an EBS-MD patient who, by the age of 30, was discovered to have asymptomatic dilated cardiomyopathy (DCM) which later progressed to right ventricle involvement including (septal) fibrosis as well as features associated with arrhythmogenic cardiomyopathy


CHA

PTER

2

(Bolling et al 2010). Very recently, left ventricular non-compaction cardiomyopathy developed in an EBS-MD patient with a homozygous plectin truncation (Villa et al). An intriguing related phenomenon was also observed in a striated-muscle-specific conditional knock-out mouse model of plectin: the mice showed progressively declining endurance performance and, by the age of 16 months, a remarkable increase in connective tissue formation in the heart, indicating cardiomyocyte degeneration (Konieczny et al). In fact, the fibrofatty replacement of cardiomyocytes is the key underlying factor for cardiac conductivity problems in arrhythmogenic cardiomyopathy, and is one of the major task force criteria for the diagnosis of the disease (Marcus et al 2010, Elliott et al). Unfortunately, no electrophysiological studies addressing potential arrhythmias had been carried out in these knock-out mice.

Based on these observations, we hypothesized that plectin might play a role in cardiac pathophysiology, and analysed a large cohort of ARVC patients for carriership of PLEC variants. In many of these patients, novel or low-frequency variants with a potential pathogenic nature were identified. Comparing the genetic variation of PLEC in the Genome of the Netherlands (GoNL) database with that in our patients suggests that novel variants in a region of the rod domain may underlie, or at least increase the susceptibility to, ARVC. Although our data does not prove a major causal role for PLEC in ARVC development, it does suggest that variants in the PLEC gene may contribute to the development of an ARVC phenotype in a disease model in which a combination of several factors, both genetic and non-genetic, are needed to reach the threshold levels above which disease development is initiated.

MATERIALS AND METHODS

Patients

Patients were clinically evaluated and the diagnosis of ARVC was based on the criteria of the consensus-based international Task Force Criteria (TFC) (Marcus et al 2010). Our Dutch Sanger-sequenced cohort included 88 patients who fulfilled these clinical criteria for ARVC (TFC+) and 19 patients who did not completely fulfil them. The British gene-panel sequenced cohort included 123 TFC+ and 235 TFC- index patients. All patients were evaluated for genetic variations in PLEC. Many of the patients had also been screened for mutations in other ARVC-related genes.


Genetic analyses

Genomic DNA was isolated from blood samples using standardized procedures. Written informed consent was obtained from the index patients and their relatives according to the participating hospitals’ medical ethics committee guidelines. For the Dutch cohort, primers for PCR amplification of the coding regions of the PLEC gene (about a 14 kb long sequence) were designed to encompass the coding exons as well as adjacent intronic sequences as described previously (Bolling et al 2013), using the sequence obtained from the GenBank database. Variant annotation is according to NM_201380.3 unless otherwise indicated. Amplifications were conducted following a standard PCR protocol and PCR products were analysed by direct Sanger sequencing. For the UK cohort, a next generation sequencing (NGS) protocol was designed to screen 2.1 Mbp of genomic DNA sequence per patient that covered the coding regions of genes known to be associated with inherited cardiomyopathies (including ARVC) and arrhythmia syndromes. The gene panel also included PLEC as a candidate gene for ARVC. The sequencing methodology has been reported in detail (Lopes et al).

Data analysis: variant classification

In this study, the pathogenic nature of the identified missense mutations were judged based on: (1) the differences in the physico-chemical properties of the affected amino acids, (2) the evolutionary conservation of the affected amino acids across orthologues, (3) the frequency of the variant in control populations and databases (such as dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), 1000Genomes (http://www.1000genomes.org/), and ESP (http://evs.gs.washington.edu/EVS/)), and (4) the predicted pathogenic or benign nature of the variant (identified using the Alamut software; Interactive Biosoftware, Rouen). Every variant was then classified as either ‘benign’, ‘likely benign’, ‘variant of unknown significance’, ‘likely pathogenic’, or ‘pathogenic’ (see chapter 4.1 for a detailed description).

Data analysis: frequency-based variant clustering

Chromosomal positions of single nucleotide variants of the PLEC gene identified in our Sanger- and gene-panel-sequenced patient cohorts were annotated using information from the 1000 Genomes (1000G), the Exome Sequencing Project (ESP) and GoNL (http://www.nlgenome.nl/) control


CHA

PTER

2

cohort databases using an in-house developed script (to be published elsewhere), and frequency information on these variants was collected. PLEC variants with an allele frequency <2% in 1000G, GoNL, and ESP (considering the European ancestry population only) were uploaded into SeattleSeq and only non-synonymous coding variants were analysed further (variants with an allele frequency ≥2% were considered as definite polymorphisms). The resulting list of low frequency and novel ‘ARVC variants’ was compared with a list of low frequency and novel coding variations extracted from the GoNL database, which was filtered for allele frequencies as described above. Regions consisting of consecutive novel or rare (<0.5%) genetic variants of the PLEC gene identified exclusively in our ARVC TFC+ or TFC- patients, but lacking novel and/or rare variants in the GoNL control population, were subsequently checked for the putative presence or absence of any variation with allele frequency of >2% in GoNL.

RESULTS

Genetic screening of plectin

As a result of the genetic analysis of 465 patients (211 TFC+ and 254 TFC-) for the coding sequences of PLEC, we identified 96 variants that were either novel (not known from control populations) or low frequency (<2% in control populations), the majority of which were missense mutations (tables 1 and 2). Next, all variants were classified, assessing their potential pathogenic nature with the help of in silico prediction tools, as described elsewhere (for the classification criteria: see chapter 4.1; for the classification outcome: see supplementary table 1). For some of the PLEC variants we studied their possible cosegregation with the disease phenotype in the families, but this yielded no conclusive results (data not shown). In order to further assess the potential involvement of the variants in the disease, we compared the localization of these variants with the localization of genetic variants of the PLEC gene in a healthy population by extracting data from the GoNL whole genome sequencing database of 500 individuals (1000 alleles/genomes). This led to the identification of clustering of consecutive novel and low frequency variants in probable and known ARVC patients and the lack of these variants in healthy controls.


Table 1. PLEC variants identified in ARVC patients from the Netherlands. Overview of all novel and low frequency (<2%) coding variants identified in the PLEC gene in Dutch ARVC patients. Frequencies in the healthy control population of the GoNL database are indicated. The variants have been classified on the basis of differences in physicochemical nature, conservation, frequency, and predicted pathogenic effect. Frequencies of variants found in patients are indicated in red, while of variants found in controls in green. Frequency-based clustering of variants in the rod domain is shown in light yellow.

variant position Sanger NGS data classificationgenomic cDNA protein ARVC GoNL freq (%)145024454 421G>A R141C 0.1071 VOUS145016664 20C>T A7V 1x - LB145016560 124G>C D42H 1x - VOUS145013602 28C>T Q10* 1x - VOUS145013561_145013560 c69_70insTAC D23_N24insY 1x - VOUS145010082 947C>T R316Q 0.1010 VOUS145009211 1204C>T V402M 0.2020 VOUS145009069 1265G>A R422Q 1x - LB145009036 1298C>T R433Q 3x 1.3655 LB145008532 1534C>T V512M 0.2045 VOUS145008497 1569G>C S523R 1x 0.2079 VOUS145007449 1745G>A A582V 0.1006 LP145007273 1836C>G Y612* 1x - LP145007235 1874A>G Y625C 1x - LP145007192 1917C>G Q639H 0.1027 LB145004124 3130G>A R1044C 0.2114 VOUS145001652 4093G>A R1365W 2x 1.0664 LB145001221 4280T>C K1427R 1x - LB145001007 4400G>C T1467R 0.2049 VOUS145000968 4439C>T R1480H 0.1014 LP145000022 4486G>A R1496C 0.4373 LP144999731 4777C>T V1593M 0.1420 LB144999571 4937G>A R1646H 1x - VOUS144999565 4943C>T R1648Q 0.2833 VOUS144999499 5009C>T R1670Q 1x 1.1152 LB144999454 5054G>A A1685V 0.2924 LP144998932 5576C>T T1859M 1x - VOUS144998885 5623C>T R1875W 1x - LP144998707 5801G>A R1934H 1x - LP144998648 5860C>G R1954G 2x - LP144998621 5887G>A R1963W 1x - LP144998620 5888G>A R1963Q 3x - VOUS144998594 5914C>G L1972V 1x - LP144998588 5920G>A E1974K 1x - LP144998576 5932G>A A1978T 1x - LP144998399 6109A>G K2037E 1x - LP144998179 6329G>A R2110Q 1x - LP144998035 6473G>A A2158V 1x 0.1761 LP144997786 6722G>A A2241V 0.6383 VOUS144997765 6743A>C V2248G 0.7143 VOUS144997681 6827G>A R2276H 1x - LP144997577 6931G>A A2311T 1x - VOUS144997252 7256G>A R2419Q 1x - LP144996830 7678C>T A2560T 2x 0.5071 LB144996320 8080G>A R2694W 0.4464 LB144996169 8231C>G A2744G 2x - LB


CHA

PTER

2

144995977 8423C>T R2808Q 0.8989 LB144995942 8458C>T A2820T 0.1096 LB144995938 8462C>T R2821Q 0.3275 LB144995788 8612G>A A2871V 1x 0.2232 VOUS144995519 8881C>T V2961M 0.2105 VOUS144995500 8900T>C Y2967C 0.8368 LB144995480 8920C>T E2974K 3x 0.5133 VOUS144995477 8923C>T E2975K 1x - VOUS144995173 9227C>T R3076Q 0.1048 VOUS144995169 9231G>C D3077E 0.9395 B144995012 9388C>T D3130N 0.1042 LB144994936 9464C>T R3155Q 0.1025 LB144994442 9958C>T D3320N 0.2053 VOUS144994064 10336G>A R3446C 1x 1.4085 VOUS144994028 10372C>T G3458R 1.2195 LB144993931 10469G>C G3490A 1.8634 LB144993491 10909G>A R3637C 0.1008 VOUS144993344 11056C>A A3686S 1.3131 LB144993119 11281C>T E3761K 0.1008 LP144993076 11324G>A A3775V 0.1008 LB144992660 11740C>T E3914K 0.3067 LB144992378 12022C>T G4008S 0.3268 VOUS144992269 12131G>A T4044M 1.0121 LB144991963 12437C>T R4146H 0.1006 LP144991958 12442C>T V4148I 0.1004 LP144991802 12598C>T V4200M 0.2020 LP144991799 12601C>T E4201K 0.2016 LP144991205 13195G>A V4399I 1x - LB

Abbreviations B: benign; LB: likely benign; LP: likely pathogenic; P: pathogenic; VOUS: variant of unknown significance

Clustering of novel, likely pathogenic variants in the rod domain

We have identified one large, potentially disease-associated cluster of novel, missense genetic variants in the rod domain of the PLEC gene in the Dutch patient cohort (variants T1859M-R2110Q, see table 1). Interestingly, another, significantly overlapping ARVC-associated region was found in the same domain in the UK cohort (variants R1688C-E2157A, see table 2). This cluster of variants was not only promising due to its presence in ARVC patients and absence in control populations, but also because all variants within the cluster were classified as ‘likely pathogenic’ or ‘variant of unknown significance’ (VOUS) (for details of the variant classification, see supplementary table 1). Therefore, on the basis of the clustering of variants in patients and their predicted pathogenicity, we considered this region as ‘potentially pathogenic’.


Table 2. PLEC variants identified in ARVC patients from the United Kingdom. Overview of all novel and low frequency (<2%) coding variants identified in the PLEC gene in British ARVC patients. Frequencies in the healthy control population of the GoNL database are indicated. The variants have been classified on the basis of differences in physicochemical nature, conservation, frequency, and predicted pathogenic effect. Frequencies of variants found in patients are indicated in red, while of variants found in controls in green. Frequency-based clustering of variants in the rod domain is shown in light yellow.

variant position NGS data classificationgenomic cDNA protein TFC+ TFC- GoNL freq (%)145024845 30G>C D10E 1x - LB145024609 266G>A P89L 1x - VOUS145024570 305C>T R102H 1x - VOUS145024454 421G>A R141C 0.1071 VOUS145024372 503G>A P168L 1x - LB145011343 743C>T R248Q 1x - VOUS145010082 947C>T R316Q 0.1010 VOUS145009211 1204C>T V402M 0.2020 VOUS145009036 1298C>T R433Q 5x 8x 1.3655 LB145008532 1534C>T V512M 0.2045 VOUS145008497 1569G>C S523R 1x 0.2079 VOUS145008206 1634C>T C545Y 1x - LP145007449 1745G>A A582V 0.1006 LP145007192 1917C>G Q639H 0.1027 LB145006317 2474G>T P825Q 1x - LP145006145 2549C>G C850S 1x - LP145004376 2959C>T A987T 1x 1x - B145004373 2962C>T V988M 1x - VOUS145004124 3130G>A R1044C 0.2114 VOUS145003722 3352G>A R1118C 1x - LB145001873 3872C>T R1291Q 1x - LB145001652 4093G>A R1365W 1.0664 LB145001482 4189G>C R1397G 1x - LB145001221 4280T>C K1427R 1x - LB145001007 4400G>C T1467R 0.2049 VOUS145000968 4439C>T R1480H 0.1014 LP145000022 4486G>A R1496C 0.4373 LP144999871 4637A>C V1546G 1x - LP144999731 4777C>T V1593M 1x 0.1420 LB144999565 4943C>T R1648Q 0.2833 VOUS144999541 4967C>T S1656L - LB144999499 5009C>T R1670Q 4x 1.1152 LB144999454 5054G>A A1685V 0.2924 LP144999446 5062G>A R1688C 2x - VOUS144999268 5240G>A A1747V 1x - LP144999224 5284C>T E1762K 2x - LP144998857 5651G>A T1884M 1x - VOUS144998782 5726G>A A1909V 1x - VOUS144998626 5882G>A A1961V 1x - LP144998621 5887G>A R1963W 2x - LP144998620 5888G>A R1963Q - VOUS144998077 6431G>A A2144V 1x - LP144998038 6470T>G E2157A 1x - VOUS144998035 6473G>A A2158V 0.1761 LP144997899 6609C>A Q2203H 1x - LB144997786 6722G>A A2241V 0.6383 VOUS144997772 6736G>A R2246W 1x - VOUS144997765 6743A>C V2248G 0.7143 VOUS


CHA

PTER

2

144997651 6857C>T R2286Q 1x - LP144997561 6947C>T R2316Q 1x - VOUS144996830 7678C>T A2560T 1x 2x 0.5071 LB144996320 8080G>A R2694W 1x 0.4464 LB144995977 8423C>T R2808Q 2x 0.8989 LB144995948 8452C>T E2818K 1x - LP144995942 8458C>T A2820T 0.1096 LB144995938 8462C>T R2821Q 1x 0.3275 LB144995807 8593T>C I2865V 1x - LB144995788 8612G>A A2871V 2x 0.2232 VOUS144995656 8744T>C K2915R 1x - VOUS144995519 8881C>T V2961M 0.2105 VOUS144995500 8900T>C Y2967C 2x 13x 0.8368 LB144995483 8917C>T D2973N 2x - VOUS144995480 8920C>T E2974K 1x 2x 0.5133 VOUS144995477 8923C>T E2975K 1x - VOUS144995459 8941C>T A2981T 1x - LB144995173 9227C>T R3076Q 0.1048 VOUS144995169 9231G>C D3077E 2x 3x 0.9395 B144995012 9388C>T D3130N 0.1042 LB144994955 9445C>T E3149K 1x - VOUS144994946 9454T>C T3152A 1x - LB144994936 9464C>T R3155Q 0.1025 LB144994442 9958C>T D3320N 0.2053 VOUS144994396 10004C>T R3335Q 1x - LB144994346 10054T>C K3352E 1x - LB144994298 10102G>A R3368C 1x - VOUS144994175 10225G>A R3409C 1x - VOUS144994064 10336G>A R3446C 1x 3x 1.4085 VOUS144994028 10372C>T G3458R 1x 6x 1.2195 LB144993946 10454C>T R3485Q 1x - LB144993931 10469G>C G3490A - LB144993859 10541C>T R3514Q 1x - LB144993653 10747A>G S3583P 1x - LP144993491 10909G>A R3637C 1x 0.1008 VOUS144993344 11056C>A A3686S 1.3131 LB144993242 11158A>G S3720P 1x - LB144993119 11281C>T E3761K 0.1008 LP144993076 11324G>A A3775V 0.1008 LB144992962 11438G>A A3813V 1x - LB144992953 11447G>A A3816V 1x - LB144992660 11740C>T E3914K 0.3067 LB144992638 11762T>A Q3921L 1x - LP144992390 12010C>G D4004H 2x 1x - B144992378 12022C>T G4008S 0.3268 VOUS144992269 12131G>A T4044M 2x 5x 1.0121 LB144991963 12437C>T R4146H 0.1006 LP144991958 12442C>T V4148I 0.1004 LP144991802 12598C>T V4200M 0.2020 LP144991799 12601C>T E4201K 1x 1x 0.2016 LP144991784 12616G>A R4206C 1x - LP144991745 12655C>T D4219N 1x - LP144991271 13129C>T A4377T 1x - LB144991172 13228G>A P4410S 1x - LP144990515 13885C>T G4629S 1x - LP144990401 13999G>A R4667C 1x - LP

Abbreviations B: benign; LB: likely benign; LP: likely pathogenic; P: pathogenic; VOUS: variant of unknown significance


Absence of frequent non-synonymous variations in the rod domain region in controls

Next, we investigated whether frequent (MAF>2%) SNPs reside in the ‘potentially pathogenic’ region in the rod domain in the GoNL control population. For this purpose, all variants of PLEC between chromosomal positions 144998038 - 144999446 were extracted from the GoNL database then uploaded to SeattleSeq for annotation with protein coding features (table 3). Only a few synonymous variants were identified, with the exception of one additional missense variant (K2047E) reported with the frequency of 8%. This variant, however, had a very low quality score, which indicated that this was a sequencing artefact rather than a true variant. Hence, it seems that missense mutations in this region are not “tolerated” without phenotypic consequences.

Further interesting regions of plectin

Based on the absence of low frequency variants in patients versus controls, we identified additional interesting, potentially ARVC-associated regions of PLEC. Notably, these were only found in the UK cohort (probably due to the larger number of patients involved in the study) and spanned much shorter regions of the gene than the one in the rod domain. One of these ARVC-associated regions was the spectrin repeat region (P825Q-V988M): though this region only contained two ‘likely pathogenic’ variants in our patients, it did not contain any non-synonymous variant in the GoNL control population (data not shown). The other two variants, despite being classified as VOUS, might also be more damaging, since this segment of the encoded plectin protein is known to be responsible for interactions with, for example, actin, nesprin, and costameric proteins. Likewise, the region of variants R3335Q-R3409C, partially residing in the intermediate filament-binding plectin-repeats of the protein, may also contribute to the development of ARVC. Though this latter region was also free of coding non-synonymous genetic variants in Dutch controls (GoNL), two of the four variants found in patients were classified as ‘likely benign’ (primarily because they were predicted to be harmless) (see classification in supplementary table 1). Additionally, the C-terminus of PLEC (R4206C-R4667C) could be potentially interesting, but one relatively frequent missense variant (T4539M, 2.381% in GoNL), which was also found in 2/107 Dutch and 18/358 British patients, resides in this otherwise likely ARVC-related plectin-repeat region.


CHA

PTER

2

The potential role of other desmosomal mutations and/or external factors: is ARVC a multifactorial disease?

Of the 30 patients who were carriers of PLEC missense variants in the potentially disease-associated region of the rod domain, the vast majority had a TFC+ cardiac phenotype (true ARVC). Of these, 14 patients were found to carry other ARVC-related potentially pathogenic mutations or VOUS in addition to their PLEC cluster variants (table 4, only likely pathogenic and pathogenic variants included), mostly in the PKP2 gene but also in DSC2, DSP, JUP, SCN5A or TMEM43 for some cases. Moreover, five patients had multiple low frequency or novel genetic variants in PLEC (four of which were additional variants in the same rod cluster). While exercise (Perrin et al, Saberniak et al) and certain viral infections (Grumbach et al) remain important contributors to the onset of an ARVC phenotype, our study indicates that a potential oligogenic inheritance might complicate the seemingly multifactorial disease background and cause variable penetrance of ARVC.

Table 3. High frequency (>2%) PLEC control variants localized in the ARVC-associated ‘potentially pathogenic’ region of the rod domain. Variants of PLEC localized in the potentially pathogenic cluster of missense variants associated with ARVC. This region was found to be enriched for missense variants in both the Dutch and UK ARVC patient cohorts and lacking low frequency (<2%) variation in the healthy population represented by GoNL. No frequent coding non-synonymous PLEC variants, except for the c.6319T>C; p.K2047E variant (highlighted in black), which most likely is an artefact, were identified in the GoNL control population. Synonymous variants are indicated in gray.

variant position dbSNP GoNL data remark

genomic cDNA protein rs number quality frequency (%)

144999417 5091C>T A1697A rs55836855 9320,24 47,1429

144998868 5640C>T A1880A - 222,26 8,1633

144998514 5994C>T A1998A - 43,31 4,1096 most likely artefact

144998369 6319T>C K2047E - 41,57 8,0645 most likely artefact

144998190 2106A>G A2106A rs2857829 7241,12 31,1037

144998169 6339C>T A2113A rs1140522 10024,93 35,9177


Tabl

e 4.

Car

rier

ship

of a

ddit

iona

l pot

enti

ally

pat

hoge

nic

vari

ants

in p

atie

nts

wit

h PL

EC v

aria

nts

in th

e A

RVC-

asso

ciat

ed c

lust

er

of th

e ro

d do

mai

n. O

f the

pat

ient

s ca

rryi

ng p

lect

in v

aria

nts

in th

e pu

tativ

ely

ARV

C-as

soci

ated

regi

on id

entifi

ed in

the

rod

dom

ain,

17

of t

he 4

3 w

ere

foun

d to

be

carr

iers

of a

dditi

onal

gen

etic

mut

atio

ns in

des

mos

omal

gen

es o

r ot

her

know

n A

RVC

gene

s. O

nly

gene

tic

varia

nts

clas

sifie

d as

pat

hoge

nic

or li

kely

pat

hoge

nic

are

incl

uded

.

pati

ent I

DTF

C st

atus

PLEC

“clu

ster

” var

iant

sot

her P

LEC

vari

ant

othe

r ARV

C-re

late

d m

utat

ion

1ot

her A

RVC-

rela

ted

mut

atio

n 2

1TF

C-R1

688C

(VO

US)

DSP

c.7

994C

>T; p

.T26

65M

(VO

US)

- 2

TFC-

R168

8C (V

OU

S) -

- 3

TFC-

R168

8C (V

OU

S)JU

P c.

902A

>G; p

.E30

1G (V

OU

S) -

4TF

C-A

1747

V (L

P)SC

N5A

c.6

65G

>A; p

.R22

2Q (P

) -

5TF

C-E1

762K

(LP)

- -

6TF

C-E1

762K

(LP)

- -

7TF

C+T1

859M

(LB)

, K20

37E

(LP)

R422

Q (L

B), A

2744

G (L

B)PK

P2 c

.206

2T>C

; S68

8P (L

P) -

8TF

C+R1

875W

(LP)

- PK

P2 c

.184

8C>A

; Y61

6X (P

) -

9TF

C+T1

884M

(VO

US)

- -

10TF

C-A

1909

V (V

OU

S)PK

P2 c

.113

2C>T

; p.Q

378X

(P)

- 11

TFC+

R193

4H (L

P) -

PKP2

del

exo

n 1-

4 (P

) -

12TF

C+R1

954G

(LP)

- PK

P2 c

.397

C>T;

p.Q

133X

(P)

TMEM

c.9

34C>

T; p

.R31

2W (V

OU

S)13

TFC-

A19

61V

(LP)

- -

14TF

C-R1

963W

(LP)

- -

15TF

C-R1

963W

(LP)

- -

16TF

C-R1

963W

(LP)

- -

17TF

C+R1

963W

(LP)

- D

SP c

.477

5A>G

; p.K

1992

R (V

OU

S) -

18TF

C-R1

963Q

(VO

US)

- -

19TF

C-R1

963Q

(VO

US)

- -

20TF

C-R1

963Q

(VO

US)

- -

21TF

C-R1

963Q

(VO

US)

SCN

5A c

.320

6C>T

; p.T

1069

M (L

P) -

22TF

C-R1

963Q

(VO

US)

- -

23TF

C+R1

963Q

(VO

US)

Q10

* (V

OU

S)PK

P2 c

.238

6T>C

; C79

6R (P

) -

24TF

C+R1

963Q

(VO

US)

, K20

99R

(LP)

D42

H (V

OU

S) -

- 25

TFC-

L197

2V (L

P)R4

33Q

(LB)

- -

26TF

C+E1

974K

(LP)

- PK

P2 c

.121

1dup

; L40

4fs

(P)

DSP

c.2

69G

>A; p

.Q90

R (V

OU

S)27

TFC+

A19

78T

(LP)

- -

- 28

TFC+

R211

0Q (L

P)A

2242

V (B

)PK

P2 c

.235

C>T;

R79

X (P

) -

29TF

C-A

2144

V (L

P)PK

P2 c

.159

7_16

00de

lATC

C; p

.P53

3fsX

561

(P)

- 30

TFC-

E215

7A (V

OU

S) -

-

Abbr

evia

tions

B: b

enig

n; L

B: li

kely

ben

ign;

LP:

like

ly p

atho

geni

c; P:

pat

hoge

nic;

TFC

– ta

sk f

orce

crit

eria

(di

agno

stic

crit

eria

of

ARVC

); VO

US:

var

iant

of

unkn

own

signi

fican

ce


CHA

PTER

2

DISCUSSION

We hypothesized that the cytolinker protein plectin, which supports the binding of intermediate filaments to the desmosomes, might carry genetic variants that contribute to the development of, or at least the susceptibility for ARVC, which is known as a ‘disease of the cardiac desmosome’. Our reasons were that (1) plectin is highly expressed in the myocardium and is physically connected to the cell junctions which are known to be involved in the pathomechanism of ARVC, (2) its knock down in various mouse models leads to cardiac pathology, and (3) late-onset cardiac symptoms have recently been reported for a couple of mutated plectin-carrying EBS patients.

Our Sanger sequencing and NGS-based analysis of the PLEC gene resulted in the identification of 96 novel or low frequency (<2%), mostly heterozygous variants in the PLEC gene in patients with ARVC. Previously, multiple homozygous or compound heterozygous truncating nonsense and frameshift variants of PLEC had been shown to lead to different manifestations of epidermolysis bullosa simplex; only a couple of missense variants were identified in unusually mild cases of EBS (Bolling et al 2013). The majority of the variants now identified are missense by nature, except for a small deletion and two heterozygous truncating variants. The Q10* variant is located in one of the multiple 5’ exon 1 sequences of the gene, and upon RNA splicing, it ends up only in one isoform (isoform 1a, transcript NM_201384.1) that was previously not found to be of importance in the heart (Fuchs et al). The same exon was shown to have a small, in-frame deletion in another patient. The possible pathogenic nature of these two variants is unclear, yet not likely. Moreover, in a third patient, we identified another truncating variant, p.Y612* in exon 14, which is part of all currently known plectin isoforms. The respective patient did not show any skin phenotype, i.e. blistering. This is consistent with heterozygous carriers of truncating PLEC mutations generally reported as being healthy, while a homozygous or compound heterozygous form causes EBS. Therefore, it seems that heterozygous truncating PLEC mutations are not disease-related, although we cannot exclude that mild phenotypes might have been overlooked.

All remaining variants (n=93) detected in our patient cohorts are missense variants of which a substantial number were classified as VOUS or likely pathogenic based on conservation, predicted effects on protein function and differences in physico-chemical properties of the respective amino acid residues. When analyzing the distribution of these missense variations, we


noted that the variants are scattered around the entire gene and do not show an obvious, exclusive clustering any smaller area or domain. Moreover, when studying the presence of novel or rare variants in the control population of the Genome of the Netherlands, various missense mutations were also identified (n=48), and of these a subset were also classified as VOUS or likely pathogenic. This raised the question whether such missense mutations are all ‘harmless’ variants or whether a subset might have disease-associated effects, as was previously shown for a couple of heterozygous missense mutations, such as the p.R2110W mutation, that led to mild forms of EBS (Bolling et al 2013). For this reason we compared the distribution of PLEC variations in affected and healthy individuals, searching for regions rich in genetic variation in patients, but that show “variant deserts” in the control group. We subsequently identified one such region in the coiled-coil rod domain (between amino acids 1688-2157). This region exhibits almost exclusively synonymous (low AND high frequency) genetic variation in the GoNL control population, but contains many missense variants in our ARVC patients. According to their conservation, frequency in SNP databases and predicted pathogenicity, the majority of these variants were classified as ‘likely pathogenic’, or, in a few cases, as VOUS. Notably, due to coverage and/or mapping difficulties for some parts of this region, variant calling was hampered and in a few cases based on less than 1000 alleles.

We identified an additional short region of the plectin-repeat domains which had ARVC-associated variants clustering in the UK patient cohort but did not see these variations in the GoNL cohort. In addition, several ‘likely pathogenic’ variants found at positions outside the clustering in the rod domain were identified. Unfortunately, it is not possible to interpret the functional consequences of these missense variants and make conclusions about their potential causative nature without performing further follow-up experiments. However, a modifier role in ARVC can still be anticipated. What our findings, however, suggest is that the homo-dimerizing rod domain, which seems to contain a well-defined region of disease-associated missense variants in both the British and Dutch patient cohorts (see also figure 1), may be of structural importance for plectin molecules. Indeed, it has been observed that the dimerized rod domains are able to form remarkably stable polymers via further lateral connections with each other (Walko et al). Moreover, in EBS-Ogna mouse and patient keratinocytes, the missense variants of the rod domain were found to increase the proteolysis of plectin


CHA

PTER

2

in the hemidesmosomes, a mechanism which was rescued by treatment with calpain inhibitors (Walko et al).

Interestingly, there are a few exceptional yet mild EBS cases recently reported to be due to heterozygous missense variants of PLEC (Bolling et al 2013). In the skin, PLEC homozygous truncations (and as a consequence the lack of plectin protein expression) seem to predominantly cause a dysfunction of the hemidesmosomes, leading to insufficient attachment of keratinocytes to the dermis at the basement membrane zone and manifesting as the basal blistering of the skin (McLean et al, Smith et al). The missense heterozygous variants of the rod domain also cause basal blistering by increasing the vulnerability of plectin to calpain-mediated proteolysis (Walko et al), yet their effect is not as drastic and the phenotype is limited to the hands and feet, areas subject to more mechanical stress.

Figure 1. Schematic illustration of regions of interest in PLEC. The marked areas were found to have a number of missense variants found in ARVC patients, yet showing

“variant deserts” in the GoNL control population. Orange-yellow colour indicates the region found in the Dutch patient cohort, while regions coloured in red were found in the British patient cohort. Exon 31 encodes the homodimerizing rod domain of plectin.

The exact mechanism by which heterozygous missense variants of PLEC could lead to cardiomyopathy are unknown. However, we know that plectin contains a number of plakin repeat domains typical of desmosomal proteins and responsible for binding intermediate filaments, as well as a highly variable N-terminal actin-binding domain. Thus we anticipate that by binding both intermediate filaments and myofilaments, PLEC may play a role in attracting the structurally robust desmosomes around the more fragile adherens junctions at the cardiac intercalated disks. Adherens junctions anchor the more dynamic and fragile thin (actin) filaments of the neighbouring cardiomyocytes together


and provide the continuity between myofibrils of these neighbouring cells. We hypothesize that the presence of missense variants leads to increased proteolysis of plectin in the heart (similar to what occurs in the skin), which could in turn decrease its interjunctional linking capacity, make the adherens junctions less supported by desmosomes and thus more sensitive to stress and damage. This could explain why mutations in PLEC do not seem to be involved in a monogenic, classical Mendelian form of ARVC, since the intact, strong desmosomes could still keep the cardiomyocytes aligned together. Thus, an additional desmosomal mutation or external stressor may be necessary to pass the thresholds for disease development. This would also fit the concept of an oligogenic/multifactorial disease mechanism of ARVC that has been suggested by Marcus et al (2013) and Perrin et al, and would better explain the low penetrance observed in ARVC. The pathogenicity of previously identified genetic variants is increasingly being questioned because they are found in healthy individuals in much larger percentage than would be expected based on the estimated 1:2000 incidence and late-onset nature of the disease. Even ‘radical mutations’ can be found in 0.5% of ostensibly healthy controls, while ‘missense mutations’ were identified in nearly 16% of controls that were screened for just the five prominent ARVC genes (Kapplinger et al). These observations would also fit in an oligogenic disease model. In this study, we have shown that about half of the patients carrying a PLEC missense variant in the ’pathogenic region’ of the rod domain are also carriers of other definitive mutations in known ARVC genes.

Certain novel missense variants identified in our ARVC patient cohort have been identified in EBS patients as well, but without any obvious overlap in clinical phenotypes. For instance, variants R1963W and p.V4399I were identified in both EBS and ARVC patients, but absent from GoNL (EBS patients; unpublished results). Moreover, the mutation p.R2110W (in dermatological context reported as p.R2000W) that was detected in seven EBS index patients (Bolling et al 2013, Kiritsi et al) affects the same amino acid as the likely pathogenic p.R2110Q variant in one of our ARVC patients. Surprisingly, however, our ARVC patients exhibited no striking skin blistering disorder or muscular dystrophy, while the respective EBS patients did not show ARVC features. It is currently unclear what could cause some patients with missense variants to develop either mild EBS or ARVC, although one might expect that the stochastic distribution of wt/wt, wt/VOUS and VOUS/VOUS plectin dimers, as well as the tissue-specific factors influencing the


CHA

PTER

2

calpain-mediated proteolysis, could to some extent explain this phenotypic variability. It is also possible that some EBS patients are suffering from an undiscovered cardiomyopathy. Another explanation is that genetic modifiers or a multifactorial background might also influence the phenotype of EBS (Padalon-Brauch et al), in which case PLEC missense mutations are not the sole cause of the disease, although they still contribute substantially to the phenotype. Importantly, EBS has much younger age of onset (and is more easily diagnosed) than ARVC, and EBS patients might still have the chance of developing cardiomyopathy later in life. For this reason, EBS patients (at least those with an identified genetic variant of PLEC) might benefit from cardiological evaluation and/or regular follow-up (Bolling et al 2010). The referral of ARVC patients to dermatologists, on the contrary, does not seem to be essential based on our current knowledge. The exact mechanical role of plectin in different tissues, as well as the mechanism via which truncating and missense mutations cause different disease phenotypes, however, requires further in-depth research in the future.

There are a few examples of patients and families suffering from a combi-nation of skin blistering and cardiac phenotypes due to PLEC mutations (Schröder et al, Bolling et al 2010). One patient had a homozygous frameshift mutation in PLEC and exhibited EBS-MD with cardiac hypertrophy, while another was compound heterozygous for a truncating mutation (p.E1724X) and a missense variant (p.R433Q). Though this missense variant was detected in 3 Dutch and 13 UK patients in this study, it was also present in relatively high frequency in GoNL controls (1.36%) and thus classified as likely benign. However, in the patient carrying both PLEC variants, the p.R433Q variant is most likely the predominant form produced due to diminished protein expression from the allele carrying the nonsense mutation, and therefore it might still contribute to the cardiac pathology in this patient. In addition to these two previous case reports, we recently also observed cardiac involvement in two brothers affected by severe EBS-MD carrying double homozygous PLEC variants (p.E1914X and p.Y2967C) (Koss-Harnes et al). Since then, both patients died due to hypertrophic dilated cardiomyopathy. Additionally, one of their non-EBS brothers suffered from sudden cardiac death, a well-known feature of ARVC, and his son was affected by DCM without any signs of skin blistering or muscular dystrophy. Unfortunately, the PLEC carriership status of these family members is unknown. Villa et al have also just reported a case of EBS-MD developing left ventricular non-compaction cardiomyopathy.


Taken together, these findings suggest that it might be advisable to perform genetic screening of PLEC in other types of cardiomyopathy as well.

The exact mechanical role of plectin in different tissues, as well as the mechanism via which truncating and missense mutations cause different disease phenotypes requires in-depth research in the future.

CONCLUSIONS

We identified 96 novel or low frequency (<2%), mostly heterozygous, missense variants of PLEC in ARVC patients. The facts that these variants were identified in addition to pathogenic desmosomal mutations in a subset of these patients, that co-segregation with disease could not be established for those variants that were analysed for this study, and that novel or rare variants with predicted pathogenic nature were also identified in the healthy GoNL cohort led us to conclude that PLEC cannot be considered the primary genetic cause of inherited ARVC. By comparing the natural genetic variation of the gene, by collecting all variants with an allele frequency <2% identified in the GoNL population with that found in patients, we were able to identify a region of probably ARVC-associated missense variants in the rod domain, which is thought to mediate homodimerization/dimerization of the protein and might become more vulnerable to proteolysis. We hypothesize that genetic variations in this domain of PLEC may make the cellular junctions more fragile, thus increasing the susceptibility to ARVC. This result underscores the previously suggested multifactorial nature of ARVC and suggests that PLEC variations, at least when present in specific regions of the protein, contribute to the number of risk factors to reach the threshold levels needed to initiate the development of this disease.

ACKNOWLEDGEMENTS

The authors would like to acknowledge Jackie Senior and Kate Mc Intyre for editing this manuscript, as well as Rudi Alberts for designing the script for the GoNL, 1000 Genomes and ESP allele frequency annotation of the genetic variants.

Part of this work was undertaken at University College London Hospital and University College London (UCLH/UCL), which received some funding from the UK Department of Health’s NIHR Biomedical Research Centres funding scheme. This study made use of data generated by the Genome of the Netherlands Project.


CHA

PTER

2

REFERENCESAndrä K, Lassmann H, Bittner R et al. Targeted inac-

tivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture. Genes Dev 1997;11:3143-56

Bao JR, Wang JZ, Yao Y et al. Screening of pathogen-ic genes in Chinese patients with arrhythmo-genic right ventricular cardiomyopathy. Chin Med J (Engl) 2013;126:4238-41

Bauce B, Nava A, Beffagna G et al. Multiple muta-tions in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomy-opathy/dysplasia. Heart Rhythm 2010;7:22-9

Bolling MC, Jongbloed JDH, Boven LG et al. Plectin mutations underlie epidermolysis bullosa sim-plex in 8% of patients. J Invest Dermatol 2013; doi: 10.1038/jid.2013.277

Bolling MC, Pas HH, de Visser M et al. PLEC1 muta-tions underlie adult-onset dilated cardiomyopa-thy in epidermolysis bullosa simplex with mus-cular dystrophy. J Invest Dermatol 2010;130:1178-81

Elliott P, Andersson B, Arbustini E et al. Classifica-tion of the cardiomyopathies: a position state-ment from the European Cociety of Cardiology Working Group on Myocardial and Pericardial Diseases. Eur Heart J 2008;29:270-6

Fuchs P, Zörer M, Rezniczek GA et al. Unusual 5’ transcript complexity of plectin isoforms: nov-el tissue-specific exons modulate actin binding activity. Hum Mol Genet 1999;8:2461-72

Grumbach IM, Heim A, Vonhof S et al. Coxsackie-virus genome in myocardium of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy. Cardiology 1998;89:241-5

Jacob KA, Noorman M, Cox MGPJ et al. Geo-graphical distribution of plakophilin-2 mutation prevalence in patients with ar-rhythmogenic cardiomyopathy. Neth Heart J 2012;20:234-9

Kapplinger JD, Landstrom AP, Salisbury BA et al. Distinguishing arrhythmogenic right ventric-ular cardiomyopathy/dysplasia-associated mu-tations from background genetic noise. J Am Coll Cardiol 2011;57:2317-27

Kiritsi D, Pigors M, Tantcheva-Poor I et al. Epider-molysis bullosa simplex ogna revisited. J Invest Dermatol 2013;133:270-3

Konieczny P, Fuchs P, Reipert S et al. Myofiber in-tegrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms. J Cell Biol 2008;181:667-81

Koss-Harnes D, Hoyheim B, Jonkman MF et al. Life-long course and molecular characterization of

the original Dutch family with epidermolysis bullosa simplex with muscular dystrophy due to a homozygous novel plectin point mutation. Acta Derm Venereol 2004;84:124-31

Lopes LR, Zekavati A, Syrris P et al. Genetic com-plexity in hypertrophic cardiomyopathy re-vealed by high-throughput sequencing. J Med Genet 2013;50(4):228-39

Marcus FI, McKenna WJ, Sherrill D et al. Diagnosis of arrhythmogenic right ventricular cardiomy-opathy/dysplasia: proposed modification of the task force criteria. Eur Heart J 2010;31:806-14

Marcus FI, Edson S, Towbin JA: Genetics of ar-rhythmogenic right ventricular cardiomyopa-thy: a practical guide for physicians. J Am Coll Cardiol 2013;61:1945-8

McKoy G, Protonotarios N, Crosby A et al. Identifi-cation of a deletion in plakoglobin in arrhyth-mogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease). Lancet 2000;355:2119-24

McLean WH, Pulkkinen L, Smith FJ et al. Loss of plectin causes epidermolysis bullosa with mus-cular dystrophy: cDNA cloning and genomic organization. Genes Dev 1996;10:1724-35

Nakajima T, Kaneko Y, Irie T et al. Compound and digenic heterozygosity in desmosome genes as a cause of arrhythmogenic right ventricular cardiomyopathy in Japanese patients. Circ J 2012;76:737-43

Norgett EE, Hatsell SJ, Carvajal-Huerta L et al. Reces-sive mutation in desmoplakin disrupts desmo-plakin–intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma. Hum Mol Genet 2000;9(18):2761-6

Padalon-Brauch G, Ben Amiati D, Vodo D et al. Di-genic inheritance in epidermolysis bullosa sim-plex. J Invest Dermatol 2012;132(12):2852-4

Perrin MJ, Angaran P, Laksman Z et al. Exercise testing in asymptomatic gene carriers exposes a latent electrical substrate of arrhythmogen-ic right ventricular cardiomyopathy. J Am Coll Cardiol 2013;62:1772-9

Rigato I, Bauce B, Rampazzo A et al. Compound and digenic heterozygosity predicts life-time arrhythmic outcome and sudden cardiac death in desmosomal gene-related arrhythmogenic right ventricular cardiomyopathy. Circ Cardio-vasc Genet 2013;6:533-42

Saberniak J, Hasselberg NE, Borqquist R et al. Vigorous physical activity impairs myocardi-al function in patients with arrhythmogenic


right ventricular cardiomyopathy and in muta-tion positive family members. Eur J Heart Fail 2014;16:1337-44

Schröder R, Kunz WS, Rouan F et al. Disorganiza-tion of the desmin cytoskeleton and mitochon-drial dysfunction in plectin-related epidermol-ysis bullosa simplex with muscular dystropyhy. J Neuropathol Exp Neurol 2002;61(6):520-30

Sen-Chowdhry S, Morgan RD, Chambers JC et al: Arrhythmogenic cardiomyopathy: etiolo-gy, diagnosis, and treatment. Annu Rev Med 2010;61:233-53

Smith FJ, Eady RA, Leigh IM et al. Plectin deficien-cy results in muscular dystrophy with epider-molysis bullosa. Nat Genet 1996;13:450-7

Sonnenberg A & Liem RKH: Plakins in de-velopment and disease. Exp Cell Res 2007;313:2189-203

Teekakirikul P, Kelly MA, Rehm HL et al. Inherit-ed cardiomyopathies: molecular genetics and clinical genetic testing in the postgenomic era. J Mol Diagn 2013;15:158-70

te Rijdt WP, Jongbloed JDH, de Boer RA et al. Clin-ical utility gene card for: arrhythmogenic right ventricular cardiomyopathy (ARVC) Eur J Hum Genet 2013; doi: 10.1038/ejhg.2013.124

Villa CR, Ryan TD, Collins JJ et al. Left ventricular non-compaction cardiomyopathy associated with epidermolysis bullosa simplex with mus-cular dystrophy and PLEC1 mutation. Neuro-muscular Disord 2015;25:165-8

Walko G, Vukasinovic N, Gross K et al. Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimick-ing the dominant skin blistering disease EBS-Ogna. PLoS Genet 2011;7(12):e1002396

Wiche G, Krepler R, Artlieb U et al. Occurrence and immunolocalization of plectin in tissues. J Cell Biol 1983;97:887-901

Winter L & Wiche G: The many faces of plectin and plectinopathies; pathology and mechanisms. Acta Neuropathol 2013;125(1):77-93

Zernig G & Wiche G: Morphological integrity of single adult cardiac myocytes isolated by col-lagenase treatment: immunolocalization of tu-bulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin. Eur J Cell Biol 1985;38(1):113-22

van Tintelen JP, Entius MM, Bhuiyan ZA et al. Pla-kophilin-2 mutations are the major determi-nant of familial arrhythmogenic right ventric-ular dysplasia/cardiomyopathy. Circulation 2006;113:1650–8

Xu T, Yang Z, Vatta M et al. Compound and digenic heterozygosity contributes to arrhythmogenic right ventricular cardiomyopathy. J Am Coll Cardiol 2010;55:587–97


CHA

PTER

2

Supp

lem

enta

ry ta

ble

1: C

lass

ifica

tion

of P

LEC

vari

ants

Abb

revi

atio

ns E

SP (E

A; A

A):

alle

le fr

eque

ncy

in E

urop

ean

Anc

estr

y an

d A

fric

an A

nces

try

indi

vidu

als

in t

he E

xom

e Se

quen

cing

Pro

ject

da

taba

se; G

oNL:

alle

le c

ount

s in

the

GoN

L da

taba

se; n

.a.:

not a

pplic

able

/ava

ilabl

e

VARI

AN

TPR

EDIC

TED

EFF

ECT

CON

TRO

L D

ATA

BASE

FRE

QU

ENCI

ESCL

ASS

IFIC

ATIO

N

coor

dina

tes

Gra

ntha

m

dist

ance

cons

erva

tion

(u

p to

...)

Phyl

oP

[-

14.1

;6.4

]A

GV

GD

Poly

Phen

SIFT

Mut

atio

nTas

ter

splic

ing

1000

G

enom

esES

P

(EA

; AA

)G

oNL

c.30

G>C

, D10

E45

wea

k (c

ow)

0.53

C0be

nign

dele

terio

uspo

lym

orph

ism

- 0/

2184

0/~1

3000

0.0%

LIKE

LY B

ENIG

Nc.

266G

>A, P

89L

98w

eak

(cow

)5.

21C0

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000.

0%V

OU

S

c.30

5C>T

, R10

2H29

wea

k (c

ow)

2.38

C0pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

4/82

96; 1

/411

60,

00%

VO

US

c.42

1G>A

, R14

1C18

0w

eak

(cow

)4.

24C0

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

10%

VO

US

c.50

3G>A

, P16

8L98

wea

k (h

orse

)0.

29C0

beni

gnto

lera

ted

dise

ase

caus

ing

- 0/

2184

0; 6

/373

20,

00%

LIKE

LY B

ENIG

Nc.

20C>

T, A

7V

(NM

_201

383.

1)64

wea

k (r

egio

n)0.

37C0

beni

gnto

lera

ted

poly

mor

phis

m -

0/21

840/

~130

000.

0%LI

KELY

BEN

IGN

c.12

4G>C

, D42

H

(NM

_201

383.

1)81

high

(zeb

rafis

h)1.

17C0

poss

ibly

da

mag

ing

dele

terio

usdi

seas

e ca

usin

gst

rong

effe

ct

(but

larg

e ex

on)0/

2184

0/~1

3000

0.0%

VO

US

c.28

C>T,

Q10

* (N

M_2

0138

4.1)

n.a.

wea

k (r

egio

n)n.

a.n.

a.n.

a.n.

a.n.

a. -

0/21

840/

~130

000.

0%V

OU

S

c.69

_70i

nsTA

C,

D23

_N24

insY

(N

M_2

0138

4.1)

n.a.

wea

k

n.a.

n.a.

n.a.

n.a.

n.a.

- 0/

2184

0/~1

3000

0.0%

VO

US

c.74

3C>T

, R24

8Q43

wea

k (c

ow)

1,82

C0be

nign

dele

terio

usdi

seas

e ca

usin

gm

inor

effe

ct0/

2184

0/~1

3000

0,00

%V

OU

Sc.

947C

>T, R

316Q

43hi

gh (z

ebra

fish)

1,58

C0be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

10%

VO

US

c.12

04C>

T, V

402M

21hi

gh (z

ebra

fish)

5,37

C0po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0; 1

/421

20,

20%

VO

US

c.12

65G

>A, R

422Q

43w

eak

(cow

)1.

42C0

beni

gnto

lera

ted

poly

mor

phis

m -

7/21

841/

8410

; 57/

4134

0.0%

LIKE

LY B

ENIG

Nc.

1298

C>T,

R43

3Q43

high

(zeb

rafis

h)5.

37C0

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

13/2

184

144/

8466

; 14/

4188

1.3%

LIKE

LY B

ENIG

N

c.15

34C>

T, V

512M

21hi

gh (z

ebra

fish)

4,08

C0po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

2/84

16; 0

0,20

%V

OU

S

c.15

69G

>C, S

523R

110

high

(zeb

rafis

h)0.

37C6

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

849/

8342

; 00.

2%V

OU

Sc.

1634

C>T,

C54

5Y19

4hi

gh (z

ebra

fish)

5,61

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

00%

LIKE

LY P

ATH

OG

ENIC

c.17

45G

>A, A

582V

64hi

gh (z

ebra

fish)

2,87

C65

beni

gnde

lete

rious

dise

ase

caus

ing

min

or e

ffect

0/21

840/

~130

000,

10%

LIKE

LY P

ATH

OG

ENIC

c.18

36C>

G, Y

612*

n.a.

high

(reg

ion)

n.a.

n.a.

n.a.

n.a.

n.a.

- 0/

2184

0/~1

3000

0.0%

LIKE

LY P

ATH

OG

ENIC

c.18

74A

>G, Y

625C

194

high

(Xen

opus

)2.

06C5

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0.0%

LIKE

LY P

ATH

OG

ENIC

c.19

17C>

G, Q

639H

24w

eak

1,42

C0be

nign

tole

rate

dpo

lym

orph

ism

min

or e

ffect

0/21

840/

~130

000,

10%

LIKE

LY B

ENIG

N


VARI

AN

TPR

EDIC

TED

EFF

ECT

CON

TRO

L D

ATA

BASE

FRE

QU

ENCI

ESCL

ASS

IFIC

ATIO

N

coor

dina

tes

Gra

ntha

m

dist

ance

cons

erva

tion

(u

p to

...)

Phyl

oP

[-

14.1

;6.4

]A

GV

GD

Poly

Phen

SIFT

Mut

atio

nTas

ter

splic

ing

1000

G

enom

esES

P

(EA

; AA

)G

oNL

c.24

74G

>T, P

825Q

76hi

gh (z

ebra

fish)

5.13

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000.

0%LI

KELY

PAT

HO

GEN

IC

c.25

49C>

G, C

850S

112

high

(zeb

rafis

h)5.

13C6

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0.0%

LIKE

LY P

ATH

OG

ENIC

c.29

59C>

T, A

987T

58w

eak

(cow

)0,

53C0

beni

gnto

lera

ted

poly

mor

phis

m -

77/2

184

3/83

70; 4

42/4

402

0,00

%BE

NIG

Nc.

2962

C>T,

V98

8M21

high

(zeb

rafis

h)2.

14C1

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

840;

13/

4186

0.0%

VO

US

c.31

30G

>A, R

1044

C18

0w

eak

(cow

)2,

22C2

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

3/84

80; 0

0,20

%V

OU

S

c.33

52G

>A, R

1118

C18

0w

eak

(cow

)0.

21C2

5be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

1/84

94; 0

0.0%

LIKE

LY B

ENIG

Nc.

3872

C>T,

R12

91Q

43w

eak

(cow

)2.

63C0

beni

gnto

lera

ted

poly

mor

phis

m -

0/21

840/

~130

000.

0%LI

KELY

BEN

IGN

c.40

93G

>A, R

1365

W10

1w

eak

0.

93C1

5be

nign

dele

terio

uspo

lym

orph

ism

min

or e

ffect

9/21

8416

/809

8; 2

/386

81.

0%LI

KELY

BEN

IGN

c.41

89G

>C, R

1397

G12

5w

eak

(cow

)1,

01C4

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

9/21

840;

23/

4302

0,00

%LI

KELY

BEN

IGN

c.42

80T>

C, K

1427

R26

high

(zeb

rafis

h)2.

22C2

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

1/21

8414

/843

6; 4

4/41

780.

0%LI

KELY

BEN

IGN

c.44

00G

>C, T

1467

R71

wea

k (c

ow)

5,61

C15

poss

ibly

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

20%

VO

US

c.44

39C>

T, R

1480

H29

high

(zeb

rafis

h)5,

61C2

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

1/85

48; 0

0,10

%LI

KELY

PAT

HO

GEN

IC

c.44

86G

>A, R

1496

C18

0hi

gh (z

ebra

fish)

0,69

C65

poss

ibly

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

40%

LIKE

LY P

ATH

OG

ENIC

c.46

37T>

G, V

1546

G10

9hi

gh (z

ebra

fish)

2,06

C65

beni

gnde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

ICc.

4777

C>T,

V15

93M

21w

eak

(cow

)0.

85C0

beni

gnde

lete

rious

poly

mor

phis

mm

inor

effe

ct6/

2184

38/7

890;

4/3

790

0.1%

LIKE

LY B

ENIG

Nc.

4937

G>A

, R16

46H

29m

oder

atel

y (o

nly

R or

K)

2.30

C0be

nign

dele

terio

usdi

seas

e ca

usin

gm

inor

effe

ct0/

2184

00.

0%V

OU

S

c.49

43C>

T, R

1648

Q43

high

(zeb

rafis

h)0,

77C3

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

20%

VO

US

c.49

67C>

T, S

1656

L14

5w

eak

1.

74C0

beni

gnto

lera

ted

poly

mor

phis

m -

6/21

840

0.0%

LIKE

LY B

ENIG

Nc.

5009

C>T,

R16

70Q

43m

oder

ate

2.06

C0be

nign

dele

terio

uspo

lym

orph

ism

- 0/

2184

26/6

166;

2/2

752

1.0%

LIKE

LY B

ENIG

Nc.

5054

G>A

, A16

85V

64hi

gh (z

ebra

fish)

5,45

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

20%

LIKE

LY P

ATH

OG

ENIC

c.50

62G

>A, R

1688

C18

0w

eak

(cow

)1,

74C4

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 10

/218

40/

~130

000,

00%

VO

US

c.52

40G

>A, A

1747

V64

high

(zeb

rafis

h)5,

53C6

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

c.52

84C>

T, E

1762

K56

high

(zeb

rafis

h)5,

53C6

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

1/21

840/

~130

000,

00%

LIKE

LY P

ATH

OG

ENIC

c.55

76C>

T, T

1859

M81

wea

k (c

ow)

3.19

C0po

ssib

ly

dam

agin

gto

lera

ted

dise

ase

caus

ing

- 0/

2184

1/63

12; 2

2/28

880.

0%V

OU

S

c.56

23C>

T, R

1875

W10

1hi

gh (z

ebra

fish)

2.63

C65

poss

ibly

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000.

0%LI

KELY

PAT

HO

GEN

IC


CHA

PTER

2

c.56

51G

>A, T

1884

M81

wea

k (c

ow)

0,93

C0pr

obab

ly

dam

agin

gde

lete

rious

poly

mor

phis

m -

0/21

840/

~130

000,

00%

VO

US

c.57

26G

>A, A

1909

V64

wea

k (c

ow)

1,82

C0po

ssib

ly

dam

agin

gde

lete

rious

poly

mor

phis

mm

inor

effe

ct0/

2184

0/~1

3000

0,00

%V

OU

S

c.58

01G

>A, R

1934

H29

high

(zeb

rafis

h)1.

82C2

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0.0%

LIKE

LY P

ATH

OG

ENIC

c.58

60C>

G, R

1954

G12

5hi

gh (z

ebra

fish)

0.85

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000.

0%LI

KELY

PAT

HO

GEN

IC

c.58

82G

>A, A

1961

V64

high

(zeb

rafis

h)2,

71C6

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

c.58

87G

>A, R

1963

W10

1hi

gh (z

ebra

fish)

1.90

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000.

0%LI

KELY

PAT

HO

GEN

IC

c.58

88G

>A, R

1963

Q43

high

(zeb

rafis

h)2.

06C3

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 19

/218

35/4

580;

4/1

900

0.0%

VO

US

c.59

14C>

G, L

1972

V32

high

(zeb

rafis

h)5.

37C2

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

00.

0%LI

KELY

PAT

HO

GEN

IC

c.59

20G

>A, E

1974

K56

high

(zeb

rafis

h)5.

37C5

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0.0%

LIKE

LY P

ATH

OG

ENIC

c.59

32G

>A, A

1978

T58

high

(zeb

rafis

h)2.

71C5

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 1/

2184

1/62

16; 6

/262

80.

0%LI

KELY

PAT

HO

GEN

IC

c.61

09A

>G, K

2037

E56

high

(zeb

rafis

h)1.

82C5

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

00.

0%LI

KELY

PAT

HO

GEN

IC

c.63

29G

>A, R

2110

Q43

high

(zeb

rafis

h)1.

90C3

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

840

0.0%

LIKE

LY P

ATH

OG

ENIC

c.64

31G

>A, A

2144

V64

high

(zeb

rafis

h)5,

21C0

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

gm

inor

effe

ct1/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

c.64

70T>

G, E

2157

A10

7hi

gh (z

ebra

fish)

4,32

C0be

nign

tole

rate

ddi

seas

e ca

usin

g -

0/21

841/

7912

; 1/3

826

0,00

%V

OU

S

c.64

73G

>A, A

2158

V64

high

(zeb

rafis

h)5.

21C0

poss

ibly

da

mag

ing

dele

terio

usdi

seas

e ca

usin

gst

rong

effe

ct

(but

larg

e ex

on)

0/21

841/

7880

; 00.

1%LI

KELY

PAT

HO

GEN

IC

c.66

09C>

A, Q

2203

H24

wea

k (c

ow)

0,45

C0be

nign

tole

rate

dpo

lym

orph

ism

-10

/218

40/

7114

; 13/

3336

0,00

%LI

KELY

BEN

IGN

c.67

22G

>A, A

2241

V64

high

(zeb

rafis

h)3,

03C0

beni

gnde

lete

rious

dise

ase

caus

ing

-5/

2184

2/76

76; 3

/356

30,

60%

VO

US

c.67

36G

>A, R

2246

W10

1w

eak

(dog

)1,

82C2

5be

nign

dele

terio

uspo

lym

orph

ism

min

or e

ffect

0/21

840/

~130

000,

00%

VO

US

c.67

43A

>C, V

2248

G10

9w

eak

(cow

)2,

3C0

beni

gnde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,70

%V

OU

Sc.

6827

G>A

, R22

76H

29hi

gh (z

ebra

fish)

4.00

C25

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

841/

8102

; 00.

0%LI

KELY

PAT

HO

GEN

IC

c.68

57C>

T, R

2286

Q43

high

(zeb

rafis

h)5,

77C3

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

c.69

31G

>A, A

2311

T58

high

(zeb

rafis

h)2.

14C0

beni

gnde

lete

rious

dise

ase

caus

ing

- 0/

2184

00.

0%V

OU

S


VARI

AN

TPR

EDIC

TED

EFF

ECT

CON

TRO

L D

ATA

BASE

FRE

QU

ENCI

ESCL

ASS

IFIC

ATIO

N

coor

dina

tes

Gra

ntha

m

dist

ance

cons

erva

tion

(u

p to

...)

Phyl

oP

[-

14.1

;6.4

]A

GV

GD

Poly

Phen

SIFT

Mut

atio

nTas

ter

splic

ing

1000

G

enom

esES

P

(EA

; AA

)G

oNL

c.69

47C>

T, R

2316

Q43

high

(fro

g)3,

43C0

beni

gnde

lete

rious

dise

ase

caus

ing

- 7/

2184

6/84

70; 1

/425

60,

00%

VO

US

c.72

56G

>A, R

2419

Q43

high

(zeb

rafis

h)4.

08C3

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

00.

0%LI

KELY

PAT

HO

GEN

IC

c.76

78C>

T, A

2560

T58

wea

k (c

ow)

0.53

C0be

nign

tole

rate

dpo

lym

orph

ism

- 5/

2184

49/8

568;

9/4

378

0.5%

LIKE

LY B

ENIG

Nc.

8080

G>A

, R26

94W

101

wea

k (m

ouse

)2,

06C0

beni

gnde

lete

rious

poly

mor

phis

mm

inor

effe

ct0/

2184

4/81

98; 1

/429

60,

40%

LIKE

LY B

ENIG

Nc.

8231

C>G

, A27

44G

60w

eak

(cow

)2.

30C0

beni

gnto

lera

ted

poly

mor

phis

m -

0/21

840

0.0%

LIKE

LY B

ENIG

Nc.

8423

C>T,

R28

08Q

43w

eak

(cow

)1,

5C0

beni

gnde

lete

rious

dise

ase

caus

ing

- 23

/218

433

/849

4; 1

31/4

296

0,90

%LI

KELY

BEN

IGN

c.84

52C>

T, E

2818

K56

high

(zeb

rafis

h)3.

68C5

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

843/

8524

; 00.

0%LI

KELY

PAT

HO

GEN

ICc.

8458

C>T,

A28

20T

58w

eak

(lem

ur)

-0.0

4C0

beni

gnto

lera

ted

poly

mor

phis

m -

0/21

840/

~130

000,

10%

LIKE

LY B

ENIG

Nc.

8462

C>T,

R28

21Q

43w

eak

(lem

ur)

0,12

C0be

nign

tole

rate

dpo

lym

orph

ism

- 2/

2184

14/8

518;

4/4

312

0,30

%LI

KELY

BEN

IGN

c.85

39T>

C, I2

865V

29w

eak

(cow

)0,

12C0

beni

gnto

lera

ted

poly

mor

phis

m -

0/21

846/

8380

; 1/4

152

0,00

%LI

KELY

BEN

IGN

c.86

12G

>a, A

2871

V64

high

(zeb

rafis

h)5.

45C6

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 1/

2184

14/8

348;

3/4

140

0.2%

VO

US

c.87

44T>

C, K

2915

R26

high

(zeb

rafis

h)-1

.09

C25

beni

gnde

lete

rious

poly

mor

phis

mm

inor

effe

ct0/

2184

0/~1

3000

0,00

%V

OU

Sc.

8881

C>T,

V29

61M

21hi

gh (z

ebra

fish)

-0.1

2C0

beni

gnde

lete

rious

poly

mor

phis

m -

0/21

840/

~130

000,

20%

VO

US

c.89

00T>

C, Y

2967

C19

4w

eak

(cow

)1.

58C0

beni

gnto

lera

ted

dise

ase

caus

ing

- 9/

2184

99/8

272;

10/

3984

0.8%

LIKE

LY B

ENIG

Nc.

8917

C>T,

D29

73N

23hi

gh (z

ebra

fish)

3.11

C15

beni

gnde

lete

rious

dise

ase

caus

ing

- 0/

2184

20/8

328;

3/4

018

0.0%

VO

US

c.89

20C>

T, E

2974

K56

mod

erat

e (z

ebra

fish)

1.66

C55

beni

gnde

lete

rious

poly

mor

phis

m -

0/21

8421

/833

4; 0

0.5%

VO

US

c.89

23C>

T, E

2975

K56

mod

erat

e 4.

16C1

5be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

844/

8332

; 00.

0%V

OU

Sc.

8941

C>T,

A29

81T

58w

eak

-0

.36

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

00.

0%LI

KELY

BEN

IGN

c.92

27C>

T, R

3076

Q43

wea

k (c

ow)

1,98

C0be

nign

dele

terio

usdi

seas

e ca

usin

g -

0/21

841/

8538

; 1/4

330

0,10

%V

OU

Sc.

9231

G>C

, D30

77E

45hi

gh (z

ebra

fish)

0,29

C35

beni

gnde

lete

rious

poly

mor

phis

m -

117/

2184

39/8

532;

927

/433

00,

90%

BEN

IGN

c.93

88C>

T, D

3130

N23

wea

k (le

mur

)0,

04C0

beni

gnto

lera

ted

poly

mor

phis

m -

0/21

841/

8206

; 00,

10%

LIKE

LY B

ENIG

Nc.

9445

C>T,

E31

49K

56hi

gh (z

ebra

fish)

2,47

C55

beni

gnde

lete

rious

dise

ase

caus

ing

- 4/

2184

13/8

308;

2/4

118

0,00

%V

OU

Sc.

9454

T>C,

T31

52A

58w

eak

-0

.76

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

00.

0%LI

KELY

BEN

IGN

c.94

64C>

T, R

3155

Q43

wea

k-0

.12

C0be

nign

tole

rate

dpo

lym

orph

ism

min

or e

ffect

1/21

840/

~130

000,

10%

LIKE

LY B

ENIG

Nc.

9958

C>T,

D33

20N

23hi

gh (z

ebra

fish)

2,55

C0be

nign

tole

rate

ddi

seas

e ca

usin

g -

0/21

840/

~130

000,

20%

VO

US

c.10

004C

>T, R

3335

Q43

wea

k (m

ouse

)-0

.12

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

0/~1

3000

0,00

%LI

KELY

BEN

IGN

c.10

054T

>C, K

3352

E56

wea

k (c

ow)

-0.3

6C0

beni

gnto

lera

ted

poly

mor

phis

mm

inor

effe

ct0/

2184

0/~1

3000

0,00

%LI

KELY

BEN

IGN

c.10

102G

>A, R

3368

C18

0w

eak

(cow

)1,

25C5

5be

nign

dele

terio

uspo

lym

orph

ism

- 0/

2184

0/~1

3000

0,00

%V

OU

Sc.

1022

5G>A

, R34

09C

180

high

(zeb

rafis

h)1,

17C0

poss

ibly

da

mag

ing

tole

rate

ddi

seas

e ca

usin

g -

0/21

842/

8540

; 00,

00%

VO

US

c.10

336G

>A, R

3446

C18

0hi

gh (z

ebra

fish)

1.09

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

7/21

8475

/841

2; 1

2/41

841.

0%V

OU

S

c.10

372C

>T, G

3458

R12

5w

eak

-1

.41

C0be

nign

tole

rate

dpo

lym

orph

ism

stro

ng e

ffect

(b

ut la

rge

exon

)

11/2

184

62/8

338;

7/3

998

1.0%

LIKE

LY B

ENIG

N


CHA

PTER

2

c.10

454C

>T, R

3485

Q43

wea

k (c

ow)

0,53

C0be

nign

dele

terio

uspo

lym

orph

ism

- 0/

2184

0/~1

3000

0,00

%LI

KELY

BEN

IGN

c.10

469G

>C, G

3490

A60

high

(zeb

rafis

h)5.

86C5

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 21

/218

413

9/83

90; 8

/417

21.

8%LI

KELY

BEN

IGN

c.10

541C

>T, R

3514

Q43

wea

k (c

ow)

2,55

C0be

nign

tole

rate

ddi

seas

e ca

usin

g -

0/21

841/

8291

; 00,

00%

LIKE

LY B

ENIG

Nc.

1074

7A>G

, S35

83P

74hi

gh (z

ebra

fish)

4,81

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

840/

~130

000,

00%

LIKE

LY P

ATH

OG

ENIC

c.10

909G

>A, R

3637

C18

0w

eak

(cow

)0.

93C3

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 1/

2184

10/8

372;

00.

1%V

OU

S

c.11

056C

>A, A

3686

S99

wea

k (m

ouse

)-0

.04

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

0/~1

3000

0,10

%LI

KELY

BEN

IGN

c.11

158A

>G, S

3720

P74

wea

k (c

ow)

0,12

C0be

nign

dele

terio

uspo

lym

orph

ism

- 0/

2184

0/~1

3000

0,00

%LI

KELY

BEN

IGN

c.11

281C

>T, E

3761

K56

high

(zeb

rafis

h)5,

29C5

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,10

%LI

KELY

PAT

HO

GEN

IC

c.11

324G

>A, A

3775

V64

wea

k (c

ow)

0,77

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

0; 1

/436

00,

10%

LIKE

LY B

ENIG

Nc.

1143

8G>A

, A38

13V

64w

eak

0,53

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

2/82

72; 0

/388

00,

00%

LIKE

LY B

ENIG

Nc.

1144

7G>A

, A38

16V

64w

eak

(lem

ur)

0,61

C0be

nign

tole

rate

dpo

lym

orph

ism

min

or e

ffect

0/21

844/

8290

; 00,

00%

LIKE

LY B

ENIG

Nc.

1174

0C>T

, E39

14K

56w

eak

(cow

)3,

51C0

beni

gnto

lera

ted

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,30

%LI

KELY

BEN

IGN

c.11

762T

>A, Q

3921

L11

3hi

gh (z

ebra

fish)

4,24

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

g -

0/21

842/

8376

; 00,

00%

LIKE

LY P

ATH

OG

ENIC

c.12

010C

>G, D

4004

H81

high

(zeb

rafis

h)1,

74C0

beni

gnto

lera

ted

poly

mor

phis

m -

82/2

184

2/80

26; 4

08/3

760

0,00

%BE

NIG

Nc.

1202

2C>T

, G40

08S

56hi

gh (z

ebra

fish)

2,47

C55

beni

gnde

lete

rious

dise

ase

caus

ing

- 0/

2184

7/76

56; 3

/354

40,

30%

VO

US

c.12

131G

>A, T

4044

M81

high

(zeb

rafis

h)2.

79C6

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 18

/218

490

/838

2; 3

/411

21.

0%LI

KELY

BEN

IGN

c.12

437C

>T, R

4146

H29

high

(zeb

rafis

h)5,

86C2

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,10

%LI

KELY

PAT

HO

GEN

IC

c.12

442C

>T, V

4148

I29

high

(zeb

rafis

h)5,

86C2

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,10

%LI

KELY

PAT

HO

GEN

IC

c.12

598C

>T, V

4200

M21

high

(zeb

rafis

h)5,

86C1

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/83

62; 1

/405

60,

20%

LIKE

LY P

ATH

OG

ENIC

c.12

601C

>T, E

4201

K56

high

(zeb

rafis

h)5,

86C1

5po

ssib

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

4/83

68; 1

/407

30,

20%

LIKE

LY P

ATH

OG

ENIC

c.12

616G

>A, R

4206

C18

0hi

gh (z

ebra

fish)

1,98

C65

prob

ably

da

mag

ing

dele

terio

usdi

seas

e ca

usin

gm

inor

effe

ct0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

c.12

655C

>T, D

4219

N23

high

(zeb

rafis

h)5,

86C1

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

c.13

129C

>T, A

4377

T58

wea

k (c

ow)

0,93

C0be

nign

tole

rate

dpo

lym

orph

ism

- 0/

2184

6/84

46; 0

0,00

%LI

KELY

BEN

IGN

c.13

195G

>A, V

4399

I29

wea

k

0.69

CC0

beni

gnto

lera

ted

poly

mor

phis

m -

12/2

184

0/83

30; 2

/402

40.

0%LI

KELY

BEN

IGN

c.13

228G

>A, P

4410

S74

high

(zeb

rafis

h)5.

86C6

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 1/

2184

0/83

04; 1

3/39

620.

0%LI

KELY

PAT

HO

GEN

IC

c.13

885C

>T, G

4629

S56

high

(zeb

rafis

h)4.

00C5

5be

nign

dele

terio

usdi

seas

e ca

usin

gm

inor

effe

ct0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

ICc.

1399

9G>A

, R46

67C

180

high

(zeb

rafis

h)2,

79C6

5pr

obab

ly

dam

agin

gde

lete

rious

dise

ase

caus

ing

- 0/

2184

0/~1

3000

0,00

%LI

KELY

PAT

HO

GEN

IC

Documents

University of Groningen Matters of the heart: genetic and ... · Peripartum cardiomyopathy (PPCM) was observed in two DCM families carrying RBM20 mutations (p.R636H and p.S637N)