The p.A897KfsX4 frameshift variation in Desmocollin-2 is not a
causative mutation in arrhythmogenic right ventricular
Marzia De Bortoli1, Giorgia Beffagna1, Barbara Bauce2, Alessandra Lorenzon1,
Gessica Smaniotto1, Ilaria Rigato2, Martina Calore1, Ilena E. A. Li Mura1, Cristina
Basso3, Gaetano Thiene3, Gerolamo Lanfranchi1,4, Gian Antonio Danieli1, Andrea
Nava2, Alessandra Rampazzo1*
1Department of Biology, University of Padua, Padua, Italy
2Department of Cardio-thoracic and Vascular Sciences, University of Padua
Medical School, Padua, Italy
3Department of Medico-Diagnostic Sciences and Special Therapies, University of
Padua Medical School, Padua, Italy
4CRIBI Biotecnology Centre, University of Padua, Padua, Italy
* corresponding author
Alessandra Rampazzo, Department of Biology, University of Padua, via U. Bassi
58/B, 35131 Padua, Italy. Tel: +39 049 8276208; Fax: +39 049 8276209;
Running title: DSC2 A897KfsX4 polymorphism in ARVC probands
Mutations in genes encoding desmosomal proteins have been reported to cause
arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), an
autosomal dominant disease characterized by progressive myocardial atrophy with
fibro-fatty replacement. We screened 112 ARVC/D probands for mutations in
desmocollin-2 (DSC2) gene and we detected two different amino acid
substitutions (p.E102K, p.I345T) and a frameshift variation (p.A897KfsX4) in 7
(6.2%) patients. DSC2a variant p.A897KfsX4, previously reported as
p.E896fsX900 mutation, was identified in five unrelated probands. Four of them
were found to carry one or two mutations in different ARVC/D genes.
Unexpectedly, p.A897KfsX4 variation was also found in 6 (1.5%) out of 400
In vitro functional studies demonstrated that, unlike wild-type DSC2a this C-
terminal mutated protein was localised in the cytoplasm. p.A897KfsX4 variation
affects the last five amino acids of the DSC2a isoform but not of DSC2b. In
contrast with what we found in other human tissues, in the heart DSC2b is more
expressed than DSC2a, suggesting that relative deficiency of DSC2a might be
compensated by isoform b.
In conclusion, DSC2 gene mutations are not frequently involved in ARVC/D.
The p.A897KfsX4 variation, identified in several Italian healthy control subjects,
which affects only one of the two DSC2 isoforms, may be considered a rare
variant, though possibly affecting phenotypic expression of concomitant ARVC/D
Keywords Sudden death, ARVC/D, desmosome, DSC2, polymorphism
Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) (MIM
#107970) is an inherited disorder characterised by progressive fibro-fatty
replacement of the right ventricular myocardium1. Clinical manifestations occur
most often between the second and fourth decade of life and are characterized by
ventricular arrhythmias, heart failure, and sudden death. The disease shows
usually autosomal dominant inheritance with reduced penetrance2, although
autosomal recessive transmission has also been reported in Naxos Syndrome3.
Eight genes have been detected so far as being independently involved in
the pathogenesis of the disease. Five of them encode major desmosomal proteins:
plakoglobin [MIM *173325]3-4, desmoplakin [MIM +125647]5, plakophilin-2
[MIM *602861]6, desmoglein-2 [MIM *125671]7 and desmocollin-2 [MIM
*125645]8-9. The involvement of such genes leaded to the current idea that
ARVC/D is a disorder caused mainly by defects in cell-cell adhesion.
Other three nondesmosomal genes have been associated with ARVC/D:
cardiac ryanodine receptor 2 (RYR2 [MIM180902])10, transforming growth factor
beta-3 (TGFβ3 [MIM 190230])11 and transmembrane protein 43 (TMEM43 [MIM
To date, five different DSC2 mutations have been reported: two frameshift
mutations p.M477fsX480 and p. E896fsX9008, a heterozygous splice acceptor site
mutation c.631-2A>G9 and two missense mutations p.E102K and p.I345T13. In
vitro functional studies demonstrated that the two point mutations affect the
intracellular localisation of desmocollin-2a, thus suggesting a potential pathogenic
effect13. More recently a homozygous mutation p.S614fsX625 in DSC2 gene
associated with autosomal recessive ARVC/D, mild palmoplantar keratoderma
and woolly hair has been described14.
In this study we investigated the frequency of DSC2 mutations in an
unselected Italian ARVC/D patient cohort. In five probands, we have identified
the frameshift p.A897KfsX4 variation (previously reported as p.E896fsX9008),
which was also detected among Italian healthy control subjects. On the basis of
genetic and functional data, we discuss here the pathogenic role of the
Materials and Methods
One hundred-twelve ARVC/D unrelated index cases were screened for DSC2
mutations by denaturing high-performance liquid chromatography (DHPLC) and
direct sequencing. This patient group includes 54 subjects already reported in a
previous study13. The coding region of DSP, PKP2 and DSG2 genes was screened
for mutations in DSC2 mutation carriers.
A control group of 200 healthy and unrelated Italian subjects (400 alleles)
was used to exclude that identified mutations could be DNA polymorphisms. All
controls were matched to the probands by ancestry and underwent ordinary
Mutation screening was performed in all available family members of index cases
in which a DSC2 mutation was detected.
Comparison of p.A897KfsX4 variant in patients and control subjects was
assessed by One-tail Fisher’s Exact test. A P value <0.05 was considered
Each patient underwent physical examination and family history, 12-lead
electrocardiography (ECG), signal-averaged ECG (SAECG), 24 hour Holter
ECG, two-dimensional echocardiography. A clinical diagnosis of ARVC/D was
based on major and minor criteria, established by the European Society of
Cardiology/International Society and Federation of Cardiology Task Force15.
Informed consent for clinical investigations and blood sampling for DNA analysis
was obtained from all participating individuals, according to the pertinent Italian
legislation and in compliance with Helsinki declaration.
DNA samples of all frameshift variation carriers were assessed for a common
haplotype, using the following microsatellite markers: D18S847, D18SH3,
D18SH4 and D18S36. Markers D18SH3 and D18SH4 are new polymorphic tri-
and di-nucleotides repeats that we identified starting respectively at position
26755775 and 26908386 on chromosome 18q12.1 (Human Genome Browser,
http://genome.ucsc.edu/). They were amplified using primers D18SH3F
5’CTCCCTTATGACCCAGGAAA3’ and D18SH4R
5’ACCATGTGGGAAACACCAAT3’ under standard PCR conditions. D18SH4
is within the DSC2 intron 12, whereas the other three markers are in close
proximity to the DSC2 locus. Forward primers were fluorescently labelled. In
particular, D18S847 and D18SH3 were labelled with TAMRA; D18SH4 with
HEX and D18S36 with FAM. Amplification products were pooled into post-
amplification panel, mixed with GeneScan ROX400 size standard. Capillary
electrophoresis was carried out on an ABI PRISM 3730XL DNA sequencer and
Genotyper V3.5 analysis software was used to analyze each amplicon.
Expression pattern of DSC2 isoforms
Expression of DSC2a and DSC2b isoforms in different human tissues was
examined by PCR amplification of cDNAs including heart, brain, placenta, lung,
skeletal muscle, liver, kidney and pancreas (Multiple Tissue cDNA panel I,
Clontech). Amplification was performed by using primers previously reported16.
The splice form “b” resulted in a PCR fragment of 500 bp, whereas splice form
“a” produced a 454 bp fragment.
PCR-based site-directed mutagenesis was performed on wild type DSC2a-
pcDNA3.1/CT-GFP, already available to the study13, to obtain three new
constructs. Each construct differs from the others for a variation introduced in
human DSC2 coding region: an insertion c.2687_2688insGA leading to the
frameshift variation p.A897KfsX4, and two nucleotide substitutions c.536A>G
(p.D179G) and c.2393G>A (p.R798Q) which resulted to be common
polymorphisms. The following mutagenic primers were used: DSC2D179G: 5’-
tccataagaggtcctggagttggccaagaacccggaatttatttt-3’; DSC2R798Q: 5’-
caccagacctcggaatcctgccagggggctggccaccatcacacc-3’; DSC2insGA: 5’-
caaatttggacactagcagagaagcatgcatgaagagacaag-3’ and DSC2frameGA: 5’-
cactagcagagaagcatgcacaagggcaattctgcagata-3’. Two steps of mutagenesis were
necessary to obtain a construct coding for p.A897KfsX4 variation, the first to
introduce the GA insertion and the second one to remove the premature stop
codon and the following 5 nucleotides to get the right frame with GFP sequence.
Thus, the mutant construct doesn’t contain additional amino acids compared to the
wild-type. The coding regions of each mutant construct were fully sequenced.
Cell culture and transfection
HL-1 cells, kindly provided by Dr. W.C. Claycomb (New Orleans), were used and
maintained as previously reported17. Cells were cultured at 37 °C in 5% CO2.
Constructs corresponding to wild-type or to a variant of human DSC2a were
transiently transfected in HL-1 cells at confluence of 70-80%, using Effectene
reagent (Qiagen) according to the manufacturer’s instructions. Transfected cells
were incubated for 48 h to allow protein expression and desmosome formation.
Immunostaining and confocal imaging
HL-1 cells were fixed with cold methanol/acetone (1:1) and immunostained for
endogenous desmoglein with the murine mAb (clone DG3.10) as previously
described in detail13. Images were acquired with a Radiance 2000 confocal
microscope (BioRad) with a 60× oil objective.
DSC2 mutation screening was performed in a series of 112 consecutive unrelated
index cases. Two different amino acid substitutions (p.E102K, p.I345T) and a
frameshift (p.A897KfsX4) have been detected in 7 (6.2%) patients (Figure 1a).
We also identified two polymorphisms (c.536A>G and c.2393G>A, allele
frequency 2.7 % and 4.7%, respectively) in exon 5 and in exon 15, resulting in
predicted p.D179G (novel) and p.R798Q (rs61731921) amino acid substitutions.
The two missense mutations p.E102K and p.I345T, previously reported by
our group13, affect the normal localization of mutant proteins in cultured
cardiomyocytes. The frameshift variation p.A897KfsX4, primarily reported as
p.E896fsX900 by Syrris et al8, causes a premature termination of the protein.
Only the last 5 amino acids of desmocollin-2a were altered, three were changed
and the last two were lost (Figure 1b). The five involved aa residues are less
conserved among mammals, in contrast with the high conservation of the
upstream region (Figure 1c). DSC2b is not affected by the p.A897KfsX4 variation
(Figure 1a), since it has a shortened C-terminal domain caused by a stop codon in
the additional exon 16.
Five unrelated ARVC/D index cases (allele frequency 2.2%) were found to
carry the p.A897KfsX4 variation, as well as 6 out of 200 control subjects (allele
A 0.11 P value, falling below the 0.05 significance threshold, indicates
that there was no evidence of overrepresentation of p.A897KfsX4 variant in
patients compared with control subjects.
Mutations in other ARVC/D genes were identified in four of these five
patients (Table 1). Proband 1 was found to carry the p.A897KfsX4 mutation and
the previously reported PKP2 p.E58D common polymorphism18. The allele
frequency of the p.E58D variant in our control population resulted to be 0.9%, in
contrast with the reported 5% in Finnish population representing a genetic
The additional detected mutations DSG2 p.Y87C and PKP2 p.V406SfsX3
have been previously reported as disease causing19,20. None of the novel identified
mutations (DSG2 p.G638R; DSP p.N375I; PKP2 p.F552C) were observed in 400
control chromosomes and occurred in residues that are highly conserved among
species (data not shown). Moreover, they all lead to a change in biochemical
properties of the amino acid involved.
In Proband 4 it has been also identified a nucleotide substitution c.45C>T
in PKP2 gene which causes a synonymous variation p.T15T. 220 control alleles
are negatives for this variation. This nucleotide substitution might be a rare
polymorphism or it might activate a cryptic splice site and produce an aberrant
Proband 1 was a 60 yrs old woman who was diagnosed as having ARVC/D due to
ECG abnormalities. Two-dimentional echocardiogram revealed a severe dilation
of the right ventricle with depressed ventricular function. Moreover, frequent
PVCs (premature ventricular contraction) with LBBB (left bundle-branch block)
morphology were present at 24-hour Holter ECG. The genetic analysis extended
to her son revealed that he carries the DSC2 frameshift variation p.A897KfsX4
and PKP2 polymorphism p.E58D as well. Clinical evaluation showed the
presence of some criteria also in the son, even if he did not fulfilled the diagnosis
Proband 2 was diagnosed at the age of 45 due to palpitations. Twenty-
four-hour Holter ECG demonstrated frequent episodes of non sustained
ventricular arrhythmias and imaging techniques showed a severe right ventricular
dilation/dysfunction. He resulted to be a double heterozygote for the DSC2
p.A897KfsX4 variation and the DSG2 p.Y87C mutation. His daughter, clinically
unaffected, is negative for both (Table 2).
Proband 3 experienced a sustained VT episode at the age of 38. Clinical
and instrumental examination revealed at that time right ventricular abnormalities
that became more evident during follow-up. She carried also a missense mutation
in DSP gene (p.N375I) and in DSG2 gene (p.G638R). The genetic study was
extended to additional family members and two subjects were double
heterozygotes for DSG2 mutation and DSC2 variation and one subject results to
carry the DSG2 mutation (Figure 2). One of the double heterozygotes (subject
3.(II,7)) carrying the DSG2 p.G638R and the DSC2 p.A897KfsX4 fulfilled the
ARVC/D diagnostic criteria, whereas the other family members were clinically
unaffected (Table 2).
Proband 4 was a female patient who underwent cardiac evaluation due to
several presyncopal episodes. The ECG showed negative T waves in V1-V4 and
in inferior leads. Late potentials were present at 40-80 Hz. At 2D echocardiogram
the right ventricle was mildly dilated with presence of kinetic alterations; the 24-
holter ECG documented the presence of frequent PVCs with LBBB morphology.
Cardiac magnetic resonance confirmed the presence of mild right ventricular
dilatation and kinetic alterations, localised on the subtricuspid region and on the
apex. The left ventricle was not involved. She was found to carry also the PKP2
p.V406SfsX3 mutation and the PKP2 p.T15T synonymous variation. The genetic
analysis extended to her daughter revealed that she carries only p.T15T variation,
in presence of negative clinical and instrumental findings.
Proband 5 underwent cardiac evaluation at the age of 45 yrs, due to the
presence of PVCs detected during a pre-operatory ECG that also showed
intraventricular conduction delay and negative T waves in V4-V6. SAECG
documented the presence of late potentials at all filters settings. Two D
echocardiogram demonstrated biventricular dimensional and kinetic
abnormalities. At the age of 49 he received an ICD (implantable cardioverter
defibrillator) due to recurrent VT episodes. He was found to carry additional
mutations in other two ARVC/D genes: p.N375I in DSP and p.F552C in PKP2.
His brother carries only the DSP mutation. The clinical and instrumental
examination demonstrated the presence of some ECG criteria, even if he did not
fulfil the diagnosis (Table 2).
In contrast with other reported DSC2 mutations 8,9,13, p.A897KfsX4 is the only
one identified in more Italian and English ARVC/D patients. Haplotype analysis
was performed using four polymorphic microsatellites, one intragenic (D18SH4)
and three in close proximity to the DSC2 gene (D18S847, D18SH3 and D18S36).
Different haplotypes segregated with the p.A897KfsX4 variation in each of the
five Italian patients demonstrating the absence of a founder effect for this
variation (data not shown). This result confirms data reported by Syrris et al. for
their three families, suggesting that p.A897KfsX4 is a recurrent variation.
Expression pattern of DSC2 isoforms
Almost all analysed human cDNA samples (heart, pancreas, lung, placenta, brain,
skeletal muscle, liver and kidney) showed expression of both DSC2 splice forms
(Figure 3). It is interesting to note that all of them showed a different expression
level of the two isoforms. In particular, in contrast with what we found in the
other tissues, in heart isoform b is more expressed than isoform a.
The PCR products for heart tissue were verified and confirmed by
sequencing (data not shown).
Functional analysis of C-terminal mutant desmocollin-2a (p.A897KfsX4)
Site-directed mutagenesis was performed on wild type construct encoding for a
human dsc2a-gfp fusion protein in order to study the functional effect of the
p.A897KfsX4 variation and of two DSC2 polymorphisms p.D179G and p.R798Q,
located in the N- and C-terminal domains of the protein, respectively.
Constructs were transfected in the desmosome-forming cell line HL-1
having a differentiated cardiomyocyte phenotype and contractile activity in vitro.
The GFP signal revealed a predominant localization at the plasma membrane of
wild type (Figure 4, panel A) and proteins carrying p.D179G and p.R798Q
polymorphisms (Figure 4, panel B and C), displaying co-localization with
endogenous dsg at the cell membrane to indicate well-assembled desmosomes
(Figure 4, panels A”, B” and C”).
By contrast protein carrying frameshift variation p.A897KfsX4 was
detected in the cytoplasm (Figure 4, panel D), loosing the proper desmosomal
localization along cell boundaries. On the contrary, endogenous dsg is normally
well distributed (Figure 4, panel D’).
Desmocollin-2 was the most recent major component of the cardiac desmosome
to be implicated in ARVC/D. DSC2 gene encodes one of desmosomal cadherins,
single-pass transmembrane glycoproteins able to mediate Ca2+-dependent cell-
cell adhesion, by interacting laterally and transcellularly with each other and by
recruiting cytoplasmic plaque proteins which facilitate attachment of intermediate
We have identified two different DSC2 mutations and one DSC2 variation
in 7 out of 112 unrelated ARVC/D index cases, two already described in our
previously report13 and p.A897KfsX4 originally referred to as a causative
mutation8. The frameshift variation was identified in five independent patients,
and four of them are carriers for one or two mutations in known ARVC/D genes.
Surprisingly we found the same variation in several Italian controls with a no
significant difference in the distribution of the variant between patients and
On the contrary, in the original paper reporting identification of
p.A897KfsX4 variation, three probands resulted to carry only this frameshift
variation, which was never identified in 200 control subjects8.
In contrast with other reported DSC2 mutations, p.A897KfsX4 is the only
one identified in several Italian and English ARVC/D patients. Haplotype analysis
excludes a founder effect in our patients as well as in English cases and confirms
that p.A897KfsX4 is a recurrent variation.
This variant occurs in exon 17 that encodes for ICS domain (intracellular
cadherin-like sequence domain) only in the splice variant “a” of DSC2 gene,
whereas the splice variant “b” has a different C-terminal domain. ICS domain
provides binding sites for other desmosomal constituents such as plakoglobin,
plakophilin and desmoplakin22,23,24. It has been demonstrated that the latest 37 aa
in Dsc1a are fundamental for plakoglobin binding in human epithelial A-431
cells23. On the other hand, in vitro functional studies on HL-1 cells have
demonstrated that DSC2a-GFP-A897KfsX4 protein localizes in the cytoplasm, in
contrast with wild type protein and DSC2a variants carrying the polymorphism
p.D179G or p.R798Q which correctly co-localize at the cell membrane with
However it is important to notice that the exon 17 is untranslated in
DSC2b isoform. Therefore, the insertion would affect only DSC2a, leaving
isoform b fully functional and possibly able to compensate in cardiac myocytes
the relative deficiency of DSC2a isoform. Furthermore in contrast with what we
found in the other human tissues, in heart DSC2b isoform is more expressed than
DSC2a supporting the hypothesis of a possible compensation by the isoform b.
Little is known about human DSC2b and in general about all Dscb
isoforms; until now only for Dsc3b it has been demonstrated a specific
desmosomal interaction with PKP325. Moreover, PKP1, for which strong in vitro
association with the ‘a’ form of Dsc1 has been reported24, was found to overlap
also with the ‘b’ form26. The possibility that Dscb isoforms contain a PKP-binding
domain should be further on investigated.
Four out of 5 patients carrying p.A897KfsX4 variation had also one or two
additional ARVC/D mutations. On the other hand, proband 1 carrying only
p.A897KfsX4 variation resulted to carry also a reported polymorphism (PKP2
p.E58D) whose pathogenic role, if any, remains to be investigated18. Moreover, Download full-text
we cannot exclude that this proband could have a large insertion, deletion or
mutation in non coding regions of ARVC/D genes or also a mutation in unknown
It may be hypothesized that p.A897KfsX4 variation could act in presence of other
ARVC/D mutations as a factor able to modify their pathogenic effect. All
probands showed moderate to severe forms of the disease, with biventricular
involvement in some cases. However, in our study group genotype-phenotype
data could not give a clear indication of the role of this variant in the disease-
phenotype. Moreover, there is not a different proportion of the variant in the
controls and affected subjects to provide evidence of an association between the
disease and the variant. Assessment of the exact significance of the p.A897KfsX4
variation requires further studies in additional families carrying mutations in other
Until now few DSC2 gene mutations were reported to cause ARVC/D.
Among them, the p.A897KfsX4 variation, identified in several Italian healthy
control subjects and altering only one of the two DSC2 isoforms, could be
considered a rare polymorphism which may affect the phenotypic expression of
concomitant ARVC/D mutations.
Further clarification of co-occurrence of mutations and rare
polymorphisms in different desmosomal proteins could be an important aspect in
accounting for the known inter- and intra-familial variable phenotypic expression.