Homozygous Desmocollin-2 Mutations and Arrhythmogenic Cardiomyopathy




Dominant mutations in desmocollin-2 ( DSC2 ) gene cause arrhythmogenic cardiomyopathy (ACM), a progressive heart muscle disease characterized by ventricular tachyarrhythmias, heart failure, and risk of juvenile sudden death. Recessive mutations are rare and are associated with a cardiac or cardiocutaneous phenotype. Here, we evaluated the impact of a homozygous founder DSC2 mutation on clinical expression of ACM. An exon-by-exon analysis of the DSC2 coding region was performed in 94 ACM index patients. The c.536A>G (p.D179G) mutation was identified in 5 patients (5.3%), 4 of which resulted to be homozygous carriers. The 5 subjects shared a conserved haplotype, strongly indicating a common founder. Genetic and clinical investigation of probands’ families revealed that p.D179G homozygous carriers displayed severe forms of biventricular cardiomyopathy without hair or skin abnormalities. The only heterozygous proband, who carried an additional variant of unknown significance in αT-catenin gene, showed a mild form of ACM without left ventricular involvement. All heterozygous family members were clinically asymptomatic. In conclusion, this is the first homozygous founder mutation in DSC2 gene identified among Italian ACM probands. Our findings provide further evidence of the occurrence of recessive DSC2 mutations in patients with ACM predominantly presenting with biventricular forms of the disease.


Arrhythmogenic cardiomyopathy (ACM; OMIM 107970 ) is a clinically and genetically heterogeneous heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death (SD). The disease is characterized by progressive fibro-fatty replacement of the ventricular myocardium. At present, mutations in desmosomal genes encoding for plakoglobin ( JUP) , desmoplakin ( DSP) , plakophilin-2 (PKP2) , desmoglein-2 (DSG2) , and desmocollin-2 ( DSC2 ) result associated with the disease in about 50% of probands. Presence of multiple mutations in desmosomal genes has been reported in a significant proportion of patients with ACM, and it is considered a powerful risk factor for lifetime major arrhythmic events and SD. Nondesmosomal disease genes, such as transmembrane protein 43 ( TMEM43 ), phospholamban ( PLN ), and αT-catenin ( CTNNA3 ), have been also identified providing evidence that ACM pathogenesis extends beyond desmosomes. Recessive ACM forms are rare. Homozygous DSC2 mutations have been associated with ACM forms with or without cutaneous manifestations. Here, we report the identification of the first homozygous founder mutation in DSC2 gene in the Italian population, segregating in ACM families with a severe cardiac-restricted phenotype.


Methods


The study included 94 unrelated probands of Italian descent diagnosed with ACM according to the revised criteria and negative for mutations in PKP2 , DSP , and DSG2 genes. Clinical evaluation in probands and family members was performed according to previously reported methods. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The study protocol was approved by the ethics committee review board of the University of Padua (Padua, Italy). All the participants provided written informed consent before inclusion in the study.


The 94 ACM index cases were screened for DSC2 mutations by denaturing high-performance liquid chromatography and direct sequencing. Sixty-two subjects had already been reported in previous studies. The coding region of JUP , CTNNA3 , and DES (encoding for desmin) genes was screened for mutations in DSC2 mutation carriers. A control group of 300 healthy and unrelated subjects (600 alleles) from the Italian population was used to exclude that identified variants could be common DNA polymorphisms. Additional 113 unrelated healthy subjects were genotyped for the presence of the DSC2 c.536A>G (p.D179G) mutation by polymerase chain reaction and enzyme digestion as the mutation abolishes a HincII restriction site. This control sample originates from Chioggia, a small city near Venice, hometown of 3 of the 5 index cases carrying the DSC2 mutation. To investigate whether DSC2 p.D179G recurrent mutation was due to a potential founder effect, haplotype analysis was carried out. DNA samples from subjects of families A and B were genotyped using 4 microsatellite markers close to DSC2 gene on chromosome 18q12.1 (D18S847, D18SH3, D18SH4, and D18S36), as previously reported. For the other patients and for control samples from Chioggia, the haplotype was reconstructed using the same markers.




Results


We performed an exon-by-exon analysis of the DSC2 gene in 94 ACM index patients who fulfilled the 2010 Task Force criteria. All patients were negative for mutations in the “big three” ACM genes PKP2 , DSP , and DSG2 . Seven nucleotide substitutions were identified in 11 index patients (11.7%). Three of them were novel: one (c.-4C>T) was in close proximity to the ATG start codon, one (c.631-10C>T) was located in intron 5, and one (c.1776G>A) was a synonymous substitution (p.A592=) in exon 12. These substitutions were considered variants of unknown significance ( Supplementary Table 1 ). The other 4 variants (p.E102K, p.D179G, p.I345T, and p.A897Kfs*4) had been previously reported. The nucleotide substitution c.536A>G in exon 5 resulting in p.D179G amino acid change ( Figure 1 ; Supplementary Table 1 ), which was previously detected in 3 index cases, was identified in 2 additional probands. Of the 5 mutation carriers (5.3%), 4 were homozygous ( Figure 1 ). One of them carries also a PKP2 rare polymorphism (c.1012A>G, p.T338A, rs139851304). Clinical findings of 5 DSC2 p.D179G index cases and their family members are reported in Table 1 .




Figure 1


The c.536A>G (p.D179G) mutation in desmocollin-2 ( DSC2 ) gene is associated with arrhythmogenic cardiomyopathy. (A) Sequence electropherograms showing the heterozygous and homozygous nucleotide substitution compared with the wild-type sequence. (B) ClustalW alignment showing evolutionary conservation of p.D179 in species. Organism names and GenBank accession numbers are reported on the sides (Bt = Bos taurus ; Cf = Canis familiaris ; Ec = Equus caballus ; Fc = Felis catus ; Gg = Gallus gallus ; Ggg = Gorilla gorilla gorilla ; Hs = Homo sapiens ; Mm = Mus musculus ; Mmu = Macaca mulatta ; Mpf = Mustela putorius furo ; Pt = Pan troglodytes ; Rn = Rattus norvegicus ). (C) Schematic representation of the DSC2 gene and protein. The involved exon and protein domain are indicated (S = signal peptide; P = proprotein; EC = extracellular cadherin domain; EA = extracellular anchor domain; TM = transmembrane region; IA = intracellular anchor domain; ICS = intracellular cytoplasmic domain). (D) Pedigrees of ACM families in which the DSC2 p.D179G mutation was detected. Numbering of the DSC2 and CTNNA3 nucleotides refers to GenBank accession number NM_024422 and NM_013266 respectively; the protein reference sequences were NP_077740 and NP_037398 .


Table 1

Clinical data of probands and family members carrying the p.D179G mutation in DSC2














































































































































































































































































































































































































































































Subject Sex Age (yrs) at diagnosis/ last investigation Family Echocardiography
RV global or regional dysfunction
and structural alterations 		Twelve-lead ECG
repolarization abnormalities 		Twelve-lead ECG
depolarization/ conduction abnormalities 		Arrhythmias LV involved ACM diagnostic criteria DSC2
p.D179G mutation
Family (M) (m) Regional RV akinesia, dyskinesia, or aneurysm and 1 of the following
(end diastole):
(M) 		Regional RV akinesia, dyskinesia, or aneurysm and 1 of the following
(end diastole):
(m) 		Inverted
T waves
in V1-V3
(>14 yrs)
(M) 		Inverted
T waves
in V1-V2 (>14 yrs) or in V4-V6
(m) 		Inverted
T waves
in V1-V4
(>14 yrs) in presence of RBBB
(m) 		Epsilon wave
(M) 		Late potentials (SAECG)
(m) 		NSVT or SVT
of LBBB morphology with superior axis
(M) 		NSVT or SVT
of RV outflow configuration or of unknown axis
(m) 		>500 ventricular extrasystoles per 24 hours (holter)
(m)
PLAX RVOT
≥32 mm; PSAX RVOT ≥36 mm;
FAC ≤33% 		PLAX RVOT
≥29 to <32 mm; PSAX RVOT ≥32 to <36 mm; FAC >33% to ≤40%
A I,2 F 81 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
II,4 F 54 0 0 + 0 0 + 0 0 + 0 0 + + 2M, 3m +/+
II,6 F 53 + 0 + 0 + 0 0 0 0 0 0 + + 3M, 1m +/+
III,1 M 51 + 0 0 0 0 0 0 0 + 0 0 0 0 1M, 1m +/-
III,2 M 43 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,3 F 46 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,4 F 21 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,5 F 20 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,6 F 17 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,7 F 12 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
B I,1 M 59 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
I,2 F 62 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
II,1 M 14 0 0 + 0 + 0 0 0 + 0 0 + + 3M, 2m +/+
C II,3 F 55 0 0 + 0 + 0 0 + + 0 0 + + 4M, 1m +/+
III,1 F 37 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,2 F 29 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
III,3 M 27 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
D I,1 M 42 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-
I,2 F 33 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/- ∗
II,1 M 11 0 0 + 0 + 0 0 + + 0 0 0 + 4M +/+ ∗
E III,1 M 20 0 0 0 + 0 + 0 0 + 0 0 + 0 4m +/- †
III,2 F 28 + 0 0 0 0 0 0 0 0 0 0 0 0 1M +/-

ACM = arrhythmogenic cardiomyopathy; DSC2 = desmocollin-2; FAC = fractional area change; LBBB = left bundle branch block; LV = left ventricle; M/m = major/minor diagnostic criterion; NSVT/SVT = not-sustained/sustained ventricular tachycardia; PLAX = parasternal long-axis view; PSAX = parasternal short-axis view; RV = right ventricle; RVOT = RV outflow tract; SAECG = signal-averaged ECG; + = present; 0 = absent; +/+ = homozygous mutation carrier; +/− = heterozygous mutation carrier.

∗ Carrying a PKP2 rare polymorphism (p.T338A).


† Carrying a CTNNA3 unclassified variant (p.V583A).



Family A consisted of 14 subjects. The proband (II,4) was diagnosed with ACM at the age of 54. She was asymptomatic and underwent cardiovascular evaluation because of the presence of negative T waves in the precordial leads. Two-dimensional echocardiogram (2D-echo) showed a moderately dilated right ventricle (RV) with reduced systolic function (ejection fraction [EF] 40%). During follow-up, the patient developed a biventricular form of the disease (left ventricle [LV] EF 30%) and received an implantable cardioverter-defibrillator (ICD) for primary prevention at the age of 74 years. Family history reported the death of a 33-year-old brother because of heart failure (II,1). The proband’s sister (II,6) was diagnosed at the age of 53 years as having a moderate form of ACM with LV involvement. To date, the remaining subjects have never shown any clinical sign of the disease.


Family B consisted of 3 subjects. The proband (II,1) was a 14-year-old boy who experienced a syncopal episode after effort. On 12-lead electrocardiogram (ECG), negative T waves in V 1 to V 3 , RV conduction delay, and low QRS voltages in limb leads were recorded; moreover, late potentials were present at all filters. Two-dimensional echo revealed a markedly dilated RV with reduced ventricular function (EF 42%) and kinetic alterations. Considering the disease extent and the syncopal episode, an ICD was implanted; however, no episodes of sustained ventricular arrhythmia were recorded during the 16 years of follow-up. In addition, the disease became biventricular with the presence of kinetic abnormalities of the LV and 12-lead ECG performed at the last follow-up showed negative T waves in V 1 to V 5 and intraventricular conduction delay with QRS fractionation ( Figure 2 ). Clinical and instrumental findings of both parents were unremarkable.




Figure 2


Instrumental findings of the homozygous proband of family B at the last follow-up. (A) Twelve-lead ECG showing negative T waves in V 1 to V 5 , intraventricular conduction delay with QRS fractionation, low QRS voltages in the limb leads, and ST-segment abnormalities in inferior leads. (B) Signal-averaged ECG results with presence of late potentials at 25 Hz. (C) Two-dimensional echocardiogram (parasternal long-axis view) showing RV outflow dilation. (D) Two-dimensional echocardiogram (apical 4-chamber view) showing RV dilation and kinetic abnormalities.

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Nov 28, 2016 | Posted by in CARDIOLOGY | Comments Off on Homozygous Desmocollin-2 Mutations and Arrhythmogenic Cardiomyopathy

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