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Indian Journal of Human Genetics, Vol. 17, No. 2, May-August, 2011, pp. 70-76 Original Article Novel mutations in arrhythmogenic right ventricular cardiomyopathy from Indian population Pranathi Rao Pamuru1, D V.N Maithili1, Khalid Mohiuddin2, Narasimhan Calambur2, Pratibha Nallari1 1 Department of Genetics, Osmania University, Hyderabad, Andhra Pradesh, India Code Number: hg11017 DOI: 10.4103/0971-6866.86182 Abstract Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive condition with right ventricular myocardium being replaced by fibro-fatty tissue. The spectrum of the expression may range from benign palpitations to the most malignant sudden death. Most of the mutations identified for the condition are localized in desmosomal proteins although three other nondesmosomal genes (cardiac ryanodine receptor-2, TGF-β3, and TMEM43) have also been implicated in ARVC. Both desmosomal and nondesmosomal genes were screened in a set of patients from local population.
Keywords: Arrhythmogenic right ventricular cardiomyopathy, fibro-fatty tissue, palpitations, sudden death Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rarer form of cardiomyopathy characterized by progressive fibro-adipocytic replacement of myocardial cells in the right ventricle. The symptoms include ventricular tachyarrhythmia, syncope, and even sudden death as an initial manifestation of the condition, with vast clinical heterogeneity. The condition is also genetically heterogenous with 11 loci been implicated and 8 genes identified. The genes identified include desmosomal (plakophilin-2 [Pkp-2], desmoplakin [Dsp], plakoglobin [JUP], desmoglein-2 [DSG-2], and desmocollin-2 [DSC-2]) and nondesmosomal genes (TGFβ-3, RyR-2, and TMEM43). With the discovery of five desmosomal genes harboring the mutations for ARVC, desmosomal disruption was proposed to be the major mechanism underlying the condition. But recent reports implicating the involvement of Wnt pathway and armadillo proteins in the process of fibro-fatty replacement have given a new direction in understanding the etiology of the ARVC. [1] This study reports three novel mutations from Indian population in cardiac ryanodine receptor (RyR-2) and Pkp-2 genes. An intronic insertion was identified in one patient in RYR2 gene. A missense mutation and a two base pair deletion in the N-terminal region were identified in three patients. This study also describes the variability in expression of the condition in these probands. Materials and Methods This study included 34 patients diagnosed using the criteria proposed by Task Force of the European Society of Cardiology/International Society and Federation of Cardiology [2] and 100 control individuals selected based on the absence of history/family history of any cardiac disorder. An informed consent was obtained from all the individuals participating in this study, and Institutional ethics committee approval from Care Hospitals Hyderabad has been obtained for clinical and genetic analyses. Diagnosis was based on 12-lead surface electrocardiograph (ECG), cardiac Doppler Echo, and history of familial condition. For genetic analysis, DNA was isolated by rapid nonenzymatic method [3] from whole blood. All the exons of Pkp-2, DSC-2, DSG-2, and JUP genes along with regulatory and promoter regions of TGFβ -3 gene, exons coding for FKBP binding domain of RyR-2 gene, and N-terminal region of Dsp were screened for mutations. The primer sequences for Pkp-2, JUP, DSC2, DSG-2, and TMEM43 genes were obtained from Gerull et al., [4] Whittock et al., [5] Syrris et al., [6] Awad et al., [7] and Merner et al., [8] respectively. For amplification of regulatory regions of transforming growth factor β-3 and exons of RyR2 and Dsp, genes were taken from http://telethon.bio.unipd.it/ARVDnet/molgen_arvd8.html. The amplified polymerase chain reaction (PCR) products were screened by single-strand conformation polymorphism (SSCP) for any variation in the sequence. The amplified PCR products with abnormal band pattern were purified using GeNei TMQuick PCR Purification Kit (Bangalore, India) and sequenced commercially (SolGent Co., Ltd., South Korea) using ABI 3730XL capillary DNA Sequencer (50 cm capillary). Results Genetic screening Screening of the previously implicated nondesmosomal gene segments, namely, regulatory regions of TGF-β3 and exon-10 of TMEM43 did not reveal any mutations, but a polymorphism in 5′ UTR (c) region of TGF-β3 gene was identified [Table - 1]. Mutational screening of exons coding for FKBP binding domain of RyR-2 which was proposed to be involved in pathogenesis of ARVC revealed an abnormal pattern in a patient and sequencing revealed an insertion of a base "G" (2487896_248797 ins G) [Figure - 1] 30 bp downstream the coding region of exon-28 [Table - 2]. Affect of this insertion mutation on splicing was analyzed using in silico splice site prediction software (http://www.fruitfly.org/cgi-bin/seq _tools /splice.pl) [Figure - 2] which revealed a formation of a cryptic splice site due to insertion, and usage of this site would lead to introduction of 10 novel amino acids in the exon-28 before splicing. Exon-28 of RyR-2 gene is involved in the formation of SPRY domain, and although the function of the domain is unknown, it is predicted to be involved in the interdomain interactions; and defects in this domain are reported in failing hearts [9] and hence, the insertion mutation in exon-28 coding for SPRY domain might alter interdomain interactions thereby leading to heart failure, which is reported in ARVC. Screening of desmosomal genes Dsp, JUP, desmsocollin-2, and DSG-2 do not reveal any patient-specific pathogenic mutations, but novel and known polymorphisms were identified [Table - 1]. Mutation screening of desmosomal gene Pkp-2 revealed an abnormal in three patients. An abnormal but similar band pattern in two unrelated patients (P1 and P2) in exon-3.1 was observed. Sequencing of these two samples revealed a heterozygous 2 base deletion (c.433_434 delCT) in exon-3.1. This deletion would affect 145 th amino acid with the addition of seven novel amino acids followed by the introduction of a premature termination codon (L145EfsX8) [Figure - 3] [Table - 2]. On screening the family members, both the daughters of P-1 were found to be carriers of the same mutation. Relatives of P-2 were unavailable for this study. Because identical mutation was identified in two patients, haplotype analysis was carried out to determine the origin of the mutation. Four repeat markers spanning 300,000 bp including the entire Pkp-2 gene were analyzed in both the patients and relatives of P-1. Primer sequences for these markers were obtained from van Tintelen et al. [10] The phase of P-1 was determined based on the analysis on three first-degree relatives. The chromosome containing the haplotype shared by both the carriers and patient possessed the deletion. Comparison of the haplotypes of both the patients shows that the mutation may have originated independently. Haplotype of all the typed individuals is given in [Figure - 4]. In addition, one patient showed an abnormal band pattern in exon-3.2 of Pkp-2 gene and sequencing revealed a heterozygous C792T missense change leading to P>L substitution at 244 th amino acid position [Table - 2]. This amino acid falls in the amino terminal region preceding the armadillo repeats. It has been reported earlier by Chen et al.[11] that N-terminal region of Pkp-2 is essential in promoting the interactions with other desmosomal components, namely, JUP, Dsp, desmoglein, and desmocollin isoforms and more importantly this region is critical for localization of Pkp-2 to the cell borders. Further, N-terminal region has also been implicated in signaling functions through its interaction with the β-catenin and upregulating the β-catenin/T-cell factor signaling activity. Clinical investigations Clinically the patient harboring RyR-2 gene mutation suffered from symptoms of dyspnea and presyncope during physical strain, an episode of syncope and swelling of limbs for the past 6-7 years and was diagnosed with ARVC at the age of 55 years. ECG evaluation revealed sinus bradycardia and low-voltage QRS complexes with QRS transition being less than/equal to V1. Repolarization abnormalities in the precordial leads with T-wave inversion beyond lead-V3 along with atrial fibrillation were also observed. 2D Doppler ECHO revealed dilatation and thinning out of right ventricular free wall with evidence of scarring on profusion imaging. This patient satisfied two major and two minor criteria [Table - 3]. The clinical profile of two patients (P-1 and P-2) harboring same Pkp-2 gene mutation in exon-3.1 was analyzed. Patient P-1 was a 29-year-old woman, presented with symptoms such as dyspnoea, palpitations, chest pain, presyncope, and syncope with occasional swelling of limbs, while patient P-2 was a 32-year-old man, suffering from palpitations and presyncope. Twelve-lead ECG of patient P-1 revealed normal sinus rhythm with heart rate of 60 bpm. There was a left axis deviation with left anterior hemiblock. There was also an epsilon wave in lead V1 with poor progression of R waves voltage in precordial leads. T-wave inversion was observed from leads V1 to V6 with low overall QRS voltage (in all 12 leads). ECG of patient P-2 also revealed normal sinus rhythm and heart rate of 60 bpm. There was a right axis deviation with right posterior hemiblock. There was an epsilon wave in lead V1 RBBB and upright R waves from leads V1 to V6 with small QR in lead V1. T-wave inversion was observed from leads V1 to V6 [Table - 3]. Cardiac Echo Doppler of patient P-1 revealed a dilated right ventricle with hypokinetic right ventricular apex, adjoining free wall with an apical clot and RV dysfunction. The mid-segment free wall of RV was also hypokinetic with normal LV function. In contrast to P-1, RV of patient P-2 was only mildly dilated. This patient showed good RV free wall motion and normal LV function. Therefore, patient P-1 showed RV dysfunction with structural alterations and also conduction abnormalities, thereby fulfilling two major criteria for diagnosis. Further the two minor criteria of inverted T waves and family history of premature sudden death (<35 years) were also fulfilled. Patient P-2 showed depolarization abnormalities and also repolarization abnormalities along with mild RV dilation, thereby fulfilling one major and two minor criteria. Two first-degree relatives of patient P-1 (both daughters) were available for further analysis. Twelve-lead ECG and Cardiac Echo Doppler showed no abnormalities. Clinically the patient with missense mutation in exon-3.2 of Pkp-2 gene was a 28-year-old man suffering with symptoms palpitations and presyncope with an average frequency of four episodes per week. A 12-lead ECG revealed a heart rate of 75 bpm with sinus rhythm and a QRS duration of 0.12 ms with left axis deviation. T-wave inversions in anterior leads V2-V3 and right bundle branch block morphology were also observed. 2D Doppler ECHO revealed conserved RV dimensions with normal systolic function, and RV morphology showed dyskinesis and right atrial enlargement. Hence, fulfilling three major and one minor standardized criteria [Table - 3]. Discussion ARVC is a rare form of cardiomyopathy, with an estimated prevalence of 1 in 5000. [12] Initial reports of ARVC are from Italian population, followed by American and now from Asian populations with mutations in similar genes underlying the condition, which underscore the significance of the pathogenic mechanism in maintaining the functional myocardium. This study, carried out for the first time in Indian population, corroborates the earlier findings of RyR-2 and Pkp-2 genes harboring mutations in individuals with ARVC. Insertion mutation in the intronic region of exon-28 of RyR-2 gene reported in this study would lead to an induction of a cryptic splice site, usage of which would lead to the introduction of 10 novel amino acids in exon-28 before splicing which in turn may affect the SPRY domain thereby altering the interdomain interactions. The deletion (433_434 delCT) in Pkp-2 gene observed in the present report would cause premature truncation resulting in the absence of armadillo repeats and C-terminal tail regions if the protein is translated. Nonsense-mediated decay may also operate causing haplo-insufficiency. This would result in improper assemblage of desmosomal complex releasing JUP free into cytosol, which could modulate Wnt/β-catenin signaling. On the other hand, because Pkp-2 is also an armadillo protein and earlier reports have demonstrated Pkp-2-dependent increase of endogenous β-catenin/TCF signaling in SW480 cells through direct interactions, haplo-insufficiency may cause JUP-independent reduction in canonical Wnt//β-catenin signaling through Tcf/Lef1. [1] Although there has been some progress made in understanding the pathogenesis at molecular level, the basis of clinical expression of the condition needs to be understood. Vast clinical heterogeneity has been observed in individuals with mutations in same genes. In this study, where both the patients harbored same mutation, patient P-1 was female who was diagnosed at the age of 29 years and patient P-2 was a male with age at diagnosis being 32 years. Comparison of the clinical expression of condition reveals severe progression of the condition in P-1 with gross RV dilation and RV hypokinesia when compared with P-2 in whom RV dimensions and contractility were highly conserved [Table - 3]. Family history of P-1 had the patient′s mother dying suddenly at the age of 25 years without any known cause. Although both the daughters of the patient have been detected to be carriers of the mutated allele, at the ages of 16 and 14 years, they did not show any kind of ECG or echocardiography abnormalities. Family history of P-2 did not have any sudden deaths. The fact that both the individuals with same mutation had such variable expression of the condition with one of them following two major and two minor (P-1) and the other one major and two minor (P-2) criteria proposed by Task Force of the European Society of Cardiology/International Society and Federation of Cardiology suggests the strong interaction of genetic and environmental factors in the expression of the condition. This observation is in conformation with earlier studies that have shown wide spectrum of expression even within the family members carrying the same mutation. [13] A study with monozygotic twins has also shown variability in expression implicating environmental factors as major triggers for the progression. [14] Therefore, specific genotype-based triggers need to be identified to prevent the severity of the progression. The third variation in Pkp-2 gene, a missense mutation C792T that would result in P244L, affects N-terminal region of the protein, which is involved in regulatory activities such as desmosomal formation and β-catenin-dependant Wnt pathway modulation. So any substitution in this region causes disturbances in the various protein-protein interactions. Conclusion ARVC is a prevalent cardiomyopathy in Indian population. Our study identified novel genetic variants (both mutations and polymorphisms) in the already implicated genes for ARVC, hence emphasizes the high genetic diversity of our population and advocates the inclusion of our population in international population-based genotypic estimation and data base studies. Drawbacks Experimentally, the mutation detection was based on the band pattern variation using SSCP technique, which is not a very sensitive. Further transcript and protein expression analyses were not performed to understand the affect of the mutations identified. References
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