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DEVELOPING A DIAGNOSTIC SERVICE FOR ARRHYTHMOGENIC RIGHT VENTRICULAR DYSPLASIA / CARDIOMYOPATHY (ARVD/C) IN SCOTLAND Silvia Borras Aberdeen TALK OUTLINE • Natural history of ARVD/C • Molecular genetics, pathogenicity model and PKP2 gene involvement • Proposed strategy of PKP2 screening • Validation study results • Conclusions • Future work • Acknowledgements NATURAL HISTORY OF ARVD/C • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibro-fatty replacement • ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV • a degree of the LV involvement is reported in up to 75% patients Prevalence: • RV 1:5000 (McKenna, 1994) with familial occurrence of 50% Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) NATURAL HISTORY OF ARVD/C • • • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibrofatty replacement ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV (taken from Thiene et al, 2007) a degree of the LV involvement is reported in up to 75% patients Prevalence: • RV 1:5000 (McKenna, 1994) with familial occurrence of 50% Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) NATURAL HISTORY OF ARVD/C • • • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibro-fatty replacement ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV a degree of the LV involvement is reported in up to 75% patients (taken from http://ourworld.compuserve.com/home pages/arvc) Prevalence: • RV 1:5000 (McKenna, 1994) with familial occurrence of 50% Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) NATURAL HISTORY OF ARVD/C • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibro-fatty replacement • ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV • a degree of the LV involvement is reported in up to 75% patients (taken from McRae et al, 2001) Prevalence: • RV 1:5000 (McKenna, 1994) with familial occurrence of 50% Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) NATURAL HISTORY OF ARVD/C • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibro-fatty replacement • ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV • a degree of the LV involvement is reported in up to 75% patients (taken from http://en.wikipedia.org/wiki/Left_ bundle_branch_block) Prevalence: • RV 1:5000 (McKenna, 1994) with familial occurrence of 50% Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) NATURAL HISTORY OF ARVD/C • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibro-fatty replacement • ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV • a degree of the LV involvement is reported in up to 75% patients Prevalence: • RV 1:5000 (McKenna, 1994) with familial occurrence of 50% Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) NATURAL HISTORY OF ARVD/C • • one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006) progressive myocardial atrophy of the RV with fibro-fatty replacement • ventricular electric instability with syncopes and palpitations due to ventricular tachycardias or fibrillations originating in the RV • a degree of the LV involvement is reported in up to 75% patients Prevalence: • RV 1:5 000 (McKenna, 1994) Regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) Veneto region of Italy – ARVD/C accounts for 20% of all sudden deaths (Thiene et al, 2001) GENETICS OF ARVD/C ARVD/C locus name MIM Gene Chromosome Inheri tance Pene trance Detection rate ARVD/C-1 107970 TGFß -3 14q23-24 AD high ARVD/C-2 600996 RYR-2 1q42-43 AD high ARVD/C-3 602086 Not identified 14q12-22 AD ARVD/C-4 602087 Not identified 2q32.1-32.3 AD ARVD/C-5 604400 LAMR1 TMEM43 3p23 AD ARVD/C-6 604401 PTPLA 10p12-14 AD ARVD/C-7 609160 DES ZASP 10q22 AD ARVD/C-8 607450 Desmoplakin (DSP) 6p24 AD ~50% 6-16% ARVD/C-9 609040 Plakophilin-2 (PKP2) 12p11 AD/AR ~30% 11-43% ARVD/C-10 610193 Desmoglein-2 (DSG-2) 18q12.1 q12.2 AD 10-12% ARVD/C-11 610476 Desmocollin (DSC-2) 18q12.1 AD 1-5% Naxos 601214 Plakoglobin (JUP) 17q21 AR Unknown 100% DESMOSOMAL MODEL OF PATHOGENICITY Defects in desmosomes >> affected signal transduction between myocytes >> myocyte detachment and apoptosis >> inflammatory process >> fibro-fatty substitution >> intraventricular conduction delay of the electrical impulse >> life-threatening arrhythmias. (adapted from www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1483166#B7, MacRae et al, 2006) PKP2 GENE INVOLVEMENT PKP2 mutation prevalence: • UK: 27% (32/120), Gerull et al (2004) • Holland: 43-52% (24/56; 43/82), van Tintelen et al (2006), van der Smagt (2007), respectively • The US: 43% (25/58), Dalal et al (2006) >> patients with PKP2 mutation present with arrhythmia earlier than the patients with a mutation in other ARVD/C genes Location 12p11 Size 126.09kb No of exons 14 Transcripts PKP2a (837aa) PKP2b (881aa) PKP2 GENE INVOLVEMENT PKP2 mutation prevalence: • UK: 27% (32/120), Gerull et al (2004) • Holland: 43-52% (24/56; 43/82), van Tintelen et al (2006), van der Smagt (2007), respectively • The US: 43% (25/58), Dalal et al (2006) >> patients with PKP2 mutation present with arrhythmia earlier than the patients with a mutation in other ARVD/C genes Most frequent mutations in PKP2 gene (taken from Awad, 2008) MATERIALS & METHODS Cohort: Males – 16 Females - 5 Aim: PKP2 Screening strategy Reason for referral 1. Introduce a molecular screening service for PKP2 Extraction of genomic DNA from blood or tissue samples gene 5% 10% Primer design 2. Validate the method on PCR amplification of all 14 exons (16 DNA fragments) previously tested samples Gel electrophoresis of aselected samples 3. Set up molecular genetic testing of PKP2 gene Sequencing of successfully amplified PCR products Sequence analysis 47% 33% 5% Sudden death Family history of ARVD/C Arrhytmia Abnormal scan Confirmation of samples with identified sequence change Unknown MLPA analysis (SALSA MLPA kit P168 PKP2, MRC Holland) RESULTS Patient 1: 20061443 • 54-year old female referred due to FH Symptoms: • light palpitations, runs of VT lasting 5-10 minutes abnormal ECHO >1000 premature beats in 24-hour Holter monitoring c.148_151delACAG; p.thr50ser fsX61 in exon 1 of PKP2 gene Patient 2: 20062522 • 43-year old male Symptoms: • exercise-induced VT age 22 extensive dilation of RV and hypokinesia age 41 c.663C>A; p.tyr221stop in exon 3 Patients 3 & 4: 20071165 & 20071255 • 55 and 50-year old males Symptoms: • two episodes of VT in their 30s c.2197_2202insGdelCACACC; A733fsX740 in exon 11 Patient 5: 20072636 • 26-year old male Symptoms: • exercise-induced VT c.1759G>A; p.val587ile in exon 8 c.209G>T; p.ser70ile in exon 1 VALIDATION STUDY SUMMARY Patient ID Result reported by Holland Result reported by Aberdeen 20032441 Wild type (Jan 2007) Wild type 20051827 Wild type (Jan 2007) Wild type 20060877 Wild type (Aug 2006) Wild type 20061443 c.148_151delACAG; p.thr50S (Aug 2006) c.148_151delACAG; p.thr50ser fsX61 20062275 Wild type (Feb 2007) Wild type 20062285 Wild type (Feb 2007) Wild type 20062522 c.663C>A; p.tyr221X (Feb 2007) c.663C>A p.tyr221stop 20071165 c.2197_2202insGdelCACACC (July 2007) c.2197_2202insGdelCACACC; p.his733ala fsX8 20071255 c.2197_2202insGdelCACACC (May 2008) c.2197_2202insGdelCACACC; p.his733ala fsX8 20072636 c.1759G>A; p.val587ile (Sept 2007) c.209G>T; p.ser70ile c.1759G>A; p.val587ile c.1097T>C; p.leu366pro SNP was detected in exon 4 in five patients and a number of intronic SNPs in fragments 6,10,12,13 and 14. CONCLUSIONS • Four pathogenic genetic changes and two UVs detected in cohort of 21 patients >> mutation pick up rate of 28.6%. • No large genomic rearrangements detected by MLPA analysis of 18 PKP2 probes and 7 DSP probes and 3 probes each for JUP, TGFß3 and RYR2 genes. • The mean age of disease onset in patients with identified PKP2 sequence variant was 32 years (22-52 years) as opposed to 39 years (7-63) in patients without a PKP2 mutation. • As expected no specific G/P correlations were found in this study. • Variable phenotypical expression of the same PKP2 mutation within a family. • July 2008 – launch of PKP2 screening service in Aberdeen. FUTURE WORK • Improvements to the existing service • 21 patients successfully genotyped since July 2008 (two pathogenic mutations and two UVs potentially pathogenic changes – pick up rate 19.4%). Where do we go from here? • Offer the service to patients in Scotland under the Consortium arrangements. • Improve the resolution of the MLPA analysis and implement MLPA testing in the routine service. • Develop mutation screening of DSG2 gene to increase mutation detection rate in patients with suspected ARVD/C diagnosis (cDNA experiments). • Prepare a gene dossier for ARVD/C genetic testing. FUTURE WORK • Improvements to the existing service • 21 patients successfully genotyped since July 2008 (two pathogenic mutations and two UVs potentially pathogenic changes – pick up rate 19.4%). Where do we go from here? • Offer the service to patients in Scotland under the Consortium arrangements. • Improve the resolution of the MLPA analysis and implement MLPA testing in the routine service. • Develop mutation screening of DSG2 gene to increase mutation detection rate in patients with suspected ARVD/C diagnosis (cDNA experiments). • Prepare a gene dossier for ARVD/C genetic testing. REFERENCES Dalal D, Molin LH, Piccini J, Tichnell C, James C, Bomma C, Prakasa K, Towbin JA, Marcus FI, Spevak PJ, Bluemke DA, Abraham T, Russell SD, Calkins H and Judge DO (2006b). Clinical features of arrhythmogenic right ventricular dysplasia/cardiomyopathy associated with mutations in Plakophilin-2. Circulation, 113:1641-1649 Fontaine G, Frank R, Vedel J, Grosgogeat Y, Cabrol C, Facquet J (1977). Stimulation studies and epicardial mapping in ventricular tachycardia: study of mechanisms and selection for surgery. In: Kulbertus HE (eds) Reentrant Arrhythmias. MTP Publishing, Lancaster, 334–350 Gerull B, Heuser A, Wichter T, Paul M, Basson CT, McDermott DA, Lerman BB, Markowitz SM, Ellinor PT, MacRae CA, Peters S, Grossmann KS, Michely B, Sasse-Klaassen S, Birchmeier W, Dietz R, Breithardt G, Schulze-Bahr E, Thierfelder L (2004). Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy. Nature Genet., 36: 1162-1164 McKenna W, Thiene G, Nava A, et al. Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy (1994). Br Heart J., 71: 215–218 Nava A, Bauce B, Basso C, Muriago M, Rampazzo A, Villanova C, Daliento L, Buja G, Corardo D, Danielli GA, Thiene G (2000). Clinical profile and long-term follow up of 37 families with right ventricular cardiomyopathy. J Am Coll Cardiol., 36:2226-2233 Nava A, Thiene G, Canciani B, Scognamiglio R, Daliento L, Buja GF, Martini B, Stritoni P, Fasoli G (1988). Familial occurrence of right ventricular dysplasia: a study involving nine families. J Am Coll Cardiol. 12:1222– 1228 Peters S (2006) Advances in the diagnostic management of arrhythmogenic right ventricular dysplasia–cardiomyopathy. Int. J. Cardiol. 113: 4-11 Thiene G, Nava A, Corrado D, Rossi L, Pennelli N (1988). Right ventricular cardiomyopathy and sudden death in young people. N Engl J Med., 318:129–133. van der Smagt JJ, Coc MG, Nelen MR, van Tintelen JP, Entius MM, Wiesfeld AC, van gelder IC, de Jong GJ, Doevendans P, Hauer RN (2007) Large genomic deletions in plakophilin-2 are a rare cause of ARVD/C and ARVD/C-like disease. Genetics and genomics of Heart Failure. Circulation 116:II_604 (Abstract). van Tintelen JP et al (2006). Plakophilin-2 mutations are the major determinant of familial arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circulation, 113:1650–1658. ACKNOWLEDGEMENTS Everyone in the Aberdeen laboratory for their support during both carrying out the experiments and writing up this project. Dawn O’Sullivan Stephen Tennant Dr Christine Bell Caroline Clark Dr Kevin Kelly Dr John Dean Thank you…