developing a diagnostic service for arrhythmogenic right ventricular dysplasia / cardiomyopathy...
TRANSCRIPT
DEVELOPING A DIAGNOSTIC SERVICE FOR DEVELOPING A DIAGNOSTIC SERVICE FOR ARRHYTHMOGENIC RIGHT VENTRICULAR ARRHYTHMOGENIC RIGHT VENTRICULAR DYSPLASIA / CARDIOMYOPATHY (ARVD/C) DYSPLASIA / CARDIOMYOPATHY (ARVD/C)
IN SCOTLANDIN SCOTLAND
Silvia Borras
Aberdeen
TALK OUTLINETALK 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/CNATURAL 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
RV
Prevalence:
• 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/CNATURAL 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
RV
Prevalence:
• 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)
(taken from Thiene et al, 2007)
NATURAL HISTORY OF ARVD/CNATURAL 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
RV
Prevalence:
• 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)
(taken from http://ourworld.compuserve.com/homepages/arvc)
NATURAL HISTORY OF ARVD/CNATURAL 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
RV
Prevalence:
• 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)
(taken from McRae et al, 2001)
NATURAL HISTORY OF ARVD/CNATURAL 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
RV
Prevalence:
• 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)
(taken from http://en.wikipedia.org/wiki/Left_bundle_branch_block)
NATURAL HISTORY OF ARVD/CNATURAL 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
RV
Prevalence:
• 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/CNATURAL 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
RV
Prevalence:
• 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/CGENETICS OF ARVD/C
ARVD/Clocus name
MIM Gene ChromosomeInheritance
Penetrance
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 604400LAMR1
TMEM433p23 AD Unknown
ARVD/C-6 604401 PTPLA 10p12-14 AD
ARVD/C-7 609160DES
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 -
q12.2AD 10-12%
ARVD/C-11 610476 Desmocollin (DSC-2) 18q12.1 AD 1-5%
Naxos 601214 Plakoglobin (JUP) 17q21 AR 100%
DESMOSOMAL MODEL OF PATHOGENICITYDESMOSOMAL 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)
PKP2PKP2 GENE INVOLVEMENT GENE INVOLVEMENT
Location 12p11
Size 126.09kb
No of exons 14
Transcripts PKP2a (837aa)
PKP2b (881aa)
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
PKP2PKP2 GENE INVOLVEMENT 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 & METHODSMATERIALS & METHODS
47%
5%
33%
5% 10%
Sudden death
Family history ofARVD/C
Arrhytmia
Abnormal scan
Unknown
Reason for referral
Cohort:
Males – 16Females - 5
Aim:
1. Introduce a molecular screening service for PKP2 gene
2. Validate the method on previously tested DNA samples
3. Set up a molecular genetic testing of PKP2 gene
PKP2 Screening strategy
Extraction of genomic DNA from blood or tissue samples
Primer design
PCR amplification of all 14 exons (16 fragments)
Gel electrophoresis of selected samples
Sequencing of successfully amplified PCR products
Sequence analysis
Confirmation of samples with identified sequence change
MLPA analysis
(SALSA MLPA kit P168 PKP2, MRC Holland)
RESULTSRESULTS
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.209G>T; p.ser70ile in exon 1
c.1759G>A; p.val587ile in exon 8
VALIDATION STUDY SUMMARYVALIDATION STUDY SUMMARY
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.
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
CONCLUSIONSCONCLUSIONS
• 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 WORKFUTURE 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 WORKFUTURE 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.
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
Everyone in the Aberdeen laboratory for their support during both carrying out the experiments and writing up this project.
Dawn O’SullivanStephen TennantDr Christine Bell
Caroline ClarkDr Kevin Kelly
Dr John Dean
Thank you…