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…