Transcript DEVELOPING A MOLECULAR GENETIC DIAGNOSTIC SERVICE …

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…