1. Silvia Borras Aberdeen
DEVELOPING A DIAGNOSTIC SERVICE FOR ARRHYTHMOGENIC RIGHT VENTRICULAR DYSPLASIA / CARDIOMYOPATHY (ARVD/C) IN SCOTLAND
Silvia Borras
Aberdeen

2. 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

3. 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:
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

4. NATURAL HISTORY OF ARVD/C
(taken from Thiene et al, 2007)
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:
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

5. 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
pages/arvc)
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

6. 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:
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

7. 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
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

8. 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:
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

9. 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:
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)
ARVD/C is one of the major causes of SCD in young and athletes.
This cardiac disorder is described pathologically as:
And clinically as: ….syncopes presenting as LBBB, epsilon waves on ECHO or premature beats on 24hr Holter monitoring.
one of the major causes of sudden cardiac death in young and athletes (Thiene et al, 1988; Peters, 2006).
Until late 1970s it was believed to be a developmental defect of the RV myocardium, hence the original name dysplasia.
Based on the key clinical and pathological findings of subsequent studies by Thiene et al. (1988) and Nava et al (1998) the disease was reclassified to a non-ischaemic cardiomyopathy with familial preponderance.
Prevalence: 1:5000 (McKenna, 1994) with familial occurrence of 30%
autosomal dominant ARVD/C is 1 in (Peters, 2006) with familial occurrence 50-70%
There are regional variations: increased incidence in the population of Greek island Naxos 0.4 – 0.8% (Thiene and Basso, 2001) and in the Veneto region of Italy – ARVC accounts for 20% of all sudden deaths (Thiene et al., 2001). RV

10. 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
ARVD/C-3
602086
Not identified
14q12-22
ARVD/C-4
602087 2q
ARVD/C-5
604400
LAMR1
TMEM43
3p23
Unknown
ARVD/C-6
604401
PTPLA
10p12-14
ARVD/C-7
609160
DES
ZASP
10q22
ARVD/C-8
607450
Desmoplakin (DSP)
6p24
~50%
6-16%
ARVD/C-9
609040
Plakophilin-2 (PKP2)
12p11
AD/AR
~30%
11-43%
ARVD/C-10
610193
Desmoglein-2 (DSG-2)
18q q12.2
10-12%
ARVD/C-11
610476
Desmocollin (DSC-2)
18q12.1
1-5%
Naxos
601214
Plakoglobin (JUP)
17q21 AR
100%
Analysis of family pedigrees show that at least 50% patients have affected relatives suggesting a familial basis of ARVD/C. Between 30% and 80% of ARVD/C cases follow an autosomal dominant inheritance pattern with variable, age-related penetrance and phenotypic expression (Nava et al, 1988; Priori et al, 1999; Corrado et al, 2000; Hamid et al, 2002).
To date molecular genetic studies have identified 11 different loci associated with ARVD/C.
The fact that aprox. 50% of patients are found to have a mutation in one of the desmosomal genes, supports the theoty of ARVD/C being a desmosomal cardiomyopathy.

11. 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 MacRae et al, 2006)
Desmosomes are multi-protein structures in cell membrane that help to resist the shearing forces. Defects in desmomal proteins affect the essential role of desmosomes that is a signal transduction between myocytes, resulting in myocyte detachment and apoptosis. A significant cell loss is accompanied by inflammatory process. Since myocyte regeneration is very limited, the healing process results in fibro-fatty replacement. Fibrous tissue plays an important role in a conduction delay of the electrical impuls in heart resulting in life-threatening arrhythmias.

12. 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)
A key departure from this UK-Italian experience and other published cohorts has been the frequency of mutations in PKP2.
PKP2 gene codes for plakophilin-2 expressed in desmosomal plague of simple and stratified epithelia and nucleus.
In the Dutch study 16 out of 23 (70%) of probands with demonstrable familial disease had mutations in PKP2 gene suggesting the gene to be a major determinant of familial ARVC in Holland, strengthening the case for genetic testing in the population.
Majority of mutations detecected in PKP2 gene are frameshift (up to 55%), followed by nonsense mutations (27%) and missense mutations (11%).
Recessive mutation causing cryptic splicing with 7bp deletion in exon 12 reported by Awad (2008).
Van der Smagt (2007) a large deletion of first four exons incl. promotor detected by MLPA (1 in 82 patients).

13. 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)
A key departure from this UK-Italian experience and other published cohorts has been the frequency of mutations in PKP2.
PKP2 gene codes for plakophilin-2 expressed in desmosomal plague of simple and stratified epithelia and nucleus.
In the Dutch study 16 out of 23 (70%) of probands with demonstrable familial disease had mutations in PKP2 gene suggesting the gene to be a major determinant of familial ARVC in Holland, strengthening the case for genetic testing in the population.
Majority of mutations detecected in PKP2 gene are frameshift (up to 55%), followed by nonsense mutations (27%) and missense mutations (11%).
Recessive mutation causing cryptic splicing with 7bp deletion in exon 12 reported by Awad (2008).
Van der Smagt (2007) a large deletion of first four exons incl. promotor detected by MLPA (1 in 82 patients).

14. PKP2 Screening strategy
MATERIALS & METHODS
Cohort:
Males – 16
Females - 5
Aim:
Introduce a molecular screening service for PKP2 gene
Validate the method on previously tested DNA samples
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)
Reason for referral
Cohort of 21 patients selected by Clinical Geneticist, 16 males and 5 females with mean age at the diagnosis of 37.5 yrs, ranging from 7 to 63yrs.

15. RESULTS Patient 1: 20061443 54-year old female referred due to FH
c.148_151delACAG; p.thr50ser fsX61 in exon 1 of PKP2 gene
Patient 1:
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
Aged 52 years, she presented with light palpitations, runs of VT lasting 5-10 minutes (*minor criterion), abnormal ECHO (*minor criterion) and >1000 premature beats in 24-hour Holter monitoring (*minor criterion). Her son, the index case, was first diagnosed with irregular pulse in his late teens during the routine medical examination prior to his entering the Army. His symptoms, abnormal ECHO (*minor criterion) and signal averaged ECG (*minor criterion) were consistent with ARVD/C diagnosis. Implanted ICD showed at least intervention during exercise. Her daughter, who also presented with syncope (*minor criterion) and tiny bulging of RV on MRI (*minor criterion), was subsequently diagnosed with ARVD/C. Both ECHO and SAECG were normal.
Frameshift mutation resulting in formation of premature stop codon detected: c.148_151delACAG; p.thr50ser fsX61 in exon 1 of PKP2 gene. Because of predicted major rearrangements in plakophilin-2, this mutation is considered to be disease- causing.

16. Patient 2: 20062522 43-year old male Symptoms:
c.663C>A; p.tyr221stop in exon 3
Patient 2:
43-year old male
Symptoms:
exercise-induced VT age 22 extensive dilation of RV and hypokinesia age 41
is a 43-year old male first suspected for ARVD/C at the age of 22 years when presenting with exercise-induced VT (*minor criterion). He has been treated with antiarrhythmic drugs since the age of 25. Regular ECHO monitoring revealed an extensive dilation of RV (*major criterion) and hypokinesia (*minor criterion) were detected in this patient. With one major and two minor criteria, this patient would qualify for ARVD/C diagnostic testing.
Heterozygous nonsense mutation c.663C>A; p.tyr221stop was identified in exon 3. Pathogenic.

17. 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
is a 55-year old male diagnosed with ARVD/C at the age of 30 years when he experienced the first episode of VT (*minor criterion). Five years later, whilst on antiarrhythmic medication, he had another episode of VT. This resulted in ICD implant that last intervened at the age 50 years.
is a 50-year old male referred for ARVD/C genetic testing due to arrhythmia. Similar to the previous case, he experienced two episodes of VT (*minor criterion) at the age of 31 whilst exercising. He has since been treated with antiarrhythmic drugs. The only clinical details available for this patient show RV involvement on ECHO (*minor criterion).
6bp deletion in exon 11 of PKP2 gene c.2197_2202insGdelCACACC; A733fsX740 was detected in both apparently unrelated cases. This frameshift mutation, which results a premature stop codon is predicted to be pathogenic.

18. 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
is a 26-year old male who presented with exercise induced monomorphic sustained VT aged 25 years (*minor criterion). ECG and ECHO investigations showed no signs of the disease.
Molecular genetic testing detected two unclassified missense variants, c.209G>T; p.ser70ile resulting in incorporation of serine (ser) instead of isoleucine (ile) at codon 70 of exon 1 and c.1759G>A; p.val587ile resulting in incorporation of valine (val) instead of isoleucine (ile) at codon 587 of exon 8.
The conservation studies demonstrate that serine and valine residues at codon 70 and 587 respectively are highly conserved throughout evolution implying their important role in maintaining the integrity and function of plakophilin-2. In silico analysis of both unclassified variant suggest that although the changes may be tolerated by protein, they are likely to affect its proper functioning.

19. VALIDATION STUDY SUMMARY
Patient ID
Result reported by Holland
Result reported by Aberdeen
Wild type (Jan 2007)
Wild type
Wild type (Aug 2006)
c.148_151delACAG; p.thr50S (Aug 2006)
c.148_151delACAG; p.thr50ser fsX61
Wild type (Feb 2007)
c.663C>A; p.tyr221X (Feb 2007)
c.663C>A p.tyr221stop
c.2197_2202insGdelCACACC (July 2007)
c.2197_2202insGdelCACACC; p.his733ala fsX8
c.2197_2202insGdelCACACC (May 2008)
c.2197_2202insGdelCACACC;
p.his733ala fsX8
c.1759G>A; p.val587ile (Sept 2007)
c.209G>T; p.ser70ile
c.1759G>A; p.val587ile
Results for 9/10 previously tested patients were consistent with the results produced by the external laboratory. However, we reported the val587ile missense variant as unclassified (as oppose to pathogenic) and also described another unclassified missense variant, ser70ile, which was not reported by the external laboratory. In accordance with new Best Practice guidelines for the Interpretation and Reporting of Unclassified Variants in Clinical Molecular Genetics (2008), as a diagnostic laboratory, we are obliged to determine the clinical significance of all variants identified in routine testing. Published literature and in silico analysis of both unclassified variants failed to produce satisfactory evidence to either support or rule out their pathogenicity, therefore further testing (i.e. segregation studies) is recommended to determine the phenotypical effect of these variants.
We speculate that less stringent guidelines for reporting UVs by external laboratories may have been responsible for the difference in interpretation of this patient’s genotype. Limited evidence available on the ARVD/C database curated by Cardiogenetics Research Group of the University Medical Center in Groningen, Holland suggests that the val587ile variant is pathogenic and lists the ser70ile sequence change as polymorphism (
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.

20. 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.
Mean age consistent with Dalal (1996): patients with a mutation in PKP2 gene present with the symptoms at earlier age.
No G/P correlation possibly due to a small sample size.
Syrris et al (2006a) reported a similar result suggesting a possible involvement of other genetic and environmental factors in ARVD/C.
Using the original McKenna criteria (McKenna et al., 1994), molecular genetic testing would be clinically relevant in only one patient with a mutation in PKP2 gene (<5%). Modified criteria for diagnosis of familial ARVD/C (Hamid et al, 2002); Sen-Chowdhry et al, 2004) would include another case of a first-degree relative of ARVD/C index case. Potentially pathogenic mutations in three remaining patients would be completely missed if the clinical guidelines for genetic screening were strictly followed in our study. This result may however been affected by lack of information about clinical diagnosis for a number of patients.

21. FUTURE WORK Where do we go from here?
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.
Introduction of tagged primers to reduce the cost of sequencing.

22. FUTURE WORK Where do we go from here?
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.
Introduction of tagged primers to reduce the cost of sequencing.

23. 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:
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:
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:
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.

24. 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

25. Thank you…