Arrhythmogenic Right Ventricular Cardiomyopathy linked to 3p25 (ARVD5): Narrowing of the Critical Region
V. French1, K. Hodgkinson1,2, L. Thierfelder3, D. Galutira1, I. Pardoe1, P. Parfrey2, S. Connors4 and T-L. Young1
1Discipline of Genetics, Memorial University, Health Sciences Centre, St. John's, Newfoundland, Canada; 2Patient Research Centre, Health Sciences Centre, St. John's, Newfoundland, Canada; 3Max-Delbrück Centrum fur Molekulare Medizin, Berlin, Germany; 4Division of Cardiology, Memorial University, St. John's, Newfoundland, Canada.

- A common haplotype segregated with the disease across generations suggesting a common founder in these 14 Newfoundland families and a common mutation causing the disease. 
- Fine mapping of the 9.93 Mb critical region allowed us to reduce the region to 2.36 Mb. 
- Twenty positional candidate genes map to this refined ARVC5 critical region.
- Direct sequencing to identify the causative gene and mutation for ARVC5 has begun in our lab.
1. McKenna WJ, Thiene G, Nava A, Fontaliran F, Blomstrom-Lundqvist C, Fontaine G, Camerini F.  Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy. Task Force of the Working Group Myocardial and Pericardial Disease of the European Society of Cardiology and of the Scientific Council on Cardiomyopathies of the International Society and Federation of Cardiology. BHJ 1994; 71(3):215-218.
2. Hodgkinson KA, Parfrey PS, Bassett AS, Kupprion C, Drenckhahn J, Norman MW, Thierfelder L, Stuckless SN, Dicks EL, McKenna WJ, Connors SP.  The impact of implantable cardioverter-defibrillator therapy on survival in autosomal-dominant arrhythmogenic right ventricular cardiomyopathy (ARVD5).  JACC 2005; 45(3):400-408.
3. Nava A, Scognamiglio R, Thiene G, et al. A polymorphic form of familial arrhythmogenic right ventricular dysplasia. Am J Cardiol 1987;59:1405–1409.
4. Ahmad F, Li D, Karibe A, Gonzalez O, Tapscott T, Hill R, Weilbaecher D, Blackie P, Furey M, Gardner M, Bachinski LL, Roberts R.  Localization of a gene responsible for arrhythmogenic right ventricular dysplasia to chromosome 3p23.  Circulation. 1998; 98(25):2791-2795.
5. Gerull B, Osterziel KJ, Witt C, Dietz R, Thierfelder L. A rapid protocol for cardiac troponin T mutation detection in familial hypertrophic cardiomyopathy. Hum Mutat 1998;11:179–82.
6. W. Marshall, M. Furey and B. Larsen et al., Right ventricular cardiomyopathy and sudden death in young people. N Engl J Med 1988; 319:174–175.
7. Syrris P, Ward D, Evans A, Asimaki A, Gandjbakhch E, Sen-Chowdhry S, McKenna WJ,  Arrhythmogenic right ventricular dysplasia/cardiomyopathy associated with mutations in the desmosomal gene desmocollin-2. Am J Hum Genet. 2006; 79(5):978-984.5. Gerull B, Osterziel KJ, Witt C, Dietz R, Thierfelder L. A rapid protocol for cardiac troponin T mutation detection in familial hypertrophic cardiomyopathy. Hum Mutat 1998;11:179–82.
Results & Discussion

- All affected members and obligate carriers of ARVC in these 14 NL families share a portion of the 3p25 region (Table 1). Therefore, we expect that a single gene and mutation is causing ARVC5 and a common ancestor of all 14 families introduced the disease-causing mutation.
- Fine mapping of the 9.93 Mb critical region revealed critical recombinations in 8 of the 14 NL families investigated (Table 2).
- These recombinations reduced the 9.93 Mb region to 2.36 Mb on chromosome 3, which is predicted to contain the ARVD5 gene.
Table 2: Recombinations identified in our assigned ancestral ARVC5 haplotype

- Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is characterized predominantly by the replacement of cardiomyocytes with fatty and fibrous tissue and an increase risk of sudden cardiac death (SCD).
- This degradation of cardiomyocytes interferes with the hearts’ beat.
- SCD occurs when the electrical impulses in the heart become rapid (ventricular tachycardia) and/or chaotic (ventricular fibrillation) or extremely slow, resulting in death. 
- ARVC has been mapped to multiple genetic loci.  Some genes have been defined, including desmosomal proteins.

ARVC in Newfoundland
- In 1988, Newfoundland (NL) family 64 was diagnosed with an autosomal-dominant form of ARVC.
6  In 1998, linkage analysis was used on this family to map ARVC to a critical region of     9.93 Mb on chromosome 3p25 (ARVD5).   An identical haplotype on 3p25 in all affected members of family 64 cosegregated with ARVC.4 
- Positional candidate gene screening yielded no pathogenic mutations for ARVD5 (Thierfelder, personal communication). 
- Since 1998, 13 additional ARVC families from NL have been linked to the ARVD5 locus at 3p25.
- In this population, SCD may be the presenting symptom.  It is usually due to ventricular tachyarrhythmia, for which an implantable cardioverter defibrillator (ICD) is the best treatment.
- Natural history data shows that males with ARVD5 who did not receive an ICD as treatment have a 50% chance of SCD by the age of 40 and an 80% chance by the age of 50.
- Natural history data shows that females with ARVD5 who did not receive an ICD as treatment have a 5% chance of SCD by the age of 40 and a 20% chance by the age of 50.
- The true penetrance of ARVD5 is not clear and variable expression is present between and within families.
- Clinical diagnosis of ARVC is difficult.  In the absence of accurate clinical tests for diagnosing ARVC, a genetic test can pre-symptomatically diagnose patients and can save lives if ICD treatment follows.

- Clinical diagnosis of ARVC was based on international diagnostic criteria.
- 33 ARVC-clinically diagnosed patients from 14 identified families, including Family 64, had venous blood collected.  DNA was extracted from peripheral lymphocytes as previously described
- For each of 18 polymorphic short tandem repeat DNA markers located in the ARVC5 9.93Mb critical region, fluorescent-labeled primers were used to amplify nucleotide repeats by the polymerase chain reaction.  Fragments were analyzed by capillary electrophoresis on a ABI 3100 DNA sequencer.  The ARVC haplotype, a set of closely linked alleles segregating with ARVC on chromosome 3, was constructed across families.
- Because not all carriers of the ARVC haplotype will experience cardiac symptoms, we used obligate carriers and affected members to identify critical recombinations in the ARVC5 haplotype (Figure 1). 
Table 1:ARVC5 associated haplotype from obligate carriers & affected individuals across 14 NL families 
Figure 1: Partial pedigree of Family 69