Petros Syrris

Clinical and Genetic Characterization of Families With Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Provides Novel Insights Into Patterns of Disease Expression

Background— According to clinical-pathological correlation studies, the natural history of arrhythmogenic right ventricular dysplasia/cardiomyopathy is purported to progress from localized to global right ventricular dysfunction, followed by left ventricular (LV) involvement and biventricular pump failure. The inevitable focus on sudden death victims and transplant recipients may, however, have created a skewed perspective of a genetic disease. We hypothesized that unbiased representation of the spectrum of disease expression in arrhythmogenic right ventricular dysplasia/cardiomyopathy would require in vivo assessment of families in a genetically heterogeneous population. Methods and Results— A cohort of 200 probands and relatives satisfying task force or modified diagnostic criteria for arrhythmogenic right ventricular dysplasia/cardiomyopathy underwent comprehensive clinical evaluation. Desmosomal mutations were identified in 39 individuals from 20 different families. Indices of structural severity correlated with advancing age and were increased in long-term endurance athletes. Fulfillment of modified criteria indicated phenotypically mild disease, whereas asymptomatic status did not. In >80%, ECG, rhythm monitoring, and/or gadolinium-enhanced cardiovascular magnetic resonance were suggestive of LV involvement, the extent of which often was marked among individuals with chain-termination mutations and/or desmoplakin disease. Three patterns of disease expression were identified: (1) classic, with isolated right ventricular disease or LV involvement in association with significant right ventricular impairment; (2) left dominant, with early and prominent LV manifestations and relatively mild right-sided disease; and (3) biventricular, characterized by parallel involvement of both ventricles. Conclusions— LV involvement in arrhythmogenic right ventricular dysplasia/cardiomyopathy may precede the onset of significant right ventricular dysfunction. Recognition of disease variants with early and/or predominant LV involvement supports adoption of the broader term arrhythmogenic cardiomyopathy.

Sudden arrhythmic death syndrome: familial evaluation identifies inheritable heart disease in the majority of families

AIMS At least 4% of sudden deaths are unexplained at autopsy [sudden arrhythmic death syndrome (SADS)] and a quarter may be due to inherited cardiac disease. We hypothesized that comprehensive clinical investigation of SADS families would identify more susceptible individuals and causes of death. METHODS AND RESULTS Fifty seven consecutively referred families with SADS death underwent evaluation including resting 12 lead, 24 h and exercise ECG and 2D echocardiography. Other investigations included signal averaged ECG, ajmaline testing, cardiac magnetic resonance imaging, and mutation analysis. First-degree relatives [184/262 (70%)] underwent evaluation, 13 (7%) reporting unexplained syncope. Seventeen (30%) families had a history of additional unexplained premature sudden death(s). Thirty families (53%) were diagnosed with inheritable heart disease: 13 definite long QT syndrome (LQTS), three possible/probable LQTS, five Brugada syndrome, five arrhythmogenic right ventricular cardiomyopathy (ARVC), and four other cardiomyopathies. One SCN5A and four KCNH2 mutations (38%) were identified in 13 definite LQTS families, one SCN5A mutation (20%) in five Brugada syndrome families and one (25%) PKP2 (plakophyllin2) mutation in four ARVC families. CONCLUSION Over half of SADS deaths were likely to be due to inherited heart disease; accurate identification is vital for appropriate prophylaxis amongst relatives who should undergo comprehensive cardiological evaluation, guided and confirmed by mutation analysis.

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Associated with Mutations in the Desmosomal Gene Desmocollin-2

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited myocardial disorder associated with arrhythmias, heart failure, and sudden death. To date, mutations in four genes encoding major desmosomal proteins (plakoglobin, desmoplakin, plakophilin-2, and desmoglein-2) have been implicated in the pathogenesis of ARVD/C. We screened 77 probands with ARVD/C for mutations in desmocollin-2 (DSC2), a gene coding for a desmosomal cadherin. Two heterozygous mutations--a deletion and an insertion--were identified in four probands. Both mutations result in frameshifts and premature truncation of the desmocollin-2 protein. For the first time, we have identified mutations in desmocollin-2 in patients with ARVD/C, a finding that is consistent with the hypothesis that ARVD/C is a disease of the desmosome.

Cardiological assessment of first-degree relatives in sudden arrhythmic death syndrome

4.1% of sudden cardiac deaths in the 16-64 age-group are unexplained. In this group, cardiac pathological findings are normal and toxicological tests are negative; termed sudden arrhythmic death syndrome (SADS). We searched for evidence of inherited cardiac disease in cases of SADS. Of 147 first-degree relatives of 32 people who died of SADS, 109 (74%) underwent cardiological assessment. Seven (22%) of the 32 families were diagnosed with inherited cardiac disease: four with long QT syndrome; one with non-structural cardiac electrophysiological disease; one with myotonic dystrophy; and one with hypertrophic cardiomyopathy. Families of people who die of SADS should be offered assessment in centres with experience of inherited cardiac disease.

Interleukin-1 Receptor Antagonist Gene Polymorphism and Coronary Artery Disease

BACKGROUND Cytokine gene variations are contributory factors in inflammatory pathology. Allele frequencies of interleukin (IL)-1 cluster genes [IL-1A(-889), IL-1B(-511), IL-1B(+3953), IL-1RN Intron 2 VNTR] and tissue necrosis factor (TNF)-alpha gene [TNFA(-308)] were measured in healthy blood donors (healthy control subjects), patients with angiographically normal coronary arteries (patient control subjects), single-vessel coronary disease (SVD), and those with multivessel coronary disease (MVD). METHODS AND RESULTS Five hundred fifty-six patients attending for coronary angiography in Sheffield were studied: 130 patient control subjects, 98 SVD, and 328 MVD. Significant associations were tested in an independent population (London) of 350: 57 SVD, 191 MVD, and 102 control subjects. IL-1RN*2 frequency in Sheffield patient control subjects was the same as in 827 healthy control subjects. IL-1RN*2 was significantly overrepresented in Sheffield SVD patients (34% vs 23% in patient control subjects); IL-1RN*2 homozygotes in the SVD population (chi2 carriage=8.490, 1 df, P=0.0036). This effect was present though not quite significant in the London population (P=0. 0603). A summary trend test of the IL-1RN SVD genotype data for Sheffield and London showed a significant association with *2 (P=0. 0024). No significant effect of genotype at IL-1RN was observed in the Sheffield or London MVD populations. Genotype distribution analysis comparing the SVD and MVD populations at IL-1RN showed a highly significant trend (P=0.0007) with the use of pooled data. No significant associations were seen for the other polymorphisms. CONCLUSIONS IL-1RN*2 was significantly associated with SVD. A difference in genetic association between SVD and MVD was also apparent.

Novel Mutation in Desmoplakin Causes Arrhythmogenic Left Ventricular Cardiomyopathy

Background—Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial heart muscle disease characterized by structural, electrical, and pathological abnormalities of the right ventricle (RV). Several disease loci have been identified. Mutations in desmoplakin have recently been isolated in both autosomal-dominant and autosomal-recessive forms of ARVC. Primary left ventricular (LV) variants of the disease are increasingly recognized. We report on a large family with autosomal-dominant left-sided ARVC. Methods and Results—The proband presented with sudden cardiac death and fibrofatty replacement of the LV myocardium. The family was evaluated. Diagnosis was based on modified diagnostic criteria for ARVC. Seven had inferior and/or lateral T-wave inversion on ECG, LV dilatation, and ventricular arrhythmia, predominantly extrasystoles of LV origin. Three had sustained ventricular tachycardia; 7 had late potentials on signal-averaged ECG. Cardiovascular magnetic resonance imaging in 4 patients revealed wall-motion abnormalities of the RV and patchy, late gadolinium enhancement in the LV, suggestive of fibrosis. Linkage confirmed cosegregation to the desmoplakin intragenic marker D6S2975. A heterozygous, single adenine insertion (2034insA) in the desmoplakin gene was identified in affected individuals only. A frameshift introducing a premature stop codon with truncation of the rod and carboxy terminus of desmoplakin was confirmed by Western blot analysis. Conclusions—We have described a new dominant mutation in desmoplakin that causes left-sided ARVC, with arrhythmias of LV origin, lateral T-wave inversion, and late gadolinium enhancement in the LV on magnetic resonance images. Truncation of the carboxy terminus of desmoplakin and consequent disruption of intermediate filament binding may account for the predominant LV phenotype.

The Influence of Pharmacogenetics on the Time to Achieve Target Tacrolimus Concentrations after Kidney Transplantation

Previously, we reported that, at 3 months after renal transplantation, individuals with CYP3AP1 genotype CYP3AP1*1 (linked to CYP3A5*1 and strongly associated with expression of CYP3A5) required twofold higher doses of tacrolimus to achieve target blood concentrations than individuals with the genotype CYP3AP1*3/*3 (CYP3A5 nonexpressors). This study assesses the relationship between concentration‐controlled dosing during the early period after transplantation, the time to achieve target concentrations and genotype in 178 renal transplant recipients (CYP3AP1*1/*3 or *1/*1: n = 53, CYP3AP1*3/*3: n = 125). Patients with CYP3AP1*1/*3 or *1/*1 had lower mean tacrolimus concentrations during the first week (Median 13.5 vs. 18.5 μg/L, p < 0.0001) with significant delay in achieving target concentrations (15–20 μg/L during week 1, then 10–15 μg/L). More CYP3AP1*3/*3 patients had tacrolimus concentrations above target during the first week (73.6% vs. 35.8%, p = 0.003). There was no difference in the rate of biopsy‐confirmed acute rejection, but rejection occurred earlier in the CYP3AP1*1/*3 or *1/*1 group (median 7 d vs. 13 d, p = 0.005). In conclusion, an initial dosing regimen for tacrolimus based on knowledge of the CYP3AP1 genotype and subsequently guided by concentration measurements has the potential to increase the proportion of patients achieving target blood concentrations early after transplantation.

Clinical Expression of Plakophilin-2 Mutations in Familial Arrhythmogenic Right Ventricular Cardiomyopathy

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by loss of cardiomyocytes and their replacement by adipose and fibrous tissue. It is considered a disease of cell adhesion because mutations in desmosomal genes, desmoplakin and plakoglobin, have been implicated in the pathogenesis of ARVC. In a recent report, mutations in plakophilin-2, a gene highly expressed in cardiac desmosomes, have been shown to cause ARVC. Methods and Results— We investigated 100 white patients with ARVC for mutations in plakophilin-2. Nine different mutations were identified by direct sequencing in 11 cases. Five of these mutations are novel (A733fsX740, L586fsX658, V570fsX576, R413X, and P533fsX561) and predicted to cause a premature truncation of the plakophilin-2 protein. Family studies showed incomplete disease expression in mutation carriers and identified a number of individuals who would be misdiagnosed with the existing International Task Force and modified diagnostic criteria for ARVC. Conclusions— In this study, we provide new evidence that mutations in the desmosomal plakophilin-2 gene can cause ARVC. A systematic clinical evaluation of mutation carriers within families demonstrated variable phenotypic expression, even among individuals with the same mutation, and highlighted the need for a more accurate set of diagnostic criteria for ARVC.

A Novel Dominant Mutation in Plakoglobin Causes Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder associated with arrhythmias and sudden death. A recessive mutation in the gene encoding plakoglobin has been shown to cause Naxos disease, a cardiocutaneous syndrome characterized by ARVC and abnormalities of hair and skin. Here, we report, for the first time, a dominant mutation in the gene encoding plakoglobin in a German family with ARVC but no cutaneous abnormalities. The mutation (S39_K40insS) is predicted to insert an extra serine residue at position 39 in the N-terminus of plakoglobin. Analysis of a biopsy sample of the right ventricle from the proband showed markedly decreased localization of plakoglobin, desmoplakin, and connexin43 at intercalated discs in cardiac myocytes. A yeast-two-hybrid screen revealed that the mutant protein established novel interactions with histidine-rich calcium-binding protein and TGF beta induced apoptosis protein 2. Immunoblotting and confocal microscopy in human embryonic kidney 293 (HEK293) cell lines transfected to stably express either wild-type or mutant plakoglobin protein showed that the mutant protein was apparently ubiquitylated and was preferentially located in the cytoplasm, suggesting that the S39_K40insS mutation may increase plakoglobin turnover via proteasomal degradation. HEK293 cells expressing mutant plakoglobin also showed higher rates of proliferation and lower rates of apoptosis than did cells expressing the wild-type protein. Electron microscopy showed smaller and fewer desmosomes in cells expressing mutant plakoglobin. Taken together, these observations suggest that the S39_K40insS mutation affects the structure and distribution of mechanical and electrical cell junctions and could interfere with regulatory mechanisms mediated by Wnt-signaling pathways. These results implicate novel molecular mechanisms in the pathogenesis of ARVC.

Atlas of the clinical genetics of human dilated cardiomyopathy

AIM Numerous genes are known to cause dilated cardiomyopathy (DCM). However, until now technological limitations have hindered elucidation of the contribution of all clinically relevant disease genes to DCM phenotypes in larger cohorts. We now utilized next-generation sequencing to overcome these limitations and screened all DCM disease genes in a large cohort. METHODS AND RESULTS In this multi-centre, multi-national study, we have enrolled 639 patients with sporadic or familial DCM. To all samples, we applied a standardized protocol for ultra-high coverage next-generation sequencing of 84 genes, leading to 99.1% coverage of the target region with at least 50-fold and a mean read depth of 2415. In this well characterized cohort, we find the highest number of known cardiomyopathy mutations in plakophilin-2, myosin-binding protein C-3, and desmoplakin. When we include yet unknown but predicted disease variants, we find titin, plakophilin-2, myosin-binding protein-C 3, desmoplakin, ryanodine receptor 2, desmocollin-2, desmoglein-2, and SCN5A variants among the most commonly mutated genes. The overlap between DCM, hypertrophic cardiomyopathy (HCM), and channelopathy causing mutations is considerably high. Of note, we find that >38% of patients have compound or combined mutations and 12.8% have three or even more mutations. When comparing patients recruited in the eight participating European countries we find remarkably little differences in mutation frequencies and affected genes. CONCLUSION This is to our knowledge, the first study that comprehensively investigated the genetics of DCM in a large-scale cohort and across a broad gene panel of the known DCM genes. Our results underline the high analytical quality and feasibility of Next-Generation Sequencing in clinical genetic diagnostics and provide a sound database of the genetic causes of DCM.