Paul A. van der Zwaag

Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of arrhythmogenic cardiomyopathy

To investigate whether phospholamban gene (PLN) mutations underlie patients diagnosed with either arrhythmogenic right ventricular cardiomyopathy (ARVC) or idiopathic dilated cardiomyopathy (DCM).

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Pathogenic Desmosome Mutations in Index-Patients Predict Outcome of Family Screening: Dutch Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Genotype-Phenotype Follow-Up Study

Background— Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited disease with incomplete penetrance and variable expression. Causative mutations in genes encoding 5 desmosomal proteins are found in ≈50% of ARVD/C index patients. Previous genotype-phenotype relation studies involved mainly overt ARVD/C index patients, so follow-up data on relatives are scarce. Methods and Results— One hundred forty-nine ARVD/C index patients (111 male patients; age, 49±13 years) according to 2010 Task Force criteria and 302 relatives from 93 families (282 asymptomatic; 135 male patients; age, 44±13 years) were clinically and genetically characterized. DNA analysis comprised sequencing of plakophilin-2 ( PKP2 ), desmocollin-2, desmoglein-2, desmoplakin, and plakoglobin and multiplex ligation-dependent probe amplification to identify large deletions in PKP2. Pathogenic mutations were found in 87 index patients (58%), mainly truncating PKP2 mutations, including 3 cases with multiple mutations. Multiplex ligation-dependent probe amplification revealed 3 PKP2 exon deletions. ARVD/C was diagnosed in 31% of initially asymptomatic mutation-carrying relatives and 5% of initially asymptomatic relatives of index patients without mutation. Prolonged terminal activation duration was observed more than negative T waves in V1 to V3, especially in mutation-carrying relatives <20 years of age. In 45% of screened families, ≥1 affected relatives were identified (90% with mutations). Conclusions— Pathogenic desmosomal gene mutations, mainly truncating PKP2 mutations, underlie ARVD/C in the majority (58%) of Dutch index patients and even 90% of familial cases. Additional multiplex ligation-dependent probe amplification analysis contributed to discovering pathogenic mutations underlying ARVD/C. Discovering pathogenic mutations in index patients enables those relatives who have a 6-fold increased risk of ARVD/C diagnosis to be identified. Prolonged terminal activation duration seems to be a first sign of ARVD/C in young asymptomatic relatives. # Clinical Perspective {#article-title-38}

A Genetic Variants Database for Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a hereditary cardiomyopathy characterized by fibrofatty replacement of cardiomyocytes, ventricular tachyarrhythmias and sudden death. ARVD/C is mainly caused by mutations in genes encoding desmosomal proteins. However, the pathogenicity of variants is not always clear. Therefore, we created an online database (www.arvcdatabase.info), providing information on variants in ARVD/C‐associated genes. We searched the literature using ARVD/C and its underlying genes as search terms. From the selected papers and our unpublished data, we collected details on the type of mutation and information provided at the protein level. A “details page” contains clinical data and references. To aid the interpretation of missense mutations, we provide data from in silico prediction methods. In May 2009 the database contained 481 variants in eight genes. A total of 144 variants are considered pathogenic, 73 are unknown/unclassified, and 264 have no known pathogenicity. The database was converted into the Leiden Open Variation Database (LOVD) format, a gene‐centered collection of DNA variations. The ARVD/C database will be useful for both researchers and clinicians. It can be searched to determine if variants have been published and whether they are considered pathogenic. External users are invited to add information to improve the quantity and quality of the data entered. Hum Mutat 30:1–6, 2009.

Outcome in phospholamban r14del carriers: Results of a large multicentre cohort study

Background— The pathogenic phospholamban R14del mutation causes dilated and arrhythmogenic right ventricular cardiomyopathies and is associated with an increased risk of malignant ventricular arrhythmias and end-stage heart failure. We performed a multicentre study to evaluate mortality, cardiac disease outcome, and risk factors for malignant ventricular arrhythmias in a cohort of phospholamban R14del mutation carriers. Methods and Results— Using the family tree mortality ratio method in a cohort of 403 phospholamban R14del mutation carriers, we found a standardized mortality ratio of 1.7 (95% confidence interval, 1.4–2.0) with significant excess mortality starting from the age of 25 years. Cardiological data were available for 295 carriers. In a median follow-up period of 42 months, 55 (19%) individuals had a first episode of malignant ventricular arrhythmias and 33 (11%) had an end-stage heart failure event. The youngest age at which a malignant ventricular arrhythmia occurred was 20 years, whereas for an end-stage heart failure event this was 31 years. Independent risk factors for malignant ventricular arrhythmias were left ventricular ejection fraction <45% and sustained or nonsustained ventricular tachycardia with hazard ratios of 4.0 (95% confidence interval, 1.9–8.1) and 2.6 (95% confidence interval, 1.5–4.5), respectively. Conclusions— Phospholamban R14del mutation carriers are at high risk for malignant ventricular arrhythmias and end-stage heart failure, with left ventricular ejection fraction <45% and sustained or nonsustained ventricular tachycardia as independent risk factors. High mortality and a poor prognosis are present from late adolescence. Genetic and cardiac screening is, therefore, advised from adolescence onwards.Background—The pathogenic phospholamban R14del mutation causes dilated and arrhythmogenic right ventricular cardiomyopathies and is associated with an increased risk of malignant ventricular arrhythmias and end-stage heart failure. We performed a multicentre study to evaluate mortality, cardiac disease outcome, and risk factors for malignant ventricular arrhythmias in a cohort of phospholamban R14del mutation carriers. Methods and Results—Using the family tree mortality ratio method in a cohort of 403 phospholamban R14del mutation carriers, we found a standardized mortality ratio of 1.7 (95% confidence interval, 1.4–2.0) with significant excess mortality starting from the age of 25 years. Cardiological data were available for 295 carriers. In a median follow-up period of 42 months, 55 (19%) individuals had a first episode of malignant ventricular arrhythmias and 33 (11%) had an end-stage heart failure event. The youngest age at which a malignant ventricular arrhythmia occurred was 20 years, whereas for an end-stage heart failure event this was 31 years. Independent risk factors for malignant ventricular arrhythmias were left ventricular ejection fraction <45% and sustained or nonsustained ventricular tachycardia with hazard ratios of 4.0 (95% confidence interval, 1.9–8.1) and 2.6 (95% confidence interval, 1.5–4.5), respectively. Conclusions—Phospholamban R14del mutation carriers are at high risk for malignant ventricular arrhythmias and end-stage heart failure, with left ventricular ejection fraction <45% and sustained or nonsustained ventricular tachycardia as independent risk factors. High mortality and a poor prognosis are present from late adolescence. Genetic and cardiac screening is, therefore, advised from adolescence onwards.

The ARVD/C Genetic Variants Database: 2014 Update

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by myocardial atrophy, fibro‐fatty replacement, and a high risk of ventricular arrhythmias that lead to sudden death. In 2009, genetic data from 57 publications were collected in the arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) Genetic Variants Database (freeware available at http://www.arvcdatabase.info), which comprised 481 variants in eight ACM‐associated genes. In recent years, deep genetic sequencing has increased our knowledge of the genetics of ACM, revealing a large spectrum of nucleotide variations for which pathogenicity needs to be assessed. As of April 20, 2014, we have updated the ARVD/C database into the ARVD/C database to contain more than 1,400 variants in 12 ACM‐related genes (PKP2, DSP, DSC2, DSG2, JUP, TGFB3, TMEM43, LMNA, DES, TTN, PLN, CTNNA3) as reported in more than 160 references. Of these, only 411 nucleotide variants have been reported as pathogenic, whereas the significance of the other approximately 1,000 variants is still unknown. This comprehensive collection of ACM genetic data represents a valuable source of information on the spectrum of ACM‐associated genes and aims to facilitate the interpretation of genetic data and genetic counseling.

Cardiovascular malformations caused by NOTCH1 mutations do not keep left: data on 428 probands with left-sided CHD and their families

Purpose:We aimed to determine the prevalence and phenotypic spectrum of NOTCH1 mutations in left-sided congenital heart disease (LS-CHD). LS-CHD includes aortic valve stenosis, a bicuspid aortic valve, coarctation of the aorta, and hypoplastic left heart syndrome.Methods:NOTCH1 was screened for mutations in 428 nonsyndromic probands with LS-CHD, and family histories were obtained for all. When a mutation was detected, relatives were also tested.Results:In 148/428 patients (35%), LS-CHD was familial. Fourteen mutations (3%; 5 RNA splicing mutations, 8 truncating mutations, 1 whole-gene deletion) were detected, 11 in familial disease (11/148 (7%)) and 3 in sporadic disease (3/280 (1%)). Forty-nine additional mutation carriers were identified among the 14 families, of whom 12 (25%) were asymptomatic. Most of these mutation carriers had LS-CHD, but 9 (18%) had right-sided congenital heart disease (RS-CHD) or conotruncal heart disease (CTD). Thoracic aortic aneurysms (TAAs) occurred in 6 mutation carriers (probands included 6/63 (10%)).Conclusion:Pathogenic mutations in NOTCH1 were identified in 7% of familial LS-CHD and in 1% of sporadic LS-CHD. The penetrance is high; a cardiovascular malformation was found in 75% of NOTCH1 mutation carriers. The phenotypic spectrum includes LS-CHD, RS-CHD, CTD, and TAA. Testing NOTCH1 for an early diagnosis in LS-CHD/RS-CHD/CTD/TAA is warranted.Genet Med 18 9, 914–923.

Left-dominant arrhythmogenic cardiomyopathy in a large family: associated desmosomal or nondesmosomal genotype?

BACKGROUND Arrhythmogenic cardiomyopathy (AC) is considered a predominantly right ventricular (RV) desmosomal disease. However, left-dominant forms due to desmosomal gene mutations, including PKP2 variant c.419C>T, have been described. Recently, a nondesmosomal phospholamban (PLN) mutation (c.40_42delAGA) has been identified, causing dilated cardiomyopathy and arrhythmias. OBJECTIVE To gain more insight into pathogenicity of the PKP2 variant c.419C>T by cosegregation analysis of the PKP2 variant c.419C>T vs the PLN mutation c.40_42delAGA. METHODS A Dutch family (13 family members, median age 49 years, range 34-71 years) with ventricular tachycardia underwent (1) meticulous phenotypic characterization and (2) screening of 5 desmosomal genes (PKP2, DSC2, DSG2, DSP, JUP) and PLN. RESULTS Six family members fulfilled 2010 AC Task Force Criteria. Seven had signs of left ventricular (LV) involvement (inverted T waves in leads V4-V6, LV wall motion abnormalities and late enhancement, and reduced LV ejection fraction), including 6 family members with proven AC. The PKP2 variant c.419C>T was found as a single variant in 3 family members, combined with the PLN mutation c.40_42delAGA in 3 others. PLN mutation was found in 9 family members, including the 6 with AC and all 7 with LV involvement. The PLN mutation c.40_42delAGA was found as a single mutation in 6, combined with the PKP2 variant c.419C>T in 3 others. A low-voltage electrocardiogram was seen in 4 of 9 PLN mutation-positive subjects. None of the family members with the single PKP2 variant showed any sign of RV or LV involvement. CONCLUSIONS The PLN mutation c.40_42delAGA cosegregates with AC and with electrocardiographic and structural LV abnormalities. In this family, there was no evidence of disease-causing contribution of the PKP2 variant c.419C>T.

Insights from a Desmoplakin Mutation Identified in Lethal Acantholytic Epidermolysis Bullosa

By linking tension-bearing intermediate filaments (IFs) to sites of robust cell-cell adhesion at desmosomes, desmoplakin (DP) has an essential role in maintaining integrity of tissues that experience mechanical stress such as skin and heart (Green and Simpson, 2007). Two DP isoforms are present in epithelia, resulting from alternative splicing of one gene. The C-terminal IF-binding domain in each isoform is identical, containing three plakin-repeat domains (A, B, and C), separated by flexible linker regions, and followed by a 68-residue tail involved in regulating DP-IF specificity. These motifs cooperate to anchor IFs to desmosomes (Stappenbeck et al., 1993; Meng et al., 1997; Choi et al., 2002; Fontao et al., 2003). Over 50 mutations have been identified throughout the desmoplakin gene (DSP) associated with autosomal-dominant or -recessive disorders affecting the skin, heart, hair, and nails (http://www.arvcdatabase.info; Bolling and Jonkman, 2009). Lethal acantholytic epidermolysis bullosa (LAEB) is an autosomal-recessive disease of severe skin and mucosal fragility caused by premature DSP termination. In the first reported LAEB case, the resultant truncated protein lacked the IF-binding domain and consequently lost keratin anchorage at the desmosomal plaque (Jonkman et al., 2005). The second LAEB case reported a 5-base pair deletion causing frameshift and premature termination in exon 20. No tissue was available for immunofluorescence or biochemical analysis so the effect on protein function is unknown (Bolling et al., 2010). Here, we describe a previously unreported DSP mutation resulting in LAEB without apparent cardiac involvement. Biochemical analysis of patient keratinocytes (KCs) sheds light on possible compensatory mechanisms that may allow embryonic survival. A full-term girl, born to consanguineous parents, showed virtually total denudement of skin (Figure 1a) and mucosae, absent fingernails and toenails (Figure 1b), and total alopecia (Figure 1c). Denuded sites re-epithelialized within days without residual scarring, but blistering recurred rapidly. Cardiac echocardiograms and chest X-rays were normal, but the airway filled with extensive sloughed mucosa. Histological sections revealed suprabasal acantholysis or cell clusters in a single acantholytic basal cell layer (arrow, Figure 1d). Immunofluorescence microscopy revealed staining for all basement membrane zone markers, with plectin and cytokeratin staining a discontinuous single row of basal KCs adherent to the basement membrane. DP staining was absent (data not shown). Based on the poor prognosis, the parents withdrew support and the baby expired on day 26 from airway obstruction. Autopsy was refused. Figure 1 Clinical features of lethal acantholytic epidermolysis bullosa (LAEB) and desmoplakin (DP) schematic highlighting sites of mutations After parental institutional review board-approved informed consent in adherence to the Declaration of Helsinki Principles, mutation screening was performed on genomic DNA isolated from peripheral blood. The complete genomic DNA coding sequence of DSP was amplified and directly sequenced as described (Whittock et al., 1999). The proband showed a homozygous single nucleotide deletion (DSP:c.7248delT) in exon 24, which was heterozygous in parental genomic DNA (data not shown). Consistent with a C-terminal mutation, an anti-DP C-terminal antibody (NW6), failed to reveal the punctate pattern characteristic of desmosome staining at cell-cell interfaces in cultured patient KCs (Figure 2a). Surprisingly, an N-terminal antibody (NW161) only rarely detected faint DP punctae between cells (Figure 2a). While the desmosomal cadherin, desmoglein-2, was absent from intercellular borders (data not shown), E-cadherin, b-catenin, and plakoglobin staining were better preserved, consistent with a less-pronounced effect on adherens junctions (Figure 2b). Figure 2 Immunostaining and biochemical analysis of lethal acantholytic epidermolysis bullosa (LAEB) keratinocytes (KCs) DP was undetectable in patient KC lysates, regardless of antibody domain specificity, without reduction of other desmosomal components (Figure 2c). The identified point mutation predicts truncation (p.Phe2416LeufsX14) within plakin-repeat domain B (Figure 1e). Although quantitative real-time RTPCR analysis indicated reduced DSP transcript levels in patient KCs compared to normal human epidermal KCs, the difference was not statistically significant (Figure 2e). These data support the idea that reduced DP expression in patient KCs is controlled either translationally or through degradation pathways, the latter that was proposed for a recent case of cutaneous disease without evidence of heart defects resulting from virtually complete loss of plakoglobin in the skin (Cabral et al., 2010). Interestingly, LAEB KCs exhibit reduced E-cadherin and elevated N-cadherin protein levels compared to multiple normal human epidermal KCs isolates of the same passage number (Figure 2d and Supplementary Figure S1 online). In addition, the extracellular matrix glycoprotein fibronectin was elevated in LAEB lysates (Figure 2d). Furthermore, plakoglobin and, to a lesser extent, β-catenin were observed in the nucleus of LAEB KCs (Figure 2b), where they may initiate transcription events upon the loss of DP expression (Garcia-Gras et al., 2006). We propose that elevated N-cadherin and fibronectin may facilitate re-epithelialization in the patients skin (Clark, 1990; Peinado et al., 2004), and further speculate that loss of DP might trigger a “cadherin switch,” a process that occurs during epithelial–mesenchymal transition (Nishimura and Takeichi, 2009), but has not previously been linked with desmosome molecules. Although it was not possible to assess whether similar elevation of N-cadherin occurred in cardiac tissue, compensation by this intercalated disc protein might help counter any potential loss of DP in the heart. A consistent genotype-phenotype correlation linking DSP mutations to disease has not yet emerged. DP truncations and/or point mutations may variably cause cardiac and/or skin syndromes (Supplementary Table S1 online). In the cases of LAEB, severe skin fragility is observed when mutations cause complete loss of DP C-terminal expression. However, less severe cases of skin fragility/woolly hair and palmoplantar keratoderma, with or without cardiomyopathy, have been reported in patients when (1) at least one copy of full-length DP I is expressed, but with a point mutation (Whittock et al., 2002; Alcalai et al., 2003; Mahoney et al., 2010); (2) DP I expression is lost, but at least one copy of full-length DP II is expressed (Uzumcu et al., 2006; Asimaki et al., 2009); or (3) homozygous DP C-terminus truncation occurs downstream of plakin-repeat domain B (Norgett et al., 2000). Thus, it seems likely that presence of at least one full-length copy of a DP isoform, whether DP I or DP II, may keep patients from developing early lethality as seen in LAEB. In contrast, near-complete loss of DP (described herein) would be predicted to result in severe impairment of both epidermal and cardiac integrity (Gallicano et al., 2001). In utero survival and the lack of observed cardiac involvement in the patient of this study could reflect compensatory mechanisms that stabilized adhesion, re-epithelialization, and cardiac integrity.

The TMEM43 Newfoundland mutation p.S358L causing ARVC-5 was imported from Europe and increases the stiffness of the cell nucleus

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic condition caused predominantly by mutations within desmosomal genes. The mutation leading to ARVC-5 was recently identified on the island of Newfoundland and caused by the fully penetrant missense mutation p.S358L in TMEM43. Although TMEM43-p.S358L mutation carriers were also found in the USA, Germany, and Denmark, the genetic relationship between North American and European patients and the disease mechanism of this mutation remained to be clarified. METHODS AND RESULTS We screened 22 unrelated ARVC patients without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. CONCLUSION The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43-p.S358L founder mutation. Due to its deleterious clinical phenotype, this mutation should be checked in any case of ARVC-related genotyping. It appears that the increased stiffness of the cell nucleus might be related to the massive loss of cardiomyocytes, which is typically found in ventricles of ARVC hearts.

Biallelic Truncating Mutations in ALPK3 Cause Severe Pediatric Cardiomyopathy

BACKGROUND Cardiomyopathies are usually inherited and predominantly affect adults, but they can also present in childhood. Although our understanding of the molecular basis of pediatric cardiomyopathy has improved, the underlying mechanism remains elusive in a substantial proportion of cases. OBJECTIVES This study aimed to identify new genes involved in pediatric cardiomyopathy. METHODS The authors performed homozygosity mapping and whole-exome sequencing in 2 consanguineous families with idiopathic pediatric cardiomyopathy. Sixty unrelated patients with pediatric cardiomyopathy were subsequently screened for mutations in a candidate gene. First-degree relatives were submitted to cardiac screening and cascade genetic testing. Myocardial samples from 2 patients were processed for histological and immunohistochemical studies. RESULTS We identified 5 patients from 3 unrelated families with pediatric cardiomyopathy caused by homozygous truncating mutations in ALPK3, a gene encoding a nuclear kinase that plays an essential role in early differentiation of cardiomyocytes. All patients with biallelic mutations presented with severe hypertrophic and/or dilated cardiomyopathy in utero, at birth, or in early childhood. Three patients died from heart failure within the first week of life. Moreover, 2 of 10 (20%) heterozygous family members showed hypertrophic cardiomyopathy with an atypical distribution of hypertrophy. Deficiency of alpha-kinase 3 has previously been associated with features of both hypertrophic and dilated cardiomyopathy in mice. Consistent with studies in knockout mice, we provide microscopic evidence for intercalated disc remodeling. CONCLUSIONS Biallelic truncating mutations in the newly identified gene ALPK3 give rise to severe, early-onset cardiomyopathy in humans. Our findings highlight the importance of transcription factor pathways in the molecular mechanisms underlying human cardiomyopathies.