Publications by authors named "Ronald H Lekanne Deprez"

28 Publications

  • Page 1 of 1

A mutation update for the FLNC gene in myopathies and cardiomyopathies.

Hum Mutat 2020 Jun 20;41(6):1091-1111. Epub 2020 Mar 20.

Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands.

Filamin C (FLNC) variants are associated with cardiac and muscular phenotypes. Originally, FLNC variants were described in myofibrillar myopathy (MFM) patients. Later, high-throughput screening in cardiomyopathy cohorts determined a prominent role for FLNC in isolated hypertrophic and dilated cardiomyopathies (HCM and DCM). FLNC variants are now among the more prevalent causes of genetic DCM. FLNC-associated DCM is associated with a malignant clinical course and a high risk of sudden cardiac death. The clinical spectrum of FLNC suggests different pathomechanisms related to variant types and their location in the gene. The appropriate functioning of FLNC is crucial for structural integrity and cell signaling of the sarcomere. The secondary protein structure of FLNC is critical to ensure this function. Truncating variants with subsequent haploinsufficiency are associated with DCM and cardiac arrhythmias. Interference with the dimerization and folding of the protein leads to aggregate formation detrimental for muscle function, as found in HCM and MFM. Variants associated with HCM are predominantly missense variants, which cluster in the ROD2 domain. This domain is important for binding to the sarcomere and to ensure appropriate cell signaling. We here review FLNC genotype-phenotype correlations based on available evidence.
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http://dx.doi.org/10.1002/humu.24004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318287PMC
June 2020

Dutch genome diagnostic laboratories accelerated and improved variant interpretation and increased accuracy by sharing data.

Hum Mutat 2019 12 3;40(12):2230-2238. Epub 2019 Sep 3.

Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.
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http://dx.doi.org/10.1002/humu.23896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900155PMC
December 2019

Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmosomal Variants Are Rarely De Novo.

Circ Genom Precis Med 2019 08 6;12(8):e002467. Epub 2019 Aug 6.

Amsterdam UMC, University of Amsterdam, Department of Clinical Genetics, the Netherlands (F.H.M.v.L., R.Z., R.H.L.D., J.P.v.T., C.A.J.).

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with pathogenic/likely pathogenic (P/LP) variants in genes encoding the cardiac desmosomal proteins. Origin of these variants, including de novo mutation rate and extent of founder versus recurrent variants has implications for variant adjudication and clinical care, yet this has never been systematically investigated.

Methods: We identified arrhythmogenic right ventricular cardiomyopathy probands who met 2010 Task Force Criteria and had undergone genotyping that included sequencing of the desmosomal genes (PKP2, DSP, DSG2, DSC2, and JUP) from 3 arrhythmogenic right ventricular cardiomyopathy registries in America and Europe. We classified the desmosomal variants, defined the contribution of unique versus nonunique (ie, not family-specific) P/LP variants, and identified the frequency and characteristics of de novo variants. Next, we haplotyped nonunique variants to determine how often they likely represent a single mutation event in a common ancestor (implied by shared haplotypes) versus multiple mutation events at the same genetic location.

Results: Of 501 arrhythmogenic right ventricular cardiomyopathy probands, 322 (64.3%) carried 327 desmosomal P/LP variants. Most variants (n=247, 75.6%, in 245 patients) were identified in more than one proband and, therefore, considered nonunique. For 212/327 variants (64.8%) genetic cascade screening was performed extensively enough to identify the parental origin of the P/LP variant. Only 3 variants were de novo, 2 of which were whole gene deletions. For 24 nonunique P/LP PKP2 variants, haplotyping was conducted in 183 available families. For all 24 variants, multiple seemingly unrelated families sharing identical haplotypes were identified, suggesting that these variants originate from common founders.

Conclusions: Most desmosomal P/LP variants are inherited, nonunique, and originate from ancient founders. Two of 3 de novo variants were large deletions. These observations inform genetic testing, cascade screening, and variant adjudication.
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http://dx.doi.org/10.1161/CIRCGEN.119.002467DOI Listing
August 2019

Mortality Risk Associated With Truncating Founder Mutations in Titin.

Circ Genom Precis Med 2019 05;12(5):e002436

Department of Genetics (M.J., A.F.B., A.S.U., H.K.P.v.A., E.A.M.H., D.D.), University Medical Center Utrecht, Utrecht University, the Netherlands.

Background Truncating titin variants (TTNtv) are the most prevalent genetic cause of dilated cardiomyopathy, found in ≤25% of familial cases. Moreover, TTNtv associated with dilated cardiomyopathy are estimated to be present in 0.5% of the general population. The prognosis of asymptomatic carriers of TTNtv is poorly understood because TTNtv are associated with a highly variable phenotype. We aim to assess the natural history and clinical relevance of TTNtv by analyzing standardized mortality ratios (SMR) in multigenerational pedigrees and in close relatives of present-day patients. Methods Haplotype and genealogical analyses were performed on 3 recurrent TTNtv. Subsequently, the family tree mortality ratio method was used to compare all-cause mortality of subjects at an a priori 50% risk of carrying TTNtv to the general Dutch population. SMRs were stratified for sex, age, and calendar period. Subgroups were compared with Poisson regression. Similarly, SMRs were calculated in parents of 128 present-day dilated cardiomyopathy probands with TTNtv using the reverse parent-offspring method. Results The TTNtv were established as founder mutations and traced to 18th century ancestors. In 20 522 person-years, overall mortality was not significantly increased (SMR, 1.06; 95% CI, 0.95-1.18; P=0.162). However, mortality was significantly increased in subjects living after 1965 (SMR, 1.27; 95% CI, 1.04-1.53; P=0.009) and aged ≥60 years (SMR, 1.17; 95% CI, 1.01-1.35; P=0.02). The reverse parent-offspring analysis showed overall excess mortality (SMR, 1.26; 95% CI, 1.07-1.48; P=0.003), driven by subjects aged ≥60 years. Conclusions The natural history of the analyzed TTNtv shows a relatively mild disease course with significant excess mortality in elderly patients. With increasing life expectancy, TTNtv-associated morbidity and mortality will likely become more prevalent.
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http://dx.doi.org/10.1161/CIRCGEN.118.002436DOI Listing
May 2019

Relevance of Titin Missense and Non-Frameshifting Insertions/Deletions Variants in Dilated Cardiomyopathy.

Sci Rep 2019 03 11;9(1):4093. Epub 2019 Mar 11.

Blueprint Genetics, Helsinki, Finland.

Recent advancements in next generation sequencing (NGS) technology have led to the identification of the giant sarcomere gene, titin (TTN), as a major human disease gene. Truncating variants of TTN (TTNtv) especially in the A-band region account for 20% of dilated cardiomyopathy (DCM) cases. Much attention has been focused on assessment and interpretation of TTNtv in human disease; however, missense and non-frameshifting insertions/deletions (NFS-INDELs) are difficult to assess and interpret in clinical diagnostic workflow. Targeted sequencing covering all exons of TTN was performed on a cohort of 530 primary DCM patients from three cardiogenetic centres across Europe. Using stringent bioinformatic filtering, twenty-nine and two rare TTN missense and NFS-INDELs variants predicted deleterious were identified in 6.98% and 0.38% of DCM patients, respectively. However, when compared with those identified in the largest available reference population database, no significant enrichment of such variants was identified in DCM patients. Moreover, DCM patients and reference individuals had comparable frequencies of splice-region missense variants with predicted splicing alteration. DCM patients and reference populations had comparable frequencies of rare predicted deleterious TTN missense variants including splice-region missense variants suggesting that these variants are not independently causative for DCM. Hence, these variants should be classified as likely benign in the clinical diagnostic workflow, although a modifier effect cannot be excluded at this stage.
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http://dx.doi.org/10.1038/s41598-019-39911-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412046PMC
March 2019

Lack of evidence for a causal role of CALR3 in monogenic cardiomyopathy.

Eur J Hum Genet 2018 11 9;26(11):1603-1610. Epub 2018 Jul 9.

Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.

The pathogenicity of previously published disease-associated genes and variants is sometimes questionable. Large-scale, population-based sequencing studies have uncovered numerous false assignments of pathogenicity. Misinterpretation of sequence variants may have serious implications for the patients and families involved, as genetic test results are increasingly being used in medical decision making. In this study, we assessed the role of the calreticulin-3 gene (CALR3) in cardiomyopathy. CALR3 has been included in several cardiomyopathy gene panels worldwide. Its inclusion is based on a single publication describing two missense variants in patients with hypertrophic cardiomyopathy. In our national cardiomyopathy cohort (n = 6154), we identified 17 unique, rare heterozygous CALR3 variants in 48 probands. Overall, our patient cohort contained a significantly higher number of rare CALR3 variants compared to the ExAC population (p = 0.0036). However, after removing a potential Dutch founder variant, no statistically significant difference was found (p = 0.89). In nine probands, the CALR3 variant was accompanied by a disease-causing variant in another, well-known cardiomyopathy gene. In three families, the CALR3 variant did not segregate with the disease. Furthermore, we could not demonstrate calreticulin-3 protein expression in myocardial tissues at various ages. On the basis of these findings, it seems highly questionable that variants in CALR3 are a monogenic cause of cardiomyopathy.
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http://dx.doi.org/10.1038/s41431-018-0208-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189092PMC
November 2018

Yield and Pitfalls of Ajmaline Testing in the Evaluation of Unexplained Cardiac Arrest and Sudden Unexplained Death: Single-Center Experience With 482 Families.

JACC Clin Electrophysiol 2017 12 28;3(12):1400-1408. Epub 2017 Jun 28.

Heart Center, Department of Clinical and Experimental Cardiology, Academic Medical Center, Amsterdam, the Netherlands. Electronic address:

Objectives: This study evaluated the yield of ajmaline testing and assessed the occurrence of confounding responses in a large cohort of families with unexplained cardiac arrest (UCA) or sudden unexplained death (SUD).

Background: Ajmaline testing to diagnose Brugada syndrome (BrS) is routinely used in the evaluation of SUD and UCA, but its yield, limitations, and appropriate dosing have not been studied in a large cohort.

Methods: We assessed ajmaline test response and genetic testing results in 637 individuals from 482 families who underwent ajmaline testing for SUD or UCA.

Results: Overall, 89 individuals (14%) from 88 families (18%) had a positive ajmaline test result. SCN5A mutations were identified in 9 of 86 ajmaline-positive cases (10%). SCN5A mutation carriers had positive test results at significantly lower ajmaline doses than noncarriers (0.75 [range: 0.64 to 0.98] mg/kg vs. 1.03 [range: 0.95 to 1.14] mg/kg, respectively; p < 0.01). In 7 of 88 families (8%), it was concluded that the positive ajmaline response was a confounder, either in the presence of an alternative genetic diagnosis accounting for UCA/SUD (5 cases) or noncosegregation of positive ajmaline response and arrhythmia (2 cases). The rate of confounding responses was significantly higher in positive ajmaline responses obtained at >1 mg/kg than in those obtained at ≤1 mg/kg (7 of 48 vs. 0 of 41 individuals; Fisher's exact test: p = 0.014).

Conclusions: In line with previous, smaller studies, a positive ajmaline response was observed in a large proportion of UCA/SUD families. Importantly, our data emphasize the potential for confounding possibly false-positive ajmaline responses in this population, particularly at high doses, which could possibly lead to a misdiagnosis. Clinicians should consider all alternative causes in UCA/SUD and avoid ajmaline doses >1 mg/kg.
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http://dx.doi.org/10.1016/j.jacep.2017.04.005DOI Listing
December 2017

Genetics, Clinical Features, and Long-Term Outcome of Noncompaction Cardiomyopathy.

J Am Coll Cardiol 2018 02;71(7):711-722

Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands. Electronic address:

Background: The clinical outcomes of noncompaction cardiomyopathy (NCCM) range from asymptomatic to heart failure, arrhythmias, and sudden cardiac death. Genetics play an important role in NCCM.

Objectives: This study investigated the correlations among genetics, clinical features, and outcomes in adults and children diagnosed with NCCM.

Methods: A retrospective multicenter study from 4 cardiogenetic centers in the Netherlands classified 327 unrelated NCCM patients into 3 categories: 1) genetic, with a mutation in 32% (81 adults; 23 children) of patients; 2) probably genetic, familial cardiomyopathy without a mutation in 16% (45 adults; 8 children) of patients; or 3) sporadic, no family history, without mutation in 52% (149 adults; 21 children) of patients. Clinical features and major adverse cardiac events (MACE) during follow-up were compared across the children and adults.

Results: MYH7, MYBPC3, and TTN mutations were the most common mutations (71%) found in genetic NCCM. The risk of having reduced left ventricular (LV) systolic dysfunction was higher for genetic patients compared with the probably genetic and sporadic cases (p = 0.024), with the highest risk in patients with multiple mutations and TTN mutations. Mutations were more frequent in children (p = 0.04) and were associated with MACE (p = 0.025). Adults were more likely to have sporadic NCCM. High risk for cardiac events in children and adults was related to LV systolic dysfunction in mutation carriers, but not in sporadic cases. Patients with MYH7 mutations had low risk for MACE (p = 0.03).

Conclusions: NCCM is a heterogeneous condition, and genetic stratification has a role in clinical care. Distinguishing genetic from nongenetic NCCM complements prediction of outcome and may lead to management and follow-up tailored to genetic status.
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http://dx.doi.org/10.1016/j.jacc.2017.12.019DOI Listing
February 2018

Truncating titin mutations are associated with a mild and treatable form of dilated cardiomyopathy.

Eur J Heart Fail 2017 04 3;19(4):512-521. Epub 2016 Nov 3.

AMC Heart Center, Department of Clinical and Experimental Cardiology, Academic Medical Center Amsterdam, University of Amsterdam, Amsterdam, The Netherlands.

Aims: Truncating titin mutations (tTTN) occur in 25% of dilated cardiomyopathy (DCM) cases, but the phenotype and severity of disease they cause have not yet been systematically studied. We studied whether tTTN variants are associated with a clinically distinguishable form of DCM.

Methods And Results: We compared clinical data on DCM probands and relatives with a tTTN mutation (n = 45, n = 73), LMNA mutation (n = 28, n = 29), and probands who tested negative for both genes [idiopathic DCM (iDCM); n = 60]. Median follow-up was at least 2.5 years in each group. TTN subjects presented with DCM at higher age than LMNA subjects (probands 47.9 vs. 40.4 years, P = 0.004; relatives 59.8 vs. 47.0 years, P = 0.01), less often developed LVEF <35% [probands hazard ratio (HR) 0.38, P = 0.002], had higher age of death (probands 70.4 vs. 59.4 years, P < 0.001; relatives 74.1 vs. 58.4 years, P = 0.008), and had better composite outcome (malignant ventricular arrhythmia, heart transplantation, or death; probands HR 0.09, P < 0.001; relatives HR 0.21, P = 0.02) than LMNA subjects and iDCM subjects (HR 0.36, P = 0.07). An LVEF increase of at least 10% occurred in 46.9% of TTN subjects after initiation of standard heart failure treatment, while this only occurred in 6.5% of LMNA subjects (P < 0.001) and 18.5% of iDCM subjects (P = 0.02). This was confirmed in families with co-segregation, in which the 10% point LVEF increase occurred in 55.6% of subjects (P = 0.003 vs. LMNA, P = 0.079 vs. iDCM).

Conclusions: This study shows that tTTN-associated DCM is less severe at presentation and more amenable to standard therapy than LMNA mutation-induced DCM or iDCM.
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http://dx.doi.org/10.1002/ejhf.673DOI Listing
April 2017

Targeted sequence capture and GS-FLX Titanium sequencing of 23 hypertrophic and dilated cardiomyopathy genes: implementation into diagnostics.

J Med Genet 2013 Sep 19;50(9):614-26. Epub 2013 Jun 19.

Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands.

Background: Genetic evaluation of cardiomyopathies poses a challenge. Multiple genes are involved but no clear genotype-phenotype correlations have been found so far. In the past, genetic evaluation for hypertrophic (HCM) and dilated (DCM) cardiomyopathies was performed by sequential screening of a very limited number of genes. Recent developments in sequencing have increased the throughput, enabling simultaneous screening of multiple genes for multiple patients in a single sequencing run.

Objective: Development and implementation of a next generation sequencing (NGS) based genetic test as replacement for Sanger sequencing.

Methods And Results: In order to increase the number of genes that can be screened in a shorter time period, we enriched all exons of 23 of the most relevant HCM and DCM related genes using on-array multiplexed sequence capture followed by massively parallel pyrosequencing on the GS-FLX Titanium. After optimisation of array based sequence capture it was feasible to reliably detect a large panel of known and unknown variants in HCM and DCM patients, whereby the unknown variants could be confirmed by Sanger sequencing.

Conclusions: The rate of detection of (pathogenic) variants in both HCM and DCM patients was increased due to a larger number of genes studied. Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing. Therefore, NGS is ready for implementation in a diagnostic setting.
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http://dx.doi.org/10.1136/jmedgenet-2012-101231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756457PMC
September 2013

Best practice guidelines for the use of next-generation sequencing applications in genome diagnostics: a national collaborative study of Dutch genome diagnostic laboratories.

Hum Mutat 2013 Oct 19;34(10):1313-21. Epub 2013 Aug 19.

Department of Clinical Genetics, VU University Medical Centre, Amsterdam, The Netherlands.

Next-generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS-based tests according to diagnostic standards is a challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of "diagnostic yield" as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete "diagnostic routing." We also propose that laboratories should use a list of "core disease genes" for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013.
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http://dx.doi.org/10.1002/humu.22368DOI Listing
October 2013

Genetic analysis in 418 index patients with idiopathic dilated cardiomyopathy: overview of 10 years' experience.

Eur J Heart Fail 2013 Jun 24;15(6):628-36. Epub 2013 Jan 24.

Department of Genetics, University of Groningen, University Medical Centre Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands.

Aims: With more than 40 dilated cardiomyopathy (DCM)-related genes known, genetic analysis of patients with idiopathic DCM is costly and time-consuming. We describe the yield from genetic analysis in DCM patients in a large Dutch cohort.

Methods And Results: We collected cardiological and neurological evaluations, family screenings, and genetic analyses for 418 index patients with idiopathic DCM. We identified 35 (putative) pathogenic mutations in 82 index patients (20%). The type of DCM influenced the yield, with mutations found in 25% of familial DCM cases, compared with 8% of sporadic DCM cases and 62% of cases where DCM was accompanied by neuromuscular disease. A PLN founder mutation (43 cases) and LMNA mutations (19 cases, 16 different mutations) were most prevalent and often demonstrated a specific phenotype. Other mutations were found in: MYH7, DES, TNNT2, DMD, TPM1, DMPK, SCN5A, SGCB (homozygous), and TNNI3. After a median follow-up of 40 months, the combined outcome of death from any cause, heart transplantation, or malignant ventricular arrhythmias in patients with a mutation was worse than in those without an identified mutation (hazard ratio 2.0, 95% confidence interval 1.4-3.0). This seems to be mainly attributable to a high prevalence of malignant ventricular arrhythmias and end-stage heart failure in LMNA and PLN mutation carriers.

Conclusion: The yield of identified mutations in DCM index patients with clinical clues, such as associated neuromuscular disease or familial occurrence, is higher compared with those without these clues. For sporadic DCM, specific clinical characteristics may be used to select cases for DNA analysis.
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http://dx.doi.org/10.1093/eurjhf/hft013DOI Listing
June 2013

A novel alpha-tropomyosin mutation associates with dilated and non-compaction cardiomyopathy and diminishes actin binding.

Biochim Biophys Acta 2013 Apr 9;1833(4):833-9. Epub 2012 Nov 9.

Department of Anatomy, Embryology & Physiology, Academic Medical Center, Amsterdam, The Netherlands.

Background: Dilated cardiomyopathy (DCM) is characterized by idiopathic dilatation and systolic contractile dysfunction of the ventricle(s) leading to an impaired systolic function. The origin of DCM is heterogeneous, but genetic transmission of the disease accounts for up to 50% of the cases. Mutations in alpha-tropomyosin (TPM1), a thin filament protein involved in structural and regulatory roles in muscle cells, are associated with hypertrophic cardiomyopathy (HCM) and very rarely with DCM.

Methods And Results: Here we present a large four-generation family in which DCM is inherited as an autosomal dominant trait. Six family members have a cardiomyopathy with the age of diagnosis ranging from 5 months to 52 years. The youngest affected was diagnosed with dilated and non-compaction cardiomyopathy (NCCM) and died at the age of five. Three additional children died young of suspected heart problems. We mapped the phenotype to chromosome 15 and subsequently identified a missense mutation in TPM1, resulting in a p.D84N amino acid substitution. In addition we sequenced 23 HCM/DCM genes using next generation sequencing. The TPM1 p.D84N was the only mutation identified. The mutation co-segregates with all clinically affected family members and significantly weakens the binding of tropomyosin to actin by 25%.

Conclusions: We show that a mutation in TPM1 is associated with DCM and a lethal, early onset form of NCCM, probably as a result of diminished actin binding caused by weakened charge-charge interactions. Consequently, the screening of TPM1 in patients and families with DCM and/or (severe, early onset forms of) NCCM is warranted. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
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http://dx.doi.org/10.1016/j.bbamcr.2012.11.003DOI Listing
April 2013

A complex double deletion in LMNA underlies progressive cardiac conduction disease, atrial arrhythmias, and sudden death.

Circ Cardiovasc Genet 2011 Jun 15;4(3):280-7. Epub 2011 Mar 15.

Heart Failure Research Center, Department of Experimental Cardiology, Amsterdam, The Netherlands.

Background: Cardiac conduction disease is a clinically and genetically heterogeneous disorder characterized by defects in electrical impulse generation and conduction and is associated with sudden cardiac death.

Methods And Results: We studied a 4-generation family with autosomal dominant progressive cardiac conduction disease, including atrioventricular conduction block and sinus bradycardia, atrial arrhythmias, and sudden death. Genome-wide linkage analysis mapped the disease locus to chromosome 1p22-q21. Multiplex ligation-dependent probe amplification analysis of the LMNA gene, which encodes the nuclear-envelope protein lamin A/C, revealed a novel gene rearrangement involving a 24-bp inversion flanked by a 3.8-kb deletion upstream and a 7.8-kb deletion downstream. The presence of short inverted sequence homologies at the breakpoint junctions suggested a mutational event involving serial replication slippage in trans during DNA replication.

Conclusions: We identified for the first time a complex LMNA gene rearrangement involving a double deletion in a 4-generation Dutch family with progressive conduction system disease. Our findings underscore the fact that if conventional polymerase chain reaction-based direct sequencing approaches for LMNA analysis are negative in suggestive pedigrees, mutation detection techniques capable of detecting gross genomic lesions involving deletions and insertions should be considered.
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http://dx.doi.org/10.1161/CIRCGENETICS.110.959221DOI Listing
June 2011

Clinical utility gene card for: hypertrophic cardiomyopathy (type 1-14).

Eur J Hum Genet 2011 Aug 26;19(8). Epub 2011 Jan 26.

Heart Failure Research Centre, Department of Cardiology, Academic Medical Centre, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1038/ejhg.2010.243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172916PMC
August 2011

A gain-of-function TBX5 mutation is associated with atypical Holt-Oram syndrome and paroxysmal atrial fibrillation.

Circ Res 2008 Jun 1;102(11):1433-42. Epub 2008 May 1.

Heart Failure Research Center, L2-108-1, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

Holt-Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 (TBX5) underlie this syndrome. Here, we describe a large atypical HOS family in which affected patients have mild skeletal deformations and paroxysmal atrial fibrillation, but few have congenital heart disease. Sequencing of TBX5 revealed a novel mutation, c.373G>A, resulting in the missense mutation p.Gly125Arg, in all investigated affected family members, cosegregating with the disease. We demonstrate that the mutation results in normal Nkx2-5 interaction, is correctly targeted to the nucleus, has significantly enhanced DNA binding and activation of both the Nppa(Anf) and Cx40 promoter, and significantly augments expression of Nppa, Cx40, Kcnj2, and Tbx3 in comparison with wild-type TBX5. Thus, contrary to previously published HOS mutations, the p.G125R TBX5 mutation results in a gain-of-function. We speculate that the gain-of-function mechanism underlies the mild skeletal phenotype and paroxysmal atrial fibrillation and suggest a possible role of TBX5 in the development of (paroxysmal) atrial fibrillation based on a gain-of-function either through a direct stimulation of target genes via TBX5 or indirectly via TBX5 stimulated TBX3. These findings may warrant a renewed look at the phenotypes of families and individuals hitherto not classified as HOS or as atypical but presenting with paroxysmal atrial fibrillation, because these may possibly be the result of additional TBX5 gain-of-function mutations.
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http://dx.doi.org/10.1161/CIRCRESAHA.107.168294DOI Listing
June 2008

Increased cardiac workload by closure of the ductus arteriosus leads to hypertrophy and apoptosis rather than to hyperplasia in the late fetal period.

Naunyn Schmiedebergs Arch Pharmacol 2004 Sep 31;370(3):193-202. Epub 2004 Aug 31.

Experimental and Molecular Cardiology Group, Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

It is generally thought that adult mammalian cardiomyocytes compensate for an increased workload by hypertrophy, whereas fetal myocardium grows by cellular proliferation. We analyzed the response of late-fetal rat hearts upon an increased workload imposed by premature constriction of the ductus arteriosus with indomethacin. Initially the fetal heart responds by proliferative growth, as both wet weight and labeling index (bromodeoxyuridine incorporation) of the ventricles increased, whereas neither a change in the fibroblast fraction, ploidy and nucleation in the ventricles is observed. However, this hyperplastic growth is abrogated by a subsequent burst in apoptosis and followed by a hypertrophic response as based on a decrease in DNA and increase in both RNA and protein concentration. This hypertrophic growth was accompanied by a 1.4-fold increase in the volume of the cardiomyocytes. Changes in the molecular phenotype characteristic of pressure-overload hypertrophic growth accompany the process. Thus, the levels of expression of beta-myosin heavy chain and atrial natriuretic factor mRNA increased, of sarcoplasmic/endoplasmic reticulum ATPase (SERCA2) mRNA decreased, and of alpha-myosin heavy chain, phospholamban, and calsequestrin mRNA did not change. In situ hybridization showed that the pattern of mRNA expression changed first in the right ventricular wall and subsequently in the left ventricular free wall as well. It is concluded that pressure-overload imposed on the late-fetal heart induces limited proliferative growth complemented by extensive hypertrophic growth.
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http://dx.doi.org/10.1007/s00210-004-0955-0DOI Listing
September 2004

Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium.

J Mol Cell Cardiol 2003 Dec;35(12):1461-72

Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Mouse embryonic stem (ES) cells easily differentiate towards the cardiac lineage making them suitable as an in vitro model to study cardiogenesis and as a potential source of transplantable cells. In this study, we show by in situ hybridisation that about 30% of the volume of cultures of differentiating ES cells consists of cardiomyocytes. RT-PCR analyses showed that the transcription factors Nkx2.5, Gata4, Mef2c and Irx4 were expressed at levels in the same order of magnitude as the levels observed in embryonic, neonatal and adult hearts. Atrial natriuretic factor and Connexin 40, associated with chamber formation in vivo, are expressed at relatively low levels, similar to those observed at early heart development in vivo. To facilitate the isolation of ES cell-derived cardiomyocytes, a cell line was constructed by stable transfection of the aminoglycoside phosphotransferase cDNA driven by the cardiac-specific distant upstream part of the Na(+)/Ca(2+) exchanger promoter. To accomplish single-copy integration, the construct was inserted into the hypoxanthine phosphoribosyltransferase locus of HM1 ES cells by homologous recombination. Cardiac-specific resistance to G418-sulphate (neomycin) allowed isolation of a pure population of cardiomyocytes. Genetically selected and unselected cell populations were characterised electrophysiologically using patch clamp. To explore whether clusters of cells have a similar differentiation profile, action potentials (APs) were measured in aggregates of differentiating ES cells, using a new method based on the voltage-dependent fluorescent dye di-4-ANEPPS. Both whole-cell recordings using patch-clamp and optical measurements with di-4-ANEPPS of the AP showed that upstroke velocity increases and AP duration decreases with differentiation time, accompanied by a decrease in AP interval, suggesting the initiation of the developmental programme underlying the formation of chamber myocardium.
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http://dx.doi.org/10.1016/j.yjmcc.2003.09.011DOI Listing
December 2003

TBX5 overexpression stimulates differentiation of chamber myocardium in P19C16 embryonic carcinoma cells.

J Muscle Res Cell Motil 2003 ;24(2-3):211-8

Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

In vitro differentiation of pluripotent embryonic cells is becoming a model system to study factors and genes involved in early developmental processes including cardiogenesis. An additional application involves the development of donor cells for treatment of diseases among which cardiac infarction. For this purpose differentiated cells should meet the functional characteristics of chamber myocardium, a requirement not convincingly reached as yet. The T-box transcription factor Tbx5 has been demonstrated to be crucial for heart formation. Using stably transfected clones of the P19C16 embryonic carcinoma cell line, reported to differentiate efficiently into the cardiac lineage, we investigated whether Tbx5 is sufficient to enhance cardiogenesis and differentiation of chamber myocardium. TBX5-transfected clones started to beat earlier, however, a relation between transgenic TBX5 mRNA levels and the number of beating foci or levels of Serca2a mRNA, a myocardial marker, could not be observed. However, TBX5-transfected clones displayed significantly higher levels of atrial natriuretic factor (Anf) and Connexin (Cx)40 mRNAs, which are associated with the formation of chamber myocardium. This indicates that Tbx5 enhances cardiac maturation within this system rather than cardiogenesis.
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http://dx.doi.org/10.1023/a:1026063409656DOI Listing
June 2004

Cardiac expression of Gal4 causes cardiomyopathy in a dose-dependent manner.

J Muscle Res Cell Motil 2003 ;24(2-3):205-9

Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Cardiac expression of a transgene is a common approach for determining the role of gene products in the processes underlying cardiomyopathy and heart failure (HF). We have generated transgenic mice that express the 'harmless' yeast transcription factor Gal4 in the heart under control of the alpha-myosin heavy chain promoter and found that expression of this gene causes cardiomyopathy and HF, the severity of which correlated with the number of copies of the transgene integrated into the genome and with the expression level. A line with a single copy of the transgene targeted to the hprt locus correctly expressed the transgene but did not develop cardiomyopathy. Our results indicate that expression of a transgene in the heart may non-specifically cause HF in a dose-dependent manner.
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http://dx.doi.org/10.1023/a:1026055612227DOI Listing
June 2004

Expression analysis of subtractively enriched libraries (EASEL): a widely applicable approach to the identification of differentially expressed genes.

J Biochem Biophys Methods 2003 Jul;57(1):17-33

Experimental and Molecular Cardiology Group, Cardiovascular Research Institute Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands.

A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.
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http://dx.doi.org/10.1016/s0165-022x(03)00083-6DOI Listing
July 2003

Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube.

Cardiovasc Res 2003 May;58(2):399-409

Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

Objective: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and quantitative analyses of cardiogenesis in vivo and in vitro.

Methods: Gene expression profiles were made by in situ hybridisation and real-time PCR and electrophysiological profiles by patch clamp analyses of cardiomyocytes derived from time series of differentiating HM1 mouse embryonic stem cells and from embryonic and adult mouse hearts.

Results: In embryoid bodies the in situ patterns of expression of alpha-myosin heavy chain, myosin light chain 2a and sarcoendoplasmic reticulum calcium ATPase 2a were similar to that of the heart muscle-specific marker gene cardiac troponin I. Myosin light chain 2v was expressed in part of the cardiac troponin I-expressing area, indicating heterogeneity within the cardiac cell population. Atrial natriuretic factor expression, indicative of the chamber-type program, could only very occasionally be detected by in situ hybridisation. Quantitative reverse transcriptase PCR showed that all cardiac genes, most notably atrial natriuretic factor, were expressed at relatively low levels, similar to those in embryonic hearts at embryonic day 8.75-9. Analysis of the electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes showed an increase of the upstroke velocity and a shorter duration of the action potential during prolonged differentiation in vitro. When embryonic mouse heart compartments of embryonic day 12.5 were used as a reference, the electrophysiological characteristics of a substantial part of the embryonic stem cell-derived cardiomyocytes were most reminiscent to those observed in the embryonic outflow tract.

Conclusion: Together, these data suggest that most cardiomyocytes acquired by differentiation of embryonic stem cells maintain a phenotype reminiscent of that of the cardiomyocytes of the primary heart tube, and hardly any myocytes develop a chamber myocardial phenotype.
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http://dx.doi.org/10.1016/s0008-6363(03)00282-7DOI Listing
May 2003

Development of heart muscle-cell diversity: a help or a hindrance for phenotyping embryonic stem cell-derived cardiomyocytes.

Cardiovasc Res 2003 May;58(2):303-12

Experimental & Molecular Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Despite the advances in cardiovascular treatment, cardiac disease remains a major cause of morbidity in all industrialized countries. The extraordinary potential of (embryonic) stem cells for therapeutic purposes has revolutionized ideas about cardiac repair of diseased cardiac muscle to exciting stages. This, in turn, has challenged research on cardiac differentiation of stem cells. For instance, cultures of mouse embryonic stem cells quite easily differentiate into the cardiogenic lineage, as assessed by their potential to beat spontaneously. However, repair of impaired cardiac muscle by spontaneously beating cardiac muscle cells might impose severe risks upon a human patient. Therefore, it is of crucial importance to understand the mechanisms that underlie the development of the distinct cardiac muscle cell types of the adult mammalian heart. In this review we tried to relate cardiac morphogenesis to the development of unique molecular phenotypes of cardiomyocytes. This relationship will provide a framework to assess the significance of the molecular phenotypes that are observed in embryonic stem cell-derived cardiomyocytes (ESDCs). Although for the phenotyping of ESDCs a comparison should be made with the phenotypes of the developing heart, so far none of the currently available markers allow unequivocal assignment of subtypes.
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http://dx.doi.org/10.1016/s0008-6363(03)00246-3DOI Listing
May 2003

Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data.

Neurosci Lett 2003 Mar;339(1):62-6

Department of Anatomy and Embryology K2-283, Experimental and Molecular Cardiology Group, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR efficiency for the amplicon of interest is constant or even, in the case of the comparative C(t) method, equal to 2. The latter method already leads to a 4-fold error when the PCR efficiencies vary over just a 0.04 range. PCR efficiencies of amplicons are usually calculated from standard curves based on either known RNA inputs or on dilution series of a reference cDNA sample. In this paper we show that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCR efficiencies for each sample. A computer program to perform this calculation is available on request (e-mail: bioinfo@amc.uva.nl; subject: LinRegPCR).
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http://dx.doi.org/10.1016/s0304-3940(02)01423-4DOI Listing
March 2003

Non-radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heart.

Microsc Res Tech 2002 Sep;58(5):387-94

Experimental and Molecular Cardiology Group, Academic Medical Center, Amsterdam, The Netherlands.

Non-radioactive in situ hybridisation is an excellent method to visualise mRNA molecules within their topographical context. Recently we have reported a new non-radioactive in situ hybridisation procedure on tissue sections that is essentially based on the whole mount in situ hybridisation procedure. This method is superior in spatial resolution and sensitivity compared to the radioactive in situ hybridisation procedure. Generally, low levels of gene expression, such as found with the developmental onset of gene expression and in differentiating embryonic stem cells, are difficult to detect by in situ hybridisation. Here an application of the protocol is presented which is based on tyramide signal amplification, which enables the detection of very low abundant mRNAs. The significance of this method is two-fold: (1) the molecular phenotype of embryonic stem cell-derived cardiomyocytes can be examined at the cellular level with high sensitivity, and (2) the number of cells that express the gene of interest can be assessed.
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http://dx.doi.org/10.1002/jemt.10154DOI Listing
September 2002

Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions.

Anal Biochem 2002 Aug;307(1):63-9

Experimental and Molecular Cardiology Group, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

The recent development of real-time PCR has offered the opportunity of sensitive and accurate quantification of mRNA levels that is crucial in biomedical research. Although reverse transcription (RT)-PCR is at present the most sensitive method available, many low abundant mRNAs are, although detectable, often not quantifiable. Here we report an improved two-step real-time RT-PCR procedure using SYBR green I and the LightCycler that better permits accurate quantification of mRNAs. Omission of dithiothreitol from the cDNA synthesis reaction was found to be crucial. This resulted in a lower cycle number at which the cDNA level is determined (C(T) value), steeper amplification curves, and removal of background fluorescence in the subsequent PCR. In addition, the choice of the cDNA priming oligo can improve detection sensitivity even further. In contrast to hexamer primer usage, both gene-specific and oligo-dT(VN) priming were very efficient and accurate, with gene-specific priming being the most sensitive. Finally, accurate quantification of mRNAs by real-time PCR using SYBR green I requires verification of the specificity of PCR by both melting curve and gel analysis.
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http://dx.doi.org/10.1016/s0003-2697(02)00021-0DOI Listing
August 2002