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Doubly Heterozygous LMNA and TTN Mutations Revealed by Exome Sequencing in a Severe Form of Dilated Cardiomyopathy

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Doubly Heterozygous LMNA and TTN Mutations Revealed by Exome Sequencing in a Severe Form of Dilated Cardiomyopathy European Journal of Human Genetics (2013) 21, 1105–1111 & 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13 www.nature.com/ejhg ARTICLE Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy Roberta Roncarati1,2, Chiara Viviani Anselmi2, Peter Krawitz3,4,5, Giovanna Lattanzi6, Yskert von Kodolitsch7, Andreas Perrot8,9, Elisa di Pasquale2,10, Laura Papa2, Paola Portararo2, Marta Columbaro6, Alberto Forni11, Giuseppe Faggian*,11, Gianluigi Condorelli*,1,12 and Peter N Robinson*,3,4,5 Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/ TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM. European Journal of Human Genetics (2013) 21, 1105–1111; doi:10.1038/ejhg.2013.16; published online 6 March 2013 Keywords: familial dilated cardiomyopathy; lamin A/C; titin; whole-exome sequencing; modifying variant INTRODUCTION family members, and by highly variable age of onset and rate of Dilated cardiomyopathy (DCM) is characterized by cardiac disease progression. The molecular correlates of these observations chamber enlargement and impaired contraction of the left ventricle; have remained largely unknown, which can make medical approximately 30–50% of affected individuals have a familial form management difficult. Recently, two or more sequence alterations of DCM. The genetics of familial DCM is complex and includes present either in the same or in different genes were demonstrated to over 30 disease-causing genes. With the exception of TTN, truncating occur in 3–5% of cases of familial HCM, associated with a greater mutations of which have been found in up to 27% of individuals clinical severity of HCM.6–8 Similarly, compound and digenic with DCM,1 a single DCM-causing gene accounts for no more than heterozygosity were identified in patients with arrhythmogenic right 6–8% of all cases. The known DCM disease genes include those ventricular cardiomyopathy, whereby several desmosome and cell- encoding for proteins of the sarcomere, the Z-disk, the cytoskeleton, junction genes were found to carry more than one mutation likely the mitochondria, RNA binding proteins, the sarcoplasmic reticulum, associated with low penetrance.9 It is to be expected that similar and the nuclear envelope.2–4 Familial DCM exhibits a remarkable modifiers exist for DCM; even though a single case of a 14-year-old degree of clinical variability with respect to severity, penetrance, boy with manifestations of DCM and mutations in both MYH7 and and age of onset.5 Like familial hypertrophic cardiomyopathy TNNT2 has been published,10 little is known about the genes and (HCM), familial DCM is often characterized by incomplete the molecular mechanisms involved in modifying the phenotype of penetrance, a high degree of variable expressivity even among familial DCM. 1Biomedical and Genetic Research Institute (IRGB), Milan Unit, National Research Council of Italy, Milan, Italy; 2Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Milan, Italy; 3Institute of Medical Genetics and Human Genetics, Charite´ Universita¨tsmedizin, Berlin, Germany; 4Berlin-Brandenburg Center for Regenerative Therapies, Charite´ Universita¨tsmedizin, Berlin, Germany; 5Max Planck Institute for Molecular Genetics, Berlin, Germany; 6National Research Council of Italy, Institute of Molecular Genetics, Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopedic Institute, Bologna, Italy; 7Centre of Cardiology and Cardiovascular Surgery, University Hospital Eppendorf, Hamburg, Germany; 8Department of Cardiology, Campus Virchow-Klinikum, Charite´ -Universita¨tsmedizin Berlin, Berlin, Germany; 9Experimental and Clinical Research Center (ECRC), Charite´ and the Max Delbru¨ ck Center for Molecular Medicine, Berlin, Germany; 10Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy; 11Division of Cardiac Surgery, University of Verona, Verona, Italy; 12Humanitas Clinical and Research Center, Milan, Italy *Correspondence: Professor G Faggian, Department of Cardiac Surgery, University of Verona, Piazzale Stefani 1, Verona 37126, Italy. Tel: þ 39 04 58121227; Fax: þ 39 04 58123308; E-mail: [email protected] or G Condorelli, Humanitas Clinical and Research Institute, Via Manzoni 56, Milan 20089, Italy. Tel: þ 39 02 82245201; E-mail: [email protected] or Professor PN Robinson, Institute of Medical Genetics and Human Genetics, Charite´ Universita¨tsmedizin, Augustenburger Platz 1 Berlin 13353, Germany. Tel: þ 49 30 450566006; Fax: þ 49 30 450569915; E-mail: [email protected] Received 11 September 2012; revised 20 December 2012; accepted 17 January 2013; published online 6 March 2013 LMNA/TTN mutations in severe DCM R Roncarati et al 1106 Whole-exome sequencing (WES), in which capture methods are Read mapping and variant analysis used to enrich the sequences of the coding regions of genes from Pair-end reads were quality trimmed and aligned with the human genome fragmented total genomic DNA, followed by massively parallel, ‘next- reference sequence (NCBI build 37/UCSC hg19) by using CLC Bio Genomics generation’ sequencing of the captured fragments, has led to a Workbench (CLC Bio, Aarhus, Denmark). Single-nucleotide polymorphisms paradigm shift in diagnostics and disease-gene discovery projects in (SNPs) and short indels were called with CLC Bio, filtering out calls with a human genetics.11 WES thus offers an interesting modality for the read coverage o8 Â and a Phred SNP quality of o20. Variants were filtered against NCBI dbSNP132, 1000 Human Genomes Project catalog, and the search for the causes of variable expressivity and non-penetrance in AVSIFT database, and their functional annotation was performed using familial DCM. In this work, we investigated an extended Italian family Annovar.13 Data were filtered for genes showing heterozygous variants in all diagnosed with DCM and cardiac conduction defects (CCM) using three sequenced relatives (V.13, V.15, and V.17) and a homozygous reference WESfollowedbytargetedSangerresequencing.In14outofthe41 sequence in the unaffected relative (VI.7). family members enrolled in the study, a mutation in the gene for lamin A/C (LMNA) was identified. Five of them, clearly showing DCM PCR and Sanger sequencing validation of LMNA and TTN variants features, were doubly heterozygous for variants in LMNA and in the The mutations LMNA:p.K219T (NM_170708:c.A656C) and TTN:p.L4855F titin gene (TTN). Four of these patients (all four older than 20 years of (NM_133378:c.C14563T) were validated using Sanger methods on the four age) displayed a particularly severe clinical involvement at early ages. subjects (three affected and one clinically healthy) that were sequenced using WES. Following this, all 41 participating family members were tested for the MATERIALS AND METHODS presence of these sequence variants by Sanger sequencing (Supplementary Patient recruitment Figures S2A and B). From May 1994 to September 2011, 10 patients affected by DCM underwent orthotopic heart transplantation at the University Hospital of Verona. Primer design and PCR set up Subsequently, these patients were found to belong to the same extended For each target, primers were designed with Primer 3 plus Software14 starting family, whose ancestors had moved from their native Verona to other regions from the sequences NM_170708 (LMNA exon 4) and NM_133378 (TTN of Italy and Northern Europe. In the course of this study, 42 living and 41 exon 59). The primers are shown in Table 1. PCR was performed with deceased family members were identified on the basis of data from parish an automated liquid handler (Tecan Freedom EVO; Tecan, Ma¨nnedorf, church registries and hospital records. A total of 41 subjects were enrolled in Switzerland) in a final volume of 7.5 mlwith1Â GoTaq Hot Start Master the study, including 18 females and 23 males aged 5–77 years (Supplementary Mix (Promega, Madison, WI, USA), 0.5 mMofeachprimer,and1.5mlofDNA Figure S3). Fourteen persons were affected by a range of cardiological previously normalized at a concentration of B5ng/ml. Amplification was symptoms, ranging from mild dyspnea
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