Accepted Manuscript A Novel Mutation in Lamin A/C Causing Familial Dilated Cardiomyopathy Associated with Sudden Cardiac Death Pérez-Serra Alexandra , Toro Rocío , Campuzano Oscar , Sarquella-Brugada Georgia , Berne Paola , Iglesias A , Mangas Alipio , Brugada Josep , Brugada Ramon PII:

S1071-9164(14)01343-8

DOI:

10.1016/j.cardfail.2014.12.003

Reference:

YJCAF 3451

To appear in:

Journal of Cardiac Failure

Received Date: 26 June 2014 Revised Date:

23 October 2014

Accepted Date: 3 December 2014

Please cite this article as: Alexandra P-S, Rocío T, Oscar C, Georgia S-B, Paola B, A I, Alipio M, Josep B, Ramon B, A Novel Mutation in Lamin A/C Causing Familial Dilated Cardiomyopathy Associated with Sudden Cardiac Death, Journal of Cardiac Failure (2015), doi: 10.1016/j.cardfail.2014.12.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT A NOVEL MUTATION IN LAMIN A/C CAUSING FAMILIAL DILATED CARDIOMYOPATHY ASSOCIATED WITH SUDDEN CARDIAC DEATH

Short title: Spanish family with a history of sudden death carries a new mutation

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associated with DCM.

AUTHORS

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Pérez-Serra Alexandra*1, Toro Rocío*2, Campuzano Oscar1, Sarquella-Brugada

*Both authors contributed equally.

AFFILIATIONS

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Georgia3, Berne Paola4, Iglesias A1, Mangas Alipio2, Brugada Josep4, Brugada Ramon1.

Cardiovascular Genetics Center, IDIBGI, University of Girona, Girona, Spain

2

School of Medicine, University of Cadiz, Cádiz, Spain

3

Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain

4

Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Spain

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CORRESPONDING AUTHOR

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Ramon Brugada Terradellas MD, PhD, FACC, FESC Dean, School of Medicine, Director of Cardiovascular Genetics Center C/ Pic de Peguera 11, 17003 Girona (Spain) Tel. +34 972 183366, Fax. +34 972 183367 [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Background: Dilated cardiomyopathy (DCM), a cardiac heterogeneous pathology characterized by left ventricular or biventricular dilatation, is a leading cause of heart failure and heart transplantation. The genetic origin of DCM remains unknown in most

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cases, but more than 50 genes have been associated with DCM. Here, we sought to identify the genetic implication and perform a genetic analysis in a Spanish family affected by DCM and sudden cardiac death.

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Methods and Results: Clinical assessment and genetic screening were performed in the index case as well as family members. Of all relatives clinically assessed, nine patients

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showed clinical symptoms related to the pathology. Genetic screening identified 20 family members who carried a novel mutation in LMNA (c.871G>A, p.E291K). Family segregation analysis indicated that all clinically affected patients carried this novel mutation. Clinical assessment of genetic carriers showed that electrical dysfunction was

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present previous to mechanical and structural abnormalities.

Conclusions: Our results report a novel pathogenic mutation associated with DCM,

pathology.

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supporting the benefits of comprehensive genetic studies of families affected by this

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Key words: dilated cardiomyopathy, lamin A/C, novel mutation, sudden cardiac death.

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ACCEPTED MANUSCRIPT INTRODUCTION Dilated cardiomyopathy (DCM) is characterized by cardiac left ventricular (LV) dilatation and subsequent abnormal contraction; dilatation of both ventricles may occur. The pathology can manifest at any age but occurs more frequently after middle age. A

range in symptoms from severe heart failure to asymptomatic1.

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leading cause of sudden cardiac death (SCD) in developed countries, cases can also

Genetic factors play a role in the pathogenesis of DCM, but the genetic basis remains

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unknown in most cases2. Currently, more than 60 genes have been reported to cause

monogenic DCM, most of them encoding for sarcomeric, cytoskeletal, and desmosomal

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proteins 3. To date, 20% of mutations associated with DCM are linked to TTN, the gene encoding titin protein; 6% are related to LMNA, the gene encoding lamin A/C protein4. Lamin A/C maintains the structural integrity of the nuclear envelope and organizes chromatin within the nucleus, thereby influencing DNA transcription5. Some reports of

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genetic studies in families affected by DCM and carrying mutations in LMNA discuss the severity of clinical findings associated with mutations in this gene in comparison with other DCM-related genes6, 7. A recent report indicated that subjects with LMNA

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mutations show prolonged PR interval in the electrocardiogram (ECG), myocardial septal fibrosis, and ventricular arrhythmias; all these symptoms are indicative of DCM8.

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We have identified a large family affected by DCM with a high incidence of SCD. We have performed genetic analyses both to identify the causative mutation and to provide phenotype-genotype correlation, providing further insight on genetic contributions to DCM pathology.

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ACCEPTED MANUSCRIPT METHODS Clinical assessment Written informed consent was obtained from all family members included in our study. Detailed clinical information was obtained from each subject, comprising family

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history, age of presentation, initial symptoms of heart failure, physical examination, serum creatine kinase, ECG, transthoracic echocardiography (TTE), Holter-ECG monitoring, treadmill testing and, when appropriate, cardiac magnetic resonance

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imaging (MRI). Routine TTE using Doppler and Tissue Doppler Imaging (TDI) were

performed according to the standards of the American Society of Echocardiography 9, 10.

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The diagnosis of DCM was based on symptoms, physical signs, ECG, and chest X-ray findings of ventricular dysfunction, supported by TTE or angiographic confirmation of global ventricular dilatation11. In this study all patients underwent clinical history, ECG, TTE using TDI, Holter ECG and exercise stress testing. Only those who showed any

Genetic analysis

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impairment in the TTE underwent a MRI.

Genomic DNA was extracted with Puregene DNA purification kit (Qiagen). DNA

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quality was assessed by spectrophotometry (optical absorbance 260/280:260/230 of a minimum of 1.8:2.2), and quantity was determined by fluorometry (Qubit, Life

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Technologies) before processing to obtain the 3 µg needed for sequencing. DNA integrity was assessed on a 0.8% agarose gel. The index patient (III-33, see Figure 1) was analyzed using NGS technology. Library preparation was performed according to the manufacturer’s instructions (SureSelect XT Custom 0.5-2.9 Mb library, Agilent Technologies, Inc). Sequencing was performed on the MiSeq system (Illumina) using 2 x150 bp reads length. We evaluated the 55 genes

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ACCEPTED MANUSCRIPT most commonly involved in SCD-related pathologies according to the literature (see Table 1). The secondary bioinformatic analysis of the data obtained included a first step trimming of the FAST-Q files. The trimmed reads were then mapped with GEM II, output was

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joined and sorted, and uniquely and properly mapping read pairs were selected. Finally, variant calls over the cleaned BAM file were performed with SAM tools v.1.18 and

GATK v2.4 to generate the first raw VCF files. Variants were annotated with dbSNP

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IDs, Exome Variant Server (EVS), the 1000 Genomes browser, in-home database IDs,

using Sanger sequencing.

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and Ensembl information, if available. Regions with poor coverage were sequenced

Tertiary analysis was then performed. For each genetic variation identified, alignment between different species was performed using the UNIPROT database12. The possible pathogenicity of challenged variants was consulted in silico, using CONsensus

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DELeteriousness score of missense single nucleotide variants (SNV) database (CONDEL)13 as well as the Protein Variation Effect Analyzer database (PROVEAN)14. Allelic frequency was determined in the Exome Variant Server15, dbSNP 16, and the

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1000 Genomes genetic variants database17.

Non-common genetic variants (minor allele frequency, MAF, A, p.E291K). In silico analysis predicted all previously

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reported SNV as neutral/benign. In contrast, the novel mutation was predicted as a novel deleterious genetic variation by in silico models (SIFT=0, PPH2=0.999,

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MASS=NA or 3.725, CONDEL=0.990, PROVEAN= -3.860).

Family segregation analysis revealed segregation of only the novel SNV in the LMNA gene; affected family members did not carry any of the 6 previously reported SNV. Hence, the SNV in LMNA was confirmed in nineteen family members (Figure 1). This

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novel mutation at nucleotide 871 in exon 5 of the LMNA gene, introduces a change from glutamic acid [Glu (E)] to lysine [Lys (K)] in the first position of codon. Amino acid 291 is highly conserved across species (Figure 4), which may be indicative of its

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relevance in the protein structure and/or function. Genetic analysis in relatives

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The pathology was clinically diagnosed in the index case (III-33) and nine other family members (II-7, III-11, III-13, III-17, III-21, III-27, III-35, III-39, and IV-70) (Table 2). All these members with DCM carried the same novel genetic variation identified in the proband. In addition, ten relatives who carried the mutation remained asymptomatic (III-41, III-45, III-46, IV-48, IV-50, IV-53, IV-54, IV-57, IV-72, and V-77) (see Figure 1). Importantly, none of the individuals without the genetic variation showed any symptom or cardiac structural abnormality related to DCM.

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ACCEPTED MANUSCRIPT ECG abnormalities preceded DCM in all cases, and first electrical alterations appeared before 20 years old (Figure 5). LAFB was observed in more than 20% of patients. At older ages, more electrical anomalies were evident. A long PR interval (of over 200 ms) may indicate a first-degree heart block; indeed, LBBB was present in patients from the

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third decade, and was associated with poor R wave progression as a characteristic finding of DCM. Electrical dysfunctions appeared previous to mechanical

abnormalities. Later, mechanical impairment emerged subtly, and was only able to be

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diagnosed with tissue imaging. Ventricular dilatation and global myocardial dysfunction (ejection fraction, EF, < 55%) were evident in all patients with LV dysfunction over 30

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years of age (45%), who showed a diastolic dysfunction pattern.

DISCUSSION

We present a phenotype-genotype correlation in a large Spanish family affected by

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DCM, significant ventricular arrhythmias, and predisposition to SCD at an early age. Genetic testing identified a novel missense heterozygous genetic variation, p.E291K, in the LMNA gene, which seems to be responsible for the pathology. It plays a dominant

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genetic role, and shows incomplete penetrance/variable expressivity, which are hallmarks of DCM.

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Clinical history of SCD with definitive DCM diagnosis was identified in nine members. In concordance with reported studies, a high heterogeneity of symptoms is commonly found in early stages of DCM3, 18. The earliest clinical markers of DCM progression were poor R progression, PR prolongation and, later, progression to LBBB in the ECG. Prolonged PR interval has been described recently as the best predictor of ventricular arrhythmias in LMNA mutation-positive subjects, with DCM developing later8. These

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ACCEPTED MANUSCRIPT data are crucial for clinical cardiologists because the ECG serves as a pragmatic and accessible tool for the diagnosis and evolution of this population. Subsequently, all individuals with confirmed diagnoses showed left ventricular involvement detected by TTE. In contrast, younger patients with normal ECG showed

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no alteration in the TTE or TDI results. TDI was useful for detecting patients in early stage DCM with normal LV diameter and left systolic ejection fraction at the lower

limit of normality by TTE; they showed an abnormal diastolic pattern when TDI was

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applied. This supports another report on how to diagnose LV dysfunction and impaired diastolic pattern19. Thus, the clinical cardiologist can perform follow-up of a similar

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population with accessible tools such as an ECG and TTE.

In six family members an ICD was implanted following the recommendations of Tracy et al.20 in relation to their clinical characteristics. Some controversy still exists with regard to risk factors for SCD with this condition. ICD is the preferred therapy for those

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with prior episodes of sustained VT or VF, and may also be considered for primary prevention for some patients with a strong family history of early mortality. Beyond the ICD implantation, we categorized our patients as presymptomatic or those patients who

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had the onset of clinical disease, and followed the suggestions included in the guidelines for clinical care of patients with heart failure21. We have integrated the clinical

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characteristics and family data as recommended by Hershberger et al., rather than using solely the genetic diagnosis 22. Familial genetic testing identified a novel genetic variation with an autosomal dominant pattern of inheritance, as for other LMNA mutations reported in DCM families23,24. The LMNA_p.E291K site is an evolutionarily conserved residue located in coil 2 of the alpha-helicoidal coiled-coil rod region of the lamin A/C protein, which contains a chromatin binding site. Lamin proteins are the major structural elements of the lamina

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ACCEPTED MANUSCRIPT network underlying and mechanically supporting the nuclear envelope25. Variations in the LMNA gene have resulted in changes in electrical activity and epigenetic chromatin modifications, indicating a relationship between transcriptional response and mechanical stress26. Several variations in LMNA were previously reported to be

genetic variation in LMNA is potentially pathogenic in DCM.

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probable causes of familial DCM3,23,24. Thus, combined with our analysis, the p.E291K

DCM patients carrying LMNA mutations are reported to have a worse prognosis than

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DCM patients carrying a pathogenic mutation in another DCM-associated gene27;

however, debate surrounds this report because, so far, few consistent family correlations

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have been published to clarify the risk stratification23,28. Furthermore, it has been suggested that early recognition of young DCM patients who carry a pathogenic mutation in the LMNA gene could delay the rate of progression of the cardiomyopathy29. Here, ten additional relatives carry the p.E291K genetic variation but

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remains asymptomatic. None of them is older than thirty.

Hence, as reported above, we noted an incomplete penetrance and variable expressivity in the family analyzed, in accordance with several reports focusing on familial DCM2.

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Numerous factors can determine penetrance and expressivity in the asymptomatic relatives who carry the novel genetic variation, including age/gender differences and

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genetic modulators18. In addition, most family members who carry the genetic variation without clinical symptoms are below 40 years of age, therefore they may not yet be affected by symptoms associated with the pathology, but may develop them in the near future. On the other hand, we have to consider that some of the deceased relatives passed away before reaching their fifties. Also, the combination of two or more genetic variants could be required to cause DCM, as also occurs in arrhythmogenic right ventricular cardiomyopathy30, hypertrophic cardiomyopathy31, and in a recently

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ACCEPTED MANUSCRIPT described family affected by DCM32. In the present study, we did not identify any other potential genetic alteration in the tested DCM-related genes, apart from reported natural variants, but this phenotype variability could be caused by genetic variations in unknown genes. Finally, recent exome genetic studies reveal several rare variants of

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unknown significance in DCM that could be potential pathogenic mutations or

phenotype modifiers33. As a corollary, further comprehensive genotype/phenotype

studies will be required to determine how commonly rare variants in unknown and

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known genes associated with DCM determine clinical course and disease outcome. Controversies among cardiologists make comprehensive genetic studies in DCM

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families necessary to be able to provide specific genetic counseling regarding risk of SCD in each family member18. Strikingly, although DCM genetic testing is already available in clinical practice, current DCM guidelines do not yet recommend genetic

LIMITATIONS

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testing to help in SCD risk stratification.

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One limitation of this study is that clinical assessment could not be completed in two carriers who refused to be evaluated. Additionally, the symptomatic ratio of our carriers

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may be biased because 70% of them are younger than thirty years old; importantly, symptoms tend to appear at the end of the second decade and the beginning of the third. Finally, although we have analyzed the main genes associated with DCM, it is possible that genetic alterations could be present in genes not analyzed in our study.

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ACCEPTED MANUSCRIPT CONCLUSION The present study identifies a novel pathogenic variation in the LMNA gene in a family affected by DCM and SCD, which supports its potential pathogenic effect as a dominant mutation with variable penetrance. Additionally, we establish a progressive sequential

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concatenation of electrical, mechanical, and structural findings exhibited by affected individuals. Genetic analysis in large families provides new insights into the

pathophysiology of DCM, allowing a better clinical management of these patients with

ACKNOWLEDGEMENTS

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a wide range of clinical presentation.

This work was supported by CNIC-Translational 2008 (CNIC-03-2008), Obra Social

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“la Caixa”, Fondo de Investigación Sanitaria (PI11/00019).

CONFLICT OF INTEREST

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Ramon Brugada is a consultant for FerrerIncode.

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ACCEPTED MANUSCRIPT LIST OF ABREVIATIONS Dilated cardiomyopathy (DCM) Left ventricular (LV) Sudden cardiac death (SCD)

Doppler and Tissue Doppler Imaging (TDI) Premature ventricular contractions (PVC) Implantable cardiac defibrillator (ICD)

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Left bundle branch block (LBBB)

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Transthoracic echocardiography (TTE)

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Electrocardiogram (ECG)

Left anterior fascicular block (LAFB) Single nucleotide variant (SNV)

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Next generation sequencing (NGS)

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ACCEPTED MANUSCRIPT REFERENCES 1. Jefferies JL, Towbin JA. Dilated cardiomyopathy. Lancet 2012; 375 (9716): 752-62. 2. Hershberger RE, Morales A, Siegfried JD. Clinical and genetic issues in dilated cardiomyopathy: a review for genetics professionals. Genet Med 2003; 5 (1): 15-20.

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3. McNally EM, Golbus JR, Puckelwartz MJ. Genetic mutations and mechanisms in dilated cardiomyopathy. J Clin Invest 2013; 123 (1): 19-26.

4. Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, et al.

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Truncations of titin causing dilated cardiomyopathy. N Engl J Med 2012; 366: 619– 28.

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5. Butin-Israeli V, Adam SA, Goldman AE, Goldman RD. Nuclear lamin functions and disease. Trends Genet 2012; 28 (9): 464-71.

6. Forissier JF, Bonne G, Bouchier C, Duboscq-Bidot L, Richard P, Wisnewski C, et al. Apical left ventricular aneurysm without atrio-ventricular block due to a lamin

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A/C gene mutation. Eur J Heart Fail 2003; 5 (6): 821-5.

7. Saj M, Dabrowski R, Labib S, Jankowska A, Szperl M, Broda G, et al. Variants of the lamin A/C (LMNA) gene in non-valvular atrial fibrillation patients: a possible

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pathogenic role of the Thr528Met mutation. Mol Diagn Ther 2012; 16 (2): 99-107. 8. Hasselber NE, Edvardsen T, Petri H. Risk prediction of ventricular arrhythmias and

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myocardial function in Lamin A/C mutation positive subjects. Europace, 2014, 16 (4): 563-71.

9. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, et al. Recommendations for the evaluation of left ventricular diastolic function by echocardiography. Eur J Echocardiogr 2009; 10 (2): 165-93.

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ACCEPTED MANUSCRIPT 10. Hill JC, Palma RA. Doppler tissue imaging for the assessment of left ventricular diastolic function: a systematic approach for the sonographer. J Am Soc Echocardiogr 2005; 18 (1): 80-8. 11. Codd MB, Sugrue DD, Gersh BJ, Melton LJ. 3rd Epidemiology of idiopathic

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dilated and hypertrophic cardiomyopathy. A population-based study in Olmsted County, Minnesota, 1975-1984. Circulation 1989; 80 (3): 564-72.

12. The UniProt Consortium. Reorganizing the protein space at the Universal Protein

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Resource (UniProt) Nucleic Acids Res 2012; 40: D71-D75.

13. Gonzalez-Perez A, Lopez-Bigas N. Improving the assessment of the outcome of

Genet 2011; 88 (4): 440-9.

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nonsynonymous SNVs with a consensus deleteriousness score, Condel. Am J Hum

14. Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PloS One 2012; 7 (10): e46688.

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http://provean.jcvi.org/index.php

15. URL. Exome Variant Server (EVS). http://evs.gs.washington.edu/EVS/. 16. URL. SNP database. http://www.ncbi.nlm.nih.gov/projects/SNP/.

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17. 1000 Genomes Project Consortium, Abecasis GR, Altshuler D et al. A map of human genome variation from population-scale sequencing. Nature 2010; 467

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(7319): 1061-73.

18. Lakdawala NK, Funke BH, Baxter S, Cirino AL, Roberts AE, Judge DP et al. Genetic Testing for Dilated Cardiomyopathy in Clinical Practice. J Card Fail 2012; 18 (4): 296-303. 19. Fatkin D, Yeoh T, Hayward CS, Benson V, Sheu A, Richmond Z, et al. Evaluation of left ventricular enlargement as a marker of early disease in familial dilated cardiomyopathy. Circ Cardiovasc Genet 2011; 4: 342-8.

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ACCEPTED MANUSCRIPT 20. Tracy CM, Epstein AE, Darbar D, DiMarco JP, Dunbar SB, Estes III M, et al. 2012 ACCF/AHA/HRS focused update incorporated into the ACCF/AHA/HRS 2008 guidelines for device-based therapy of cardiac rhythm abnormalities: a report of the American College of Cardiology Foundation/American Heart Association

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Task Force on Practice Guidelines and the Heart Rhythm Society. JACC 2013; 61 (3): e6-e75.

21. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG, et al.

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2009 Focused update incorporated into the ACC/AHA 2005 Guidelines for the

Diagnosis and management of Heart Failure in Adults A Report of the American

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College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines Developed in Collaboration With the International Society for Heart and Lung Transplantation. JACC 2009; 53 (15): e1-e90. 22. Hershberger RE, Lindenfeld J, Mestroni L, Seidman CE, Taylor MR, Towbin JA;

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Heart Failure Society of America. Genetic evaluation of cardiomyopathy-a Heart Failure Society of America practice guideline. J Cardiac Fail 2009; 2: 83-97. 23. Małek LA, Labib S, Mazurkiewicz L, Saj M, Płoski R, Tesson F et al. A new

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c.1621 C > G, p.R541G lamin A/C mutation in a family with DCM and regional wall motion abnormalities (akinesis/dyskinesis): genotype-phenotype correlation. J

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Hum Genet 2011; 56 (1): 83-6

24. Chen W, Huo J, Ma A, Bai L, Liu P. A novel mutation of the LMNA gene in a family with dilated cardiomyopathy, conduction system disease, and sudden cardiac death of young females. Mol Cell Biochem 2013; 382 (1-2): 307-11. 25. Mounkes LC, Burke B, Stewart CL. The A-type lamins: nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases. Trends Cardiovasc Med 2001; 11 (7): 280-5.

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ACCEPTED MANUSCRIPT 26. Raharjo WH, Enarson P, Sullivan T, Stewart CL, Burke B. Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy. J Cell Sci 2001; 114 (Pt 24): 4447-57. 27. van Berlo JH, de Voogt WG, van der Kooi AJ, van Tintelen JP, Bonne G, Yaou

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RB, et al. Meta-analysis of clinical characteristics of 299 carriers of LMNA gene mutations: do lamin A/C mutations portend a high risk of sudden death? J Mol Med (Berl) 2005; 83 (1): 79-83.

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28. Saj M, Dabrowski R, Labib S, Jankowska A, Szperl M, Broda G et al. Variants of the lamin A/C (LMNA) gene in non-valvular atrial fibrillation patients: a possible

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pathogenic role of the Thr528Met mutation. Mol Diagn Ther 2012; 16 (2): 99-107. 29. Volpi L, Ricci G, Passino C, Di Pierri E, Alì G, Maccherini M et al. Prevalent cardiac phenotype resulting in heart transplantation in a novel LMNA gene duplication. Neuromuscul Disord 2010; 20 (8): 512-6.

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30. Nakajima T, Kaneko Y, Irie T, Takahashi R, Kato T, Iijima T, et al. Compound and digenic heterozygosity in desmosome genes as a cause of arrhythmogenic right ventricular cardiomyopathy in Japanese patients. Circ J 2012; 76 (3): 737-43.

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31. Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, et al. Contemporary definitions and classification of the cardiomyopathies: an American

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Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 2006; 113 (14): 1807-16.

32. Roncarati R, Viviani Anselmi C, Krawitz P, Lattanzi G, von Kodolitsch Y, Perrot A, et al. Doubly heterozygous LMNA and TTN mutations revealed by exome

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ACCEPTED MANUSCRIPT sequencing in a severe form of dilated cardiomyopathy. Eur J Hum Genet 2013; 21 (10): 1105-11. 33. Norton N, Robertson PD, Rieder MJ, Züchner S, Rampersaud E, Martin E, et al.

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era. Circ Cardiovasc 2012: 5 (2): 167-74.

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Evaluating pathogenicity of rare variants from dilated cardiomyopathy in the exome

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ACCEPTED MANUSCRIPT

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Isoform NM_005159 NM_001103 NM_001148 NM_001129827 NM_201596 NM_001232 NM_033337 NM_001885 NM_003476 NM_001927 NM_004006 NM_024422 NM_001943 NM_004415 NM_000117 NM_000138.4 NM_000169 NM_015141 NM_005477 NM_002230 NM_020433 NM_000219 NM_172201 NM_000238 NM_000891 NM_000218 NM_002294 NM_001080116 NM_170707 NM_000256 NM_002471 NM_000257 NM_000432 NM_000258 NM_016599 NM_014476 NM_004572 NM_002667 NM_016203 NM_001035 NM_174934 NM_198056 NM_000023 NM_000232 NM_001128209 NM_003673 NM_003280 NM_000363 NM_001001430 NM_001018005 NM_133378 NM_000116 NM_003239 NM_014000 NM_001024847

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Gene ACTC1 ACTN2 ANK2 CACNA1C CACNB2 CASQ2 CAV3 CRYAB CSRP3 DES DMD DSC2 DSG2 DSP EMD FBN1 GLA GPD1L HCN4 JUP JPH2 KCNE1 KCNE2 KCNH2 KCNJ2 KCNQ1 LAMP2 LDB3 LMNA MYBPC3 MYH6 MYH7 MYL2 MYL3 MYOZ2 PDLIM3 PKP2 PLN PRKAG2 RYR2 SCN4B SCN5A SGCA SGCB SGCD TCAP TNNC1 TNNI3 TNNT2 TPM1 TTN TAZ TGFB3 VCL TGFBR2

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

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TABLES

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ACCEPTED MANUSCRIPT Table 1. Panel of genes screened in the study. All isoforms described in the UCSC Genome browser were included in the design. The final size was 432,512 kb of encoding regions and UTR boundaries. The coordinates of the sequence data were

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based on NCBI build 37 (UCSC Hg 19).

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81

Female

No

+ , PM

II-3

╬

Female

No

SCD at 62

II-5

╬

Female

No

SCD at 52

II-6

╬

Male

No

III-13

III-17

III-21

45

43

29

26

No

SCD at 52

Diagnosis

E/A

0.56

Cerebrovascular

Male

Male

Female

Female

Male

Male

death at 56

Yes. At 50 years

+

No

SCD at 42

No

No

No

No

+

+

+

+

Diastolic Pattern

Type I

+

-

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III-11

╬

Male

ECG

+

0.74

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II-9

60

Clinical

+

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II-7

Heart Transplantatio n

+

+

EF %

ECC +

Cardio-MRI

TDI

Confirmation

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I-2

Gende r

55

-

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I-1

Age at genetic diagnosis (years) ╬

Subject Code

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ACCEPTED MANUSCRIPT

1.54

Type I

61

-

ICD

ECG findings

results c.871G>A

-

c.871G>A c.871G>A c.871G>A c.871G>A

previously

+

+

+

c.871G>A

LAFB+ RBBB PR>200 ms

PM at 37

c.871G>A LAFB to non-

56

-

-

-

c.871G>A

complete LBBB PR> 200

Type II

48

+

+

+

LAFB+RBBB

Type I

53

+

-

-

c.871G>A

PR LAFB to non

0.70

Genetic

c.871G>A

complete LBBB PR

0.70

Type I

64

+

Waiting MRI

No

LAFB to non

c.871G>A

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ACCEPTED MANUSCRIPT

complete LBBB

╬

Male

No

SCD at 31

44

Female

No

+

+

0.67

Type I

44

III-33*

37

Male

No

+

+

1.42

Type II

47

III-35

35

Female

No

+

+

1.35

Type II

III-39

25

Female

No

-

-

0.70

III-41

32

Female

No

-

-

III-45

24

Female

No

-

III-46

16

No

-

III-48

14

Female Female

No

-

IV-50

20

Female

No

-

IV-53

12

Female

No

-

IV-54

8

Male

No

IV-57

2

Female

No

IV-70

5

Male

No

IV-72

3

Female

V-77

1.5

Female

LBBB

c.871G>A

+

+

+

+

+

+

48

+

+

+

Type I

45

+

NE

+

1.63

Normal

63

-

c.871G>A

-

1.48

Normal

62

-

c.871G>A

-

1.72

Normal

64

-

c.871G>A

-

1.57

Normal

59

-

c.871G>A

-

1.36

Normal

62

-

c.871G>A

-

1.94

Normal

66

-

c.871G>A

AC C

EP

TE D

M AN U

III-27

c.871G>A

SC

III-26

RI PT

PR160

PR>200

LBBB

c.871G>A

PR>200 LBBB

c.871G>A

PR>200 LBBB

c.871G>A

PR 160

-

-

1.87

Normal

64

-

c.871G>A

-

-

1.77

Normal

65

-

c.871G>A

+

+

1.82

Normal

63

-

c.871G>A

No

-

-

2.02

Normal

67

-

c.871G>A

No

-

-

2.14

Normal

68

-

c.871G>A

25

ACCEPTED MANUSCRIPT

SC

RI PT

Table key: ╬ death, + showed alteration, - not showed alteration, ECG: electrocardiogram, EF%: ejection fraction, ICD: defibrillator, MRI: magnetic resonance imaging, NE: non evaluated, PM: pacemaker, LAFB: left anterior fascicular block, LBBB: left bundle branch block, RBBB: right bundle branch block, SCD: sudden cardiac death,SR: respiratory sinus arrhythmia

AC C

EP

TE D

M AN U

Table 2. Clinical findings in mutation carriers.

26

ACCEPTED MANUSCRIPT FIGURES

Figure 1. Family pedigree. The generations are indicated in the left column and all individuals are identified with a pedigree number. The proband is indicated by arrow

RI PT

(III-33). Mutation carriers are shown in black, and relatives without mutation are white. Slashed line indicates individuals are deceased. Clinically diagnosed patients are shown

AC C

EP

TE D

M AN U

SC

with a plus sign.

27

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

28

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Figure 2. ECG of index patient. The figure shows a trifascicular block, prolonged PR segment, and complete left bundle branch block. In addition, there is a premature ventricular contraction.

29

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

Figure 3. Clinical cardiac MR image. In diastolic basal short axis SSFP technique (Steady State Free Precession), the index patient shows a slightly dilated left ventricle

AC C

EP

and no other associated findings.

30

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 4. A) Chromatogram. Left, control DNA sequence. Right, heterozygous

TE D

variation in exon 5 of the LMNA gene. B) Sequence conservation of specific twenty amino acid residues in coil 2 of the lamin A/C protein. The amino acid residues conserved throughout the seven species are shown in bold in contrast to non-conserved

EP

residues. Arrow indicates the site of the amino acid substitution identified in this study.

AC C

Reference sequence accession number is on the left.

31

M AN U

SC

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ACCEPTED MANUSCRIPT

Figure 5. Sequential findings in electrical and mechanical dysfunction. Electrical

AC C

EP

TE D

dysfunctions are previous in time to mechanical abnormalities.

32

ACCEPTED MANUSCRIPT HIGHLIGHTS A novel mutation was found in the LMNA gene, which can explain the DCM pathology in a large Spanish family. We present a sequential concatenation of electrical cardiac dysfunctions and mechanical

RI PT

and structural alterations that are present in all carriers but not in non-mutation carriers ECG monitoring of patients proved to be a useful clinical tool together with genetic screening.

AC C

EP

TE D

M AN U

SC

After genetic confirmation, these patients can be managed with ECG and TTE.