- Email: [email protected]
Genetic basis of familial dilated cardiomyopathy patients undergoing heart transplantation
Author's Accepted Manuscript
Genetic Basis of Familial Dilated Cardiomyopathy Undergoing Heart Transplantation Sofia Cuenca, Maria J. Ruiz-Cano, Juan Ramón Gimeno-Blanes, Alfonso Jurado, Clara Salas, Iria Gomez-Diaz, Laura Padron-Barthe, Jose Javier Grillo, Carlos Vilches, Javier Segovia, Domingo Pascual-Figal, Enrique Lara-Pezzi, Lorenzo Monserrat, Luis Alonso-Pulpon, Pablo Garcia-Pavia, for the Inherited Cardiac Diseases program of the Spanish Cardiovascular Research Network (Red Investigación Cardiovascular)
PII: DOI: Reference:
S1053-2498(16)00009-7 http://dx.doi.org/10.1016/j.healun.2015.12.014 HEALUN6146
To appear in:
J Heart Lung Transplant
http://www.jhltonline.org
Cite this article as: Sofia Cuenca, Maria J. Ruiz-Cano, Juan Ramón Gimeno-Blanes, Alfonso Jurado, Clara Salas, Iria Gomez-Diaz, Laura Padron-Barthe, Jose Javier Grillo, Carlos Vilches, Javier Segovia, Domingo Pascual-Figal, Enrique Lara-Pezzi, Lorenzo Monserrat, Luis Alonso-Pulpon, Pablo Garcia-Pavia, for the Inherited Cardiac Diseases program of the Spanish Cardiovascular Research Network (Red Investigación Cardiovascular), Genetic Basis of Familial Dilated Cardiomyopathy Undergoing Heart Transplantation, J Heart Lung Transplant, http://dx.doi.org/10.1016/j. healun.2015.12.014 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 galley proof before it is published in its final citable 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.
1 GENETIC BASIS OF FAMILIAL DILATED CARDIOMYOPATHY UNDERGOING HEART TRANSPLANTATION Sofia Cuenca1; Maria J. Ruiz-Cano2; Juan Ramón Gimeno-Blanes3; Alfonso Jurado2; Clara Salas4; Iria Gomez-Diaz5; Laura Padron-Barthe6; Jose Javier Grillo7; Carlos Vilches8; Javier Segovia1; Domingo Pascual-Figal3; Enrique Lara-Pezzi6; Lorenzo Monserrat5; Luis Alonso-Pulpon1; and Pablo Garcia-Pavia1,6 for the Inherited Cardiac Diseases program of the Spanish Cardiovascular Research Network (Red Investigación Cardiovascular).
Affiliation: 1
Heart Failure and Inherited Cardiac Diseases Unit. Department of Cardiology. Hospital
Universitario Puerta de Hierro, Madrid, Spain. 2
Heart failure and Heart Transplantation Unit. Department of Cardiology. Hospital
Universitario 12 de Octubre, Madrid, Spain. 3
Department of Cardiology Hospital Universitario Virgen de la Arrixaca, Murcia, Spain.
4
Department of Pathology. Hospital Universitario Puerta de Hierro, Madrid, Spain.
5
Health In Code, A Coruña, Spain.
6
Myocardial Biology Programme, Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid, Spain. 7
Department of Cardiology. Hospital Univ. Nuestra Señora de Candelaria, Tenerife, Spain.
8
Department of Immunology. Hospital Universitario Puerta de Hierro, Madrid, Spain.
Corresponding author: Pablo Garcia-Pavia, MD, PhD. Department of Cardiology. Hospital Universitario Puerta de Hierro. Manuel de Falla, 2. Majadahonda, Madrid, 28222, Spain. Email: [email protected]
Background: Dilated cardiomyopathy (DCM) is the most frequent cause of heart transplantation (HTx). The genetic basis of DCM among patients undergoing HTx is poorly characterized. We sought to determine the genetic basis of heart-transplanted familial DCM
2 and to establish the yield of modern Next Generation Sequencing (NGS) technologies in this setting.
Methods: Fifty-two heart-transplanted patients due to familial DCM underwent NGS genetic evaluation with a panel of 126 genes related to cardiac conditions (59 associated with DCM). Genetic variants were initially classified as pathogenic mutations or as variants of uncertain significance (VUS). Final pathogenicity status was determined by familial cosegregation studies.
Results: Initially, 24 pathogenic mutations were found in 21 patients (40%), 25 patients (48%) carried 19 VUS and 6 (12%) did not show any genetic variant. Familial evaluation of 220 relatives from 36 of the 46 families with genetic variants confirmed pathogenicity in 14 patients and allowed reclassification of VUS as pathogenic in 17 patients, and as nonpathogenic in 3 cases. At the end of the study, the DCM-causing mutation was identified in 38 patients (73%) and 5 patients (10%) harbored only VUS. No genetic variants were identified in 9 cases (17%).
Conclusions: The genetic spectrum of familial DCM undergoing heart transplantation is heterogeneous and involves multiple genes. NGS technology plus detailed familial studies allow identification of causative mutations in the vast majority of familial DCM cases. Detailed familial studies remain critical to determine the pathogenicity of underlying genetic defects in a substantial number of cases.
Keywords:
Familial
transplantation.
dilated
cardiomyopathy;
Next
Generation
Sequencing;
heart
3
Introduction Dilated cardiomyopathy (DCM), defined as dilatation and systolic impairment of the left or both ventricles in the absence of abnormal loading conditions or coronary artery disease(1), is the most common cause of heart failure in the young and the most frequent indication for heart transplantation (HTx) in adults and in children older than 1 year(2-4). Family studies in predominantly stable outpatients suggest that up to 20-50% of DCM patients have a familial predisposition to the disease(5-7). However, the genetic basis of this disease in most patients is still unknown(8), particularly in those with advanced disease in which there are few genetic studies(9,10). More than 50 genes have been associated so far with familial DCM; however, the precise contribution of mutations in each of these genes to this condition is unknown(8,11). The large number of genes related to familial DCM has made the study of the genetics of patients with DCM arduous, and has limited the application of genetic screening strategies to everyday clinical practice. Recently, there have been major advances in the field of genomics with the emergence of new generation sequencing techniques (Next Generation Sequencing, NGS), which enable the study of a large number of genes simultaneously in a short period of time and at a relatively low cost(12-15). A large study recently published has been the first to provide data of genetic findings in DCM patients through NGS(16). This study demonstrates the potential of NGS technology applied to the DCM field, but also illustrates the drawback of identifying rare variants without performing corroborative familial studies. To date, no major studies with complete familial evaluation have provided robust data on the genetic testing yield of NGS in DCM.
4 The purpose of this study was two-fold: to determine the genetic basis of heart transplanted DCM, and to establish the yield of modern NGS technologies plus detailed familial studies in this setting. Methods Study Population: This study was approved by the ethics committee of the participant centers and complies with the Declaration of Helsinki. A total of 52 non-related heart transplanted patients due to familial DCM at 3 Spanish heart transplant centers (Hospital Universitario Puerta de Hierro, Hospital Universitario 12 de Octubre, and Hospital Universitario Virgen de la Arrixaca) were included. DCM was defined as familial if one or more relatives (in addition to the proband) had DCM during life or at postmortem examination, or presented unexplained sudden cardiac death (SCD) before the age of 35(17). Only those patients fulfilling World Health Organization/International Society and Federation of Cardiology Task Force clinical criteria for DCM(18) at time of heart transplant were included. Patients with significant coronary artery disease or myocarditis at pathological examination of the explanted heart were excluded. Physicians blinded to the genetic results reviewed patients’ records retrospectively. Clinical data and results from the first pre-transplant evaluation at each center were collected.
Genetic evaluation DNA was obtained from blood samples extracted from the probands and stored at -70 0C. DNA samples were analyzed by NGS with a panel of 126 genes related to inherited cardiovascular diseases and 59 specifically associated with DCM (Table S1 from Supplementary material 1), including all coding exons and flanking intronic regions. Targeted enrichment was done with SureSelect (Agilent) and the design of the capture baits was carried out using eArray (Agilent).
5 DNA was sequenced in an Illumina HiSeq 1500 platform. Bioinformatic analysis was performed with a previously validated in-house pipeline. Mean coverage was 456 times and >99% of the fragments had coverage >15 times, and >98% had coverage >30 times. Sanger sequencing was used to evaluate regions of low coverage (<15 times) and to confirm the genetic variants found. Sequence variants were initially classified as pathogenic mutations (PM) or variants of uncertain significance (VUS). Variants were considered PM if they were found in a DCMassociated gene, had not been found in controls, and had been reported previously as pathogenic
in
literature
or
in
online
(http://www.ncbi.nlm.nih.gov/clinvar/)
and
international The
Human
databases Gene
such
Mutation
as
ClinVar Database
(http://www.hgmd.cf.ac.uk), or if they were novel sequence variants in a previously DCMassociated gene not found in controls that predicted a premature truncation, frameshift or abnormal splicing of the protein. Variants were classified as VUS if they were novel missense variants in a previously DCM-associated gene not found in controls, or if they were variants reported previously as pathogenic in online international databases that had been also found in controls. Other relevant genetic variants in genes not related to DCM were noted although they were excluded from the present study. VUS were reclassified according to results of familial evaluation (Figure 1). If VUS cosegregated with DCM on familial evaluation, they were annotated as a PM. In contrast, if VUS did not cosegregate with DCM, they were considered as non-pathogenic variants. Finally, VUS without corroborative family screening data or which cosegregated with another VUS were not reclassified and remained as VUS. Genetic databases used to determine the presence of variants among controls were dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and the NHLBI GO Exome Sequencing Project database (http://evs.gs.washington.edu/EVS/).
6 Conservation of amino acid residues affected by genetic variants found was determined using AlaMut (version 2.4.5; Interactive Biosoftware, Rouen, France). Conservation was graded very high, high, medium or low as previously reported(19).
Familial evaluation All relatives >16-years-old of patients with PM/VUS were offered clinical and genetic evaluation. Clinical evaluation included physical examination, ECG and an echocardiogram. Family screening was considered positive if one or more relatives had DCM and the same genetic defect as the proband (Supplemental material 2).
Haplotype analysis of emerin c.77T>C mutation carriers Three polymorphic microsatellite markers and six SNPs were genotyped in 9 mutation carriers. Polymorphic microsatellite markers flanking the emerin (EMD) gene were chosen using the Marshfield
genetic
maps
(http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp) and the UCSC genome browser (http://genome.ucsc.edu/). The SNPs were chosen from NCBI SNP database. The 9 markers (2 upstream and 7 downstream of the mutation) were located in a region of 2.87 Mb (3.35 cM) on chromosome X. PCR fragments from the amplification of the polymorphic markers were fluorescently labeled using a labeled oligonucleotide. Fragment analyses were performed on an ABI3730 automated sequencer using software package GeneMapper 4.0. Allele sizes of patients are inferred from the allele sizes present in the CEPH database (http://www.cephb.fr/). SNP genotyping was undertaken by specific PCR amplification of the region of interest and DNA sequencing with Big Dye terminator 3.1 in an AB3730 automated sequencer.
7
Statistical analysis Continuous variables are expressed as mean value ± SD. Discrete variables are shown as percentages. All data were analyzed using SPSS software (version 16.0).
Results The study cohort comprised 52 patients (mean age at first evaluation 39.9±14.3 years, range 11 to 64; 92% males). Clinical and familial characteristics are presented in Table S2 from Supplementary material 1. Fifty-one patients (98%) had known family history of DCM and 20 (38%) had family history of SCD (7 in a relative under the age of 35). Pedigrees of the 52 families are provided (Supplementary material 2). Initially, 24 PM were found in 21 patients (40%)(Table 1). Three patients harbored two PM in different genes and 6 patients presented also VUS. A total of 18 PM (75%) were novel stop codon mutations. One was a previously described mutation, causing abnormal splicing and a premature stop codon, 3 were previously described missense variants not reported in controls and 2 were previously described stop codon mutations. Twenty-five patients (48%) carried 19 VUS (Table 2). Twelve were novel missense variants not reported in controls and 7 were previously reported DCM-causing mutations also described in controls (Table 2). Finally, 6 patients (12%) did not show any genetic variations in analyzed DCM-related genes. Several relevant genetic variants in other cardiovascular-related genes were also identified (Table S3. Supplementary material 1). Familial evaluation included clinical and genetic study of 220 relatives from 36 of the 46 families with PM/VUS (78%)(Supplementary material 2). Familial study confirmed pathogenicity of the mutations in 14 patients with PM (Figure 1 and Supplementary material 2). Among patients with VUS, familial evaluation allowed reclassification of VUS as PM in 17
8 families and as non-pathogenic in 3 cases (Supplemental material 2). Familial evaluation was not possible in 3 patients with VUS and it was inconclusive in 2 patients. Among the 17 families with VUS which were reclassified as PM, 13 harbored the same missense mutation in the EMD gene (c.77T>C). These subjects were apparently unrelated males, without muscular phenotype, originating from the same geographical area (Tenerife island). A haplotype study in 9 patients with the EMD - c.77T>C mutation was consistent with a shared haplotype (Figure 2) and therefore a new founder mutation was described. After completing familial evaluation, 5 of the 17 variants classified as VUS showed a minor allele frequency (MAF) >1%, despite having previously been reported as DCM-causing mutations (Table 2). Given their MAF frequency, it is unlikely that these variants are DCMcausing by themselves, but we could not completely discard a modifier role that these variants could play in the presence of other aggressive mutations. At the end of the study, the causal DCM mutation was identified in 38 patients (73%)(Figure 3). Mutated genes included: EMD (13 patients), TTN (10), BAG3 (4), LMNA (3), FLNC (2), TNNT2 (2), DMD, DSC2, DSP, MYH7, PKP2, ABCC9 and TPM1. A total of 5 patients (10%) harbored only VUS in the following genes: PSEN2 (2), DSC2, DSG2, MYBPC3, MYH6, MYPN, and TNNC1. Finally, no genetic variants were identified in 9 cases (17%).
Discussion This study examines, for the first time, the genetic basis of familial DCM in heart-transplanted individuals. It is also the first to analyze the yield of modern NGS technology plus detailed familial studies in this setting. The results of the study show that the genetic spectrum of familial DCM undergoing HTx is heterogeneous and that several genes are involved in the pathogenesis of the disease. It also reveals that current NGS genetic studies when combined with detailed familial evaluation allow identification of the disease-causing mutation in up to 73% of individuals.
9
Genetic basis of heart transplanted DCM Although the genetic basis of DCM has been known for more than 30 years and DCM is the leading cause of HTx worldwide(2-4), very few studies have examined the genetic characteristics of heart transplant recipients(9,10). Genetic studies performed to date have been confined to small group of patients and to a limited number of genes. In 2005, Karkkainen et al.(9) investigated the prevalence of lamin A/C mutations among 66 DCM heart transplanted recipients and found mutations in 6 (9%) cases. Later, our group examined 89 heart-transplanted individuals for mutations in 5 genes coding for desmosomal proteins and found PM in 13 patients (15%)(10). Taken together, both studies suggested that several genes could be implicated in the pathogenesis of the condition and that diverse genes could predispose to a poor clinical course; however, this had never been explored in detail. The results of present study illustrate the genetic heterogeneity of heart transplanted DCM as demonstrated by the fact that PM were found in >10 different genes. Not surprisingly, the large TTN gene was one of the principal genes in which mutations were found. Interestingly, disease-causing mutations were recurrently found in other less well-characterized genes e.g. BAG3 and FLNC. Previous studies in the field also raised the question of whether the adverse clinical course in DCM could be related to the effect of simultaneous multiple mutations in several DCM-causing genes. When systematic screening of DCM-associated genes has been applied to large DCM cohorts, possible or likely disease-causing genetic variants have been described in up to 27% of patients (20-22). Surprisingly, little information has been provided, up to now, in relation to the coexistence of ≥2 pathogenic mutations in multiple genes(20-22). Compound heterozygosity has been described in ARVC and HCM patients, in whom the presence of more than one mutation caused earlier onset and more severe disease(23,24). In our study, 11 patients (21.2%) showed a combination of PM with a second PM (5.8%) or with VUS (15.4%).
10 Excluding patients with the EMD - c.77T>C founder mutation increased the rate of patients with multiple genetic variants to a striking 23.1%. This is much higher than the rate of multiple mutations found among patients with other inherited cardiomyopathies and poor clinical course(24,25). Patients with multiple genetic variants did not show any specific clinical or histological finding in our cohort (Table 4S. Supplementary material 1), but all the patients included in our study showed a severe phenotype with end-stage heart failure. We hypothesize that the combination of multiple mutations could lead to a worse clinical course in patients with DCM. Further studies with a larger number of patients with diverse clinical courses, and robust genetic data, are needed to confirm if this genetic “multi-hit” damage determines prognosis in familial DCM. If confirmed, routine genetic testing with NGS could become of immense help in predicting the patients’ clinical course.
Yield of NGS analysis and importance of familial evaluation Over the last 2 decades, mutations in more than 50 genes have been associated with DCM(68). Despite these advances, the use of genetic screening in daily practice has been limited by the low yield of genetic testing strategies. In this report, we have used NGS technology to gain further insight into familial DCM genetics and to evaluate the yield of NGS studies in this setting. Applying NGS technology, we were able to analyze simultaneously 59 DCM-related genes, and we could determine the causal PM in 40% of our patients considering only the NGS data. Moreover, after performing detailed familial evaluation studies, the causal PM could be identified in 73% of patients, highlighting the crucial role of familial cosegregation studies in this disease. Patients in whom PM were identified showed less enlarged LV dimensions and higher prevalence of ventricular arrhythmias, SCD and family history of SCD, compared with patients in whom PM were not found (Table 5S. Supplementary material 1).
11 Previous genetic studies analyzing a mean of 6 to 12 genes by Sanger sequencing revealed a genetic testing yield ranging from 17% to 27% and around 25% when considering only familial DCM cases(19-22). Our results reflect a substantial increase in the yield obtained by modern NGS techniques. Nevertheless, NGS technologies also have the drawback that multiple genetic variants of unknown significance might be identified in genes both related and apparently unrelated to the investigated disease that would need further investigation(12-15). The landmark study of Hass and colleagues(16) reported a significant number of mutations in genes such as PKP2 and TTN that are known to have a high number of genetic variants that are difficult to classify(26,27). In the present study, we were extremely cautious in classifying the causal PM and VUS and used familial evaluation to confirm/discard their pathogenicity. Furthermore, we found that 25% of our patients exhibited relevant genetic variants in genes related to other cardiovascular diseases and some of them were indeed disease-causing mutations (Table 3S. Supplementary material 1). Expanded genetic studies with careful phenotypic and familial studies will help to classify genetic variants in the future, but we expect that this issue will remain one of the major challenges in the DCM field because the majority of mutations belong only to one or a few families, and functional data are often lacking.
Clinical implications The fact that approximately 20-25% of heart transplant patients have direct evidence of familial disease illustrates the importance of providing genetic counseling and prompt clinical evaluation to the relatives of patients with end-stage DCM. As shown in this study, new genetic testing techniques plus detailed familial evaluation have a strong potential to identify the causal PM in end-stage familial DCM. The genetic yield observed is higher than that reported in other genetically determined diseases such as HCM, in which genetic testing is advocated in guidelines with a class I recommendation(28). Incorporating genetic testing to
12 routine clinical practice requires a change in most heart failure/HTx units and will involve adopting a different approach to patients and families. In particular, genetic testing should always be offered to DCM patients undergoing HTx as it might have an impact on their relatives’ clinical management (possibility of providing genetic counselling). For those patients who are reluctant to undergo genetic testing, special emphasis should be made in favor of performing at least clinical screening in first-degree relatives. Identification of other relatives with overt DCM could facilitate genetic testing in that family. Regardless, genetic information should be carefully managed and we recommend that specific protocols should be adopted in advance in order to manage difficult situations (genetic testing in minors, intellectually disabled, post-mortem genetic studies, etc.). Finally, as some studies have shown differential clinical course in DCM based on the underlying genetic defect(29-31), additional genetic studies in DCM HTx recipients will help to define the clinical course and natural history of this complex disease.
Conclusions The genetic spectrum of familial DCM undergoing HTx is heterogeneous and involves multiple genes. Current NGS technology plus detailed familial studies allow the identification of causative mutations in the majority of familial DCM cases. Careful interpretation of genetic results plus detailed familial studies are critical to determine the pathogenicity of genetic variants. Genetic testing should always be offered to end-stage familial DCM patients.
Acknowledgments We gratefully acknowledge SECUGEN for help with the haplotype analysis and Kenneth McCreath for English editing.
13 Funding This work was supported by the Instituto de Salud Carlos III [grants PI11/0699, RD012/0042/0015, RD012/0042/0049, RD012/0042/0066 and RD12/0042/0069] and the Spanish Ministry of Science and Innovation [SAF2010-22153-C03-03]. Grants are supported by the Plan Nacional de I+D+I 2008-2011 and the Plan Estatal de I+D+I 2013-2016 – European Regional Development Fund (FEDER) “A way of making Europe”.
Disclosures IG-D is an employee of Health in Code. LM is shareholder of Health in Code. SC, LP-B, EL-P and PG-P have a patent on diagnostic testing. The other authors have no conflicts of interest to declare.
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Hershberger RE, Hedges DJ, Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol 2013;10:531-47.
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Kärkkäinen S, Reissell E, Heliö T, et al. Novel mutations in the lamin A/C gene in heart transplant recipients with end stage dilated cardiomyopathy. Heart 2006;92:524-6.
10. Garcia-Pavia P, Syrris P, Salas C, et al. Desmosomal protein gene mutations in patients with idiopathic dilated cardiomyopathy undergoing cardiac transplantation: A clinicopathological study. Heart 2011;97:1744-52. 11. Garcia-Pavia P, Cobo-Marcos M, Guzzo-Merello G, et al. Genetics in Dilated Cardiomyopathy. Biomark Med 2013;7:517-33. 12. George AL. Use of Contemporary Genetics in Cardiovascular Diagnosis. Circulation 2014;130:1971-80. 13. Sikkema-Raddatz B, Johansson LF, de Boer EN, et al. Targeted next-generation sequencing can
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2013;34:1035-42. 14. Biswas A, Rao VR, Seth S, Maulik SK. Next generation sequencing in cardiomyopathy: towards personalized genomics and medicine. Mol Biol Rep 2014;41:4881-8. 15. D'Argenio V, Frisso G, Precone V, et al. DNA sequence capture and next-generation sequencing for the molecular diagnosis of genetic cardiomyopathies. J Mol Diagn 2014;16:32-44.
15 16. Haas J, Frese KS, Peil B, et al. Atlas of the clinical genetics of human dilated cardiomyopathy. Eur Heart J 2015;36:1123-35. 17. Mestroni L, Maisch B, McKenna WJ, et al. Guidelines for the study of familial dilated cardiomyopathies. Collaborative Research Group of the European Human and Capital Mobility Project on Familial Dilated Cardiomyopathy. Eur Heart J 1999;20:93-102. 18. Richardson P, McKenna W, Bristow M, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 1996;93:841-2. 19. van Spaendonck-Zwarts KY, van Rijsingen IA, van den Berg MP, et al. Genetic analysis in 418 index patients with idiopathic dilated cardiomyopathy: overview of 10 years’ experience. Eur J Heart Fail 2013;15:628-36. 20. Hershberger RE, Norton N, Morales A, Li D, Siegfried JD, Gonzalez-Quintana J. Coding Sequence Rare Variants Identified in MYBPC3, MYH6, TPM1, TNNC1 and TNNI3 from 312 Patients with Familial or Idiopathic Dilated Cardiomyopathy. Circ Cardiovasc Genet 2010;3:155-61. 21. Hershberger RE, Parks SB, Kushner JD, et al. Coding sequence mutations identified in MYH7, TNNT2, SCN5A, CSRP3, LBD3, and TCAP from 313 patients with familial or idiopathic dilated cardiomyopathy. Clin Transl Sci 2008; 1:21-6. 22. Lakdawala NK, Funke BH, Baxter S, et al. Genetic testing for dilated cardiomyopathy in clinical practice. J Card Fail 2012;18:296-303. 23. Girolami F, Ho CY, Semsarian C, et al. Clinical Features and Outcome of Hypertrophic Cardiomyopathy Associated With Triple Sarcomere Protein Gene Mutations. J Am Coll Cardiol 2010;55:1444-53. 24. Quarta G, Muir A, Pantazis A, et al. Familial Evaluation in Arrhythmogenic Right Ventricular Cardiomyopathy / Clinical Perspective: Impact of Genetics and Revised Task Force Criteria. Circulation 2011;123:2701-9.
16 25. Garcia-Pavia P, Vázquez ME, Segovia J, et al. Genetic basis of end-stage Hypertrophic Cardiomyopathy. Eur J Heart Fail 2011;13:1193-201. 26. Kapplinger JD1, Landstrom AP, Salisbury BA, et al. Distinguishing arrhythmogenic right ventricular cardiomyopathy/dysplasia-associated mutations from background genetic noise. J Am Coll Cardiol 2011;57:2317-27.
27. Roberts AM, Ware JS, Herman DS, et al. Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease. Sci Transl Med 2015;7:270ra6. 28. Elliott PM, Anastasakis A, Borger MA, et al. 2014 ESC Guidelines on diagnosis and management of hypertrophic cardiomyopathy: The Task Force for the Diagnosis and Management of Hypertrophic Cardiomyopathy of the European Society of Cardiology (ESC). Eur Heart J 2014;14;35:2733-79. 29. Van Rijsingen IA, Arbustini E, Elliott PM, et al. Risk factors for malignant ventricular arrhythmias in lamin a/c mutation carriers a European cohort study. J Am Coll Cardiol 2012;59:493-500. 30. Diegoli M, Grasso M, Favalli V, et al. Diagnostic work-up and risk stratification in X-linked dilated cardiomyopathies caused by distrophin defects. J Am Coll Cardiol 2011;58:925-34. 31. Van Rijsingen IA, van der Zwaag PA, Groeneweg JA, et al. Outcome in phospholamban R14del carriers: results of a large multicentre cohort study. Circ Cardiovasc Genet 2014;7:455-65.
Tables Table 1. Genetic variants identified and initially classified as pathogenic mutations. Table 2. Genetic variants identified and initially classified as variants of unknown significance.
17
Figures Figure 1. Family trees from 3 selected families (Family 21, top left; Family 1, top right; Family 37, bottom). Family 21: Genetic variant was initially classified as a pathogenic mutation. Family evaluation confirmed its pathogenicity. Family 1: Genetic variant was initially classified as a variant of unknown significance. Family evaluation showed absence of cosegregation and the variant was reclassified as non-pathogenic. Family 37: Genetic variant was initially classified as a variant of unknown significance. Family evaluation confirmed cosegregation and the variant was reclassified as pathogenic. Squares and circles indicate male and female family members, respectively. Symbols with a single slash mark are deceased family members. Arrows indicate probands. Solid symbols are affected individuals. Symbols containing a dot are unaffected carriers. Symbols containing an “N” are unaffected noncarriers. The ages stated refer to age at the time of death for deceased family members and current age for living family members. HTx indicates heart transplant; y, years; CE, clinical evaluation (including EKG and echocardiogram); SCD, sudden cardiac death; ICD, implantable cardioverter-defibrillator; HF, heart failure; AMI, acute myocardial infarction; DCM, dilated cardiomyopathy; CVA, cerebrovascular accident.
Figure 2. Shared haplotype surrounding the EMD gene in c.77T>C mutation carrying patients. The table shows constructed haplotypes among 9 different heart transplanted individuals. The shared markers between different families are depicted in grey.
Figure 3. Final genetic results in 52 heart transplanted patients due to familial DCM.
18 Table 1. Genetic variants identified and initially classified as pathogenic mutations.
G en e
A BC C9
B A G 3
B A G 3
Mutation*
Coddin g effect
NM_005691:c.4570_457 2delTTAinsAAAT
Leu152 4Lysfs* 5
NM_004281.3:c.361C>T
Arg121 *
NM_004281.3:c.382_383 insAG
Ala128 Glufs* 84
Pa tie nt
7
11
12
Pre vio usly des crib ed
Yes
No
No
Des crib ed in con trol s
No
No
No
Cons erva tion
-
-
-
SIF T pre dict ion (sco re)
-
-
-
Pol yph en2 pre dict ion (sco re)
-
-
-
Mut atio n Tast er (sco re)
-
-
-
Cons eque nce
Trun catin g fram eshif t Nons ense muta tion Trun catin g fram eshif t
Cose grega tion
Final Class ificat ion
Not dem onstr ated
Path ogen ic muta tion
Not dem onstr ated
Path ogen ic muta tion
Yes
Path ogen ic muta tion
Yes
Path ogen ic muta tion
Yes
Path ogen ic muta tion
B A G 3
NM_004281.3:c.1316_13 17delTA
Val439 Glyfs* 4
13
No
No
-
-
-
Trun catin g fram eshif t
B A G 3
NM_004281.3:c.903delG
Arg301 Serfs* 6
15
No
No
-
-
-
D M D
NM_004006.2:c.4069G> T
Glu135 7*
6
No
No
-
-
-
DS P
NM_004415.2:c.5851C>T
Arg195 1*
41
No
No
-
-
-
FL N C FL N
NM_001458.4:c.6231del T
Ser207 7Argfs *50
10
No
No
NM_001458.4:c.4106_41 07insA
Asn13 69Lysf
27
No
No
-
-
-
-
-
-
-
Trun catin g fram eshif t Nons ense muta tion Nons ense muta tion Trun catin g fram eshif t Trun catin
Yes
Not dem onstr ated
Path ogen ic muta tion Path ogen ic muta tion
Yes†
Path ogen ic muta tion
Not dem
Path ogen
19 C
L M N A
L M N A
L M N A
s*36
NM_170707.3:c.569G>A
Arg190 Gln
9
Yes
No
High
Aff ects pro tein fun ctio n (0.0 0)
Pro bab ly Da ma gin g (0.9 98)
Pres uma bly dise ase caus ing (1.0 00)
NM_170707.3:c.961C>T
NM_170707.3:c.568C>T
Arg321 *
Arg190 Trp
23
52
Yes
Yes
T N N T2
TT N
TT N
TT N
TT N
NM_004572.3:c.223+1G >A
NM_001001430.1:c.422 G>A
NM_003319.4:c.39059C >A
NM_003319.4:c.13000A >T
16
Arg141 Gln
Ser130 20*
Arg433 4*
NM_003319.4:c.75042_7 5043delAG
Arg250 14Serf s*4
NM_003319.4:c.59900de lC
Pro199 67Leuf s*8
17
51
2
5
7
Yes
Yes
No
No
No
No
onstr ated
Miss ense muta tion
Not dem onstr ated
Path ogen ic muta tion
Yes
Path ogen ic muta tion
Nons ense muta tion
ic muta tion
No
-
-
-
No
Aff ects pro tein fun ctio n (0.0 0)
Pro bab ly Da ma gin g (0.9 58)
Pres uma bly dise ase caus ing (1.0 00)
Miss ense muta tion
Not dem onstr ated
Path ogen ic muta tion
Not dem onstr ated
Path ogen ic muta tion
Yes†
Path ogen ic muta tion
High
PK P2
g fram eshif t
No
No
No
No
No
No
High
-
-
-
-
-
-
-
Abno rmal splici ng
Tol erat ed (0.2 2)
Pro bab ly Da ma gin g (1.0 00)
Pres uma bly dise ase caus ing (1.0 00)
Miss ense muta tion
-
-
-
-
-
-
-
-
-
--
-
-
Nons ense muta tion Nons ense muta tion Trun catin g fram eshif t Trun catin g fram eshif t
Yes
Not dem onstr ated
Path ogen ic muta tion Path ogen ic muta tion
Yes
Path ogen ic muta tion
Not dem onstr ated
Path ogen ic muta tion
20
TT N
TT N
TT N
TT N
TT N
TT N
NM_003319.4:c.60310_6 0314delCAAGT
NM_003319.4:c.12052G >T
NM_003319.4:c.56232_5 6233insTTGTCGAAAAAC GATAAACTACA
NM_003319.4:c.48240G >A
NM_003319.4:c.53521C >T
NM_003319.4:c.23997_2 3998insT
Gln201 04Tyrf s*17
Glu401 8*
Arg187 45Leuf s*69
Trp160 80*
Arg178 41*
Lys800 0*
9
17
18
19
21
50
No
No
No
No
No
No
* Variants are referred to (GRCh37.p13). † Cosegregation along with other mutations
No
No
No
No
No
No
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Trun catin g fram eshif t Nons ense muta tion Trun catin g fram eshif t Nons ense muta tion Nons ense muta tion Trun catin g fram eshif t
Yes†
Path ogen ic muta tion
Yes†
Path ogen ic muta tion
Yes†
Path ogen ic muta tion
Yes
Yes
Not dem onstr ated
Path ogen ic muta tion Path ogen ic muta tion Path ogen ic muta tion
21 Table 2. Genetic variants identified and initially classified as variants of unknown significance.
Ge ne
AC TN 2
AC TN 2
Mutation*
NM_001103 .2:c.1040C> T
Codd ing effec t
Thr3 47M et
NM_001103 .2:c.26A>G
Gln9 Arg
BA G3
NM_004281 .3:c.343C>T
Pro1 15Se r
DM D
NM_004006 .2:c.2057C> T
Thr6 86Ile
DS C2
NM_024422 .3:c.2194T> G
Leu7 32Va l
DS C2
NM_024422 .3:c.13C>T
Arg5 Cys
Pa tie nt
11
20
43
52
24
33
Prev ious ly desc ribe d
No
Yes
Yes
No
Yes
No
DS G2
NM_001943 .3:c.1174G> A
Val3 92Ile
24
Yes
DS
NM_004415
Arg9
9
No
Des crib ed in cont rols
M AF (% )
No
-
Yes
0. 05 38
Yes
-
No
-
Yes
0. 10 76
No
-
Yes
0. 15 87
No
-
Poly phe n-2 pre dicti on (sco re) Pro babl y dam agin g (0.9 89)
Cons ervati on
SIFT pre dicti on (sco re)
High
Affe cts prot ein func tion (0.0 0)
High
Tole rate d (0.1 3)
Beni gn (0.0 00)
High
Tole rate d (1.0 0)
Beni gn (0.0 01)
High
Tole rate d (0.1 0)
Beni gn (0.1 47)
Medi um
Tole rate d (0.1 5)
Beni gn (0.0 05)
Weak
Tole rate d (0.1 3)
Poss ibly dam agin g (0.6 42)
Weak
Tole rate d (0.1 4)
Beni gn (0.0 01)
High
Affe
Beni
Mutat ion Taster (score ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (0.576 ) Polym orphis m (1.000 ) Presu mably diseas e causin g (1.000 ) Polym orphis m (0.989 ) Polym orphis m (1.000 ) Presu mably diseas e causin g (0.000 ) Presu
Cons eque nce
Coseg regati on
Final classi ficati on
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
No
Not patho genic
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Yes
Patho genic muta tion
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse
Yes†
VUS
22 P
.2:c.2720G> A
07Hi s
DS P
NM_004415 .2:c.6881C> G
Ala2 294G ly
DS P
NM_004415 .2:c.2186T> C
Met 729T hr
45
Val2 6Ala
14, 28, 31, 32, 35, 39, 40, 42, 43, 44, 45, 46, 47
EM D
FLN C
NM_000117 .2:c.77T>C
NM_001458 .4:c.14126C>G
-
FLN C
NM_001458 .4:c.6617G> A
Arg2 206G ln
MY BP C3
NM_000256 .3:c.594C>A
Asp1 98Gl u
MY BP C3
NM_000256 .3:c.3742G> A
Gly1 248A rg
10
1
18
34
29
Yes
No
No
No
No
No
Yes
Yes
No
No
No
No
0. 02 31
-
-
-
-
No
-
Yes
0. 02 45
High
Medi um
High
-
High
High
High
cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 1)
Tole rate d (0.0 7)
-
Affe cts prot ein func tion (0.0 4) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func
gn (0.0 08)
Pro babl y dam agin g (0.9 20)
mably diseas e causin g (0.999 ) Presu mably diseas e causin g (1.000 )
nse mutat ion
Misse nse mutat ion
Yes†
VUS
Not demo nstrat ed
VUS
Beni gn (0.0 08)
Polym orphis m (0.667 )
Misse nse mutat ion
Poss ibly dam agin g (0.5 95)
Polym orphis m (0.999 )
Misse nse mutat ion
Yes
Patho genic muta tion
-
Poten tial abnor mal splici ng
No
Not patho genic
Misse nse mutat ion
Yes†
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
No
Not patho genic
-
Beni gn (0.1 05)
Beni gn (0.1 94)
Poss ibly dam agin g
Presu mably diseas e causin g (0.995 ) Presu mably diseas e causin g (0.994 ) Presu mably diseas e causin
23
MY H6
NM_002471 .3:c.49C>T
Arg1 7Cys
MY H7
NM_000257 .2:c.1618T> C
Phe5 40Le u
MY PN
PK P2
PSE N2
PSE N2
NM_032578 .3:c.59A>G
NM_004572 .3:c.156G>C
NM_000447 .2:c.21+5G>C
NM_000447 .2:c.389C>T
Tyr2 0Cys
Lys5 2Asn
-
Ser1 30Le u
TN NC 1
NM_003280 .2:c.400G>A
Glu1 34Ly s
TN NT 2
NM_001001 430.1:c.586 C>T
Arg1 96Tr p
25
37
3
16
3
26
24
22
No
No
Yes
No
No
Yes
No
No
No
No
Yes
No
No
Yes
No
No
-
-
0. 09 23
-
-
0. 06 92
Medi um
High
High
Weak
-
Very high
-
High
-
Medi um
tion (0.0 0) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 0) Tole rate d (0.1 4)
-
Affe cts prot ein func tion (0.0 2) Affe cts prot ein func tion (0.0 3) Affe cts prot ein func tion
(0.5 63) Pro babl y dam agin g (0.9 55)
Beni gn (0.0 30)
Pro babl y dam agin g (0.9 98) Poss ibly dam agin g (0.7 80)
-
Poss ibly dam agin g (0.5 23) Pro babl y dam agin g (0.9 57) Pro babl y dam agin g
g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (0.613 )
-
Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Yes
Patho genic muta tion
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Poten tial abnor mal splici ng
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Yes
Patho genic muta tion
Misse nse mutat ion
24
TP M1
VCL
NM_001018 005.1:c.337 C>G
NM_014000 .2:c.1433A> G
Leu1 13Va l
Asn4 78Se r
48
33
No
No
No
No
-
-
* Variants are referred to (GRCh37.p13). † Cosegregation along with other mutations
Medi um
Very high
(0.0 1) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 4)
(0.9 78) Poss ibly dam agin g (0.6 53) Poss ibly dam agin g (0.7 55)
(0.999 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 )
Misse nse mutat ion
Yes
Patho genic muta tion
Misse nse mutat ion
Yes†
VUS
25
Genetic Basis of Familial Dilated Cardiomyopathy Undergoing Heart Transplantation Sofia Cuenca, Maria J. Ruiz-Cano, Juan Ramón Gimeno-Blanes, Alfonso Jurado, Clara Salas, Iria Gomez-Diaz, Laura Padron-Barthe, Jose Javier Grillo, Carlos Vilches, Javier Segovia, Domingo Pascual-Figal, Enrique Lara-Pezzi, Lorenzo Monserrat, Luis Alonso-Pulpon, Pablo Garcia-Pavia, for the Inherited Cardiac Diseases program of the Spanish Cardiovascular Research Network (Red Investigación Cardiovascular)
PII: DOI: Reference:
S1053-2498(16)00009-7 http://dx.doi.org/10.1016/j.healun.2015.12.014 HEALUN6146
To appear in:
J Heart Lung Transplant
http://www.jhltonline.org
Cite this article as: Sofia Cuenca, Maria J. Ruiz-Cano, Juan Ramón Gimeno-Blanes, Alfonso Jurado, Clara Salas, Iria Gomez-Diaz, Laura Padron-Barthe, Jose Javier Grillo, Carlos Vilches, Javier Segovia, Domingo Pascual-Figal, Enrique Lara-Pezzi, Lorenzo Monserrat, Luis Alonso-Pulpon, Pablo Garcia-Pavia, for the Inherited Cardiac Diseases program of the Spanish Cardiovascular Research Network (Red Investigación Cardiovascular), Genetic Basis of Familial Dilated Cardiomyopathy Undergoing Heart Transplantation, J Heart Lung Transplant, http://dx.doi.org/10.1016/j. healun.2015.12.014 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 galley proof before it is published in its final citable 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.
1 GENETIC BASIS OF FAMILIAL DILATED CARDIOMYOPATHY UNDERGOING HEART TRANSPLANTATION Sofia Cuenca1; Maria J. Ruiz-Cano2; Juan Ramón Gimeno-Blanes3; Alfonso Jurado2; Clara Salas4; Iria Gomez-Diaz5; Laura Padron-Barthe6; Jose Javier Grillo7; Carlos Vilches8; Javier Segovia1; Domingo Pascual-Figal3; Enrique Lara-Pezzi6; Lorenzo Monserrat5; Luis Alonso-Pulpon1; and Pablo Garcia-Pavia1,6 for the Inherited Cardiac Diseases program of the Spanish Cardiovascular Research Network (Red Investigación Cardiovascular).
Affiliation: 1
Heart Failure and Inherited Cardiac Diseases Unit. Department of Cardiology. Hospital
Universitario Puerta de Hierro, Madrid, Spain. 2
Heart failure and Heart Transplantation Unit. Department of Cardiology. Hospital
Universitario 12 de Octubre, Madrid, Spain. 3
Department of Cardiology Hospital Universitario Virgen de la Arrixaca, Murcia, Spain.
4
Department of Pathology. Hospital Universitario Puerta de Hierro, Madrid, Spain.
5
Health In Code, A Coruña, Spain.
6
Myocardial Biology Programme, Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid, Spain. 7
Department of Cardiology. Hospital Univ. Nuestra Señora de Candelaria, Tenerife, Spain.
8
Department of Immunology. Hospital Universitario Puerta de Hierro, Madrid, Spain.
Corresponding author: Pablo Garcia-Pavia, MD, PhD. Department of Cardiology. Hospital Universitario Puerta de Hierro. Manuel de Falla, 2. Majadahonda, Madrid, 28222, Spain. Email: [email protected]
Background: Dilated cardiomyopathy (DCM) is the most frequent cause of heart transplantation (HTx). The genetic basis of DCM among patients undergoing HTx is poorly characterized. We sought to determine the genetic basis of heart-transplanted familial DCM
2 and to establish the yield of modern Next Generation Sequencing (NGS) technologies in this setting.
Methods: Fifty-two heart-transplanted patients due to familial DCM underwent NGS genetic evaluation with a panel of 126 genes related to cardiac conditions (59 associated with DCM). Genetic variants were initially classified as pathogenic mutations or as variants of uncertain significance (VUS). Final pathogenicity status was determined by familial cosegregation studies.
Results: Initially, 24 pathogenic mutations were found in 21 patients (40%), 25 patients (48%) carried 19 VUS and 6 (12%) did not show any genetic variant. Familial evaluation of 220 relatives from 36 of the 46 families with genetic variants confirmed pathogenicity in 14 patients and allowed reclassification of VUS as pathogenic in 17 patients, and as nonpathogenic in 3 cases. At the end of the study, the DCM-causing mutation was identified in 38 patients (73%) and 5 patients (10%) harbored only VUS. No genetic variants were identified in 9 cases (17%).
Conclusions: The genetic spectrum of familial DCM undergoing heart transplantation is heterogeneous and involves multiple genes. NGS technology plus detailed familial studies allow identification of causative mutations in the vast majority of familial DCM cases. Detailed familial studies remain critical to determine the pathogenicity of underlying genetic defects in a substantial number of cases.
Keywords:
Familial
transplantation.
dilated
cardiomyopathy;
Next
Generation
Sequencing;
heart
3
Introduction Dilated cardiomyopathy (DCM), defined as dilatation and systolic impairment of the left or both ventricles in the absence of abnormal loading conditions or coronary artery disease(1), is the most common cause of heart failure in the young and the most frequent indication for heart transplantation (HTx) in adults and in children older than 1 year(2-4). Family studies in predominantly stable outpatients suggest that up to 20-50% of DCM patients have a familial predisposition to the disease(5-7). However, the genetic basis of this disease in most patients is still unknown(8), particularly in those with advanced disease in which there are few genetic studies(9,10). More than 50 genes have been associated so far with familial DCM; however, the precise contribution of mutations in each of these genes to this condition is unknown(8,11). The large number of genes related to familial DCM has made the study of the genetics of patients with DCM arduous, and has limited the application of genetic screening strategies to everyday clinical practice. Recently, there have been major advances in the field of genomics with the emergence of new generation sequencing techniques (Next Generation Sequencing, NGS), which enable the study of a large number of genes simultaneously in a short period of time and at a relatively low cost(12-15). A large study recently published has been the first to provide data of genetic findings in DCM patients through NGS(16). This study demonstrates the potential of NGS technology applied to the DCM field, but also illustrates the drawback of identifying rare variants without performing corroborative familial studies. To date, no major studies with complete familial evaluation have provided robust data on the genetic testing yield of NGS in DCM.
4 The purpose of this study was two-fold: to determine the genetic basis of heart transplanted DCM, and to establish the yield of modern NGS technologies plus detailed familial studies in this setting. Methods Study Population: This study was approved by the ethics committee of the participant centers and complies with the Declaration of Helsinki. A total of 52 non-related heart transplanted patients due to familial DCM at 3 Spanish heart transplant centers (Hospital Universitario Puerta de Hierro, Hospital Universitario 12 de Octubre, and Hospital Universitario Virgen de la Arrixaca) were included. DCM was defined as familial if one or more relatives (in addition to the proband) had DCM during life or at postmortem examination, or presented unexplained sudden cardiac death (SCD) before the age of 35(17). Only those patients fulfilling World Health Organization/International Society and Federation of Cardiology Task Force clinical criteria for DCM(18) at time of heart transplant were included. Patients with significant coronary artery disease or myocarditis at pathological examination of the explanted heart were excluded. Physicians blinded to the genetic results reviewed patients’ records retrospectively. Clinical data and results from the first pre-transplant evaluation at each center were collected.
Genetic evaluation DNA was obtained from blood samples extracted from the probands and stored at -70 0C. DNA samples were analyzed by NGS with a panel of 126 genes related to inherited cardiovascular diseases and 59 specifically associated with DCM (Table S1 from Supplementary material 1), including all coding exons and flanking intronic regions. Targeted enrichment was done with SureSelect (Agilent) and the design of the capture baits was carried out using eArray (Agilent).
5 DNA was sequenced in an Illumina HiSeq 1500 platform. Bioinformatic analysis was performed with a previously validated in-house pipeline. Mean coverage was 456 times and >99% of the fragments had coverage >15 times, and >98% had coverage >30 times. Sanger sequencing was used to evaluate regions of low coverage (<15 times) and to confirm the genetic variants found. Sequence variants were initially classified as pathogenic mutations (PM) or variants of uncertain significance (VUS). Variants were considered PM if they were found in a DCMassociated gene, had not been found in controls, and had been reported previously as pathogenic
in
literature
or
in
online
(http://www.ncbi.nlm.nih.gov/clinvar/)
and
international The
Human
databases Gene
such
Mutation
as
ClinVar Database
(http://www.hgmd.cf.ac.uk), or if they were novel sequence variants in a previously DCMassociated gene not found in controls that predicted a premature truncation, frameshift or abnormal splicing of the protein. Variants were classified as VUS if they were novel missense variants in a previously DCM-associated gene not found in controls, or if they were variants reported previously as pathogenic in online international databases that had been also found in controls. Other relevant genetic variants in genes not related to DCM were noted although they were excluded from the present study. VUS were reclassified according to results of familial evaluation (Figure 1). If VUS cosegregated with DCM on familial evaluation, they were annotated as a PM. In contrast, if VUS did not cosegregate with DCM, they were considered as non-pathogenic variants. Finally, VUS without corroborative family screening data or which cosegregated with another VUS were not reclassified and remained as VUS. Genetic databases used to determine the presence of variants among controls were dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and the NHLBI GO Exome Sequencing Project database (http://evs.gs.washington.edu/EVS/).
6 Conservation of amino acid residues affected by genetic variants found was determined using AlaMut (version 2.4.5; Interactive Biosoftware, Rouen, France). Conservation was graded very high, high, medium or low as previously reported(19).
Familial evaluation All relatives >16-years-old of patients with PM/VUS were offered clinical and genetic evaluation. Clinical evaluation included physical examination, ECG and an echocardiogram. Family screening was considered positive if one or more relatives had DCM and the same genetic defect as the proband (Supplemental material 2).
Haplotype analysis of emerin c.77T>C mutation carriers Three polymorphic microsatellite markers and six SNPs were genotyped in 9 mutation carriers. Polymorphic microsatellite markers flanking the emerin (EMD) gene were chosen using the Marshfield
genetic
maps
(http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp) and the UCSC genome browser (http://genome.ucsc.edu/). The SNPs were chosen from NCBI SNP database. The 9 markers (2 upstream and 7 downstream of the mutation) were located in a region of 2.87 Mb (3.35 cM) on chromosome X. PCR fragments from the amplification of the polymorphic markers were fluorescently labeled using a labeled oligonucleotide. Fragment analyses were performed on an ABI3730 automated sequencer using software package GeneMapper 4.0. Allele sizes of patients are inferred from the allele sizes present in the CEPH database (http://www.cephb.fr/). SNP genotyping was undertaken by specific PCR amplification of the region of interest and DNA sequencing with Big Dye terminator 3.1 in an AB3730 automated sequencer.
7
Statistical analysis Continuous variables are expressed as mean value ± SD. Discrete variables are shown as percentages. All data were analyzed using SPSS software (version 16.0).
Results The study cohort comprised 52 patients (mean age at first evaluation 39.9±14.3 years, range 11 to 64; 92% males). Clinical and familial characteristics are presented in Table S2 from Supplementary material 1. Fifty-one patients (98%) had known family history of DCM and 20 (38%) had family history of SCD (7 in a relative under the age of 35). Pedigrees of the 52 families are provided (Supplementary material 2). Initially, 24 PM were found in 21 patients (40%)(Table 1). Three patients harbored two PM in different genes and 6 patients presented also VUS. A total of 18 PM (75%) were novel stop codon mutations. One was a previously described mutation, causing abnormal splicing and a premature stop codon, 3 were previously described missense variants not reported in controls and 2 were previously described stop codon mutations. Twenty-five patients (48%) carried 19 VUS (Table 2). Twelve were novel missense variants not reported in controls and 7 were previously reported DCM-causing mutations also described in controls (Table 2). Finally, 6 patients (12%) did not show any genetic variations in analyzed DCM-related genes. Several relevant genetic variants in other cardiovascular-related genes were also identified (Table S3. Supplementary material 1). Familial evaluation included clinical and genetic study of 220 relatives from 36 of the 46 families with PM/VUS (78%)(Supplementary material 2). Familial study confirmed pathogenicity of the mutations in 14 patients with PM (Figure 1 and Supplementary material 2). Among patients with VUS, familial evaluation allowed reclassification of VUS as PM in 17
8 families and as non-pathogenic in 3 cases (Supplemental material 2). Familial evaluation was not possible in 3 patients with VUS and it was inconclusive in 2 patients. Among the 17 families with VUS which were reclassified as PM, 13 harbored the same missense mutation in the EMD gene (c.77T>C). These subjects were apparently unrelated males, without muscular phenotype, originating from the same geographical area (Tenerife island). A haplotype study in 9 patients with the EMD - c.77T>C mutation was consistent with a shared haplotype (Figure 2) and therefore a new founder mutation was described. After completing familial evaluation, 5 of the 17 variants classified as VUS showed a minor allele frequency (MAF) >1%, despite having previously been reported as DCM-causing mutations (Table 2). Given their MAF frequency, it is unlikely that these variants are DCMcausing by themselves, but we could not completely discard a modifier role that these variants could play in the presence of other aggressive mutations. At the end of the study, the causal DCM mutation was identified in 38 patients (73%)(Figure 3). Mutated genes included: EMD (13 patients), TTN (10), BAG3 (4), LMNA (3), FLNC (2), TNNT2 (2), DMD, DSC2, DSP, MYH7, PKP2, ABCC9 and TPM1. A total of 5 patients (10%) harbored only VUS in the following genes: PSEN2 (2), DSC2, DSG2, MYBPC3, MYH6, MYPN, and TNNC1. Finally, no genetic variants were identified in 9 cases (17%).
Discussion This study examines, for the first time, the genetic basis of familial DCM in heart-transplanted individuals. It is also the first to analyze the yield of modern NGS technology plus detailed familial studies in this setting. The results of the study show that the genetic spectrum of familial DCM undergoing HTx is heterogeneous and that several genes are involved in the pathogenesis of the disease. It also reveals that current NGS genetic studies when combined with detailed familial evaluation allow identification of the disease-causing mutation in up to 73% of individuals.
9
Genetic basis of heart transplanted DCM Although the genetic basis of DCM has been known for more than 30 years and DCM is the leading cause of HTx worldwide(2-4), very few studies have examined the genetic characteristics of heart transplant recipients(9,10). Genetic studies performed to date have been confined to small group of patients and to a limited number of genes. In 2005, Karkkainen et al.(9) investigated the prevalence of lamin A/C mutations among 66 DCM heart transplanted recipients and found mutations in 6 (9%) cases. Later, our group examined 89 heart-transplanted individuals for mutations in 5 genes coding for desmosomal proteins and found PM in 13 patients (15%)(10). Taken together, both studies suggested that several genes could be implicated in the pathogenesis of the condition and that diverse genes could predispose to a poor clinical course; however, this had never been explored in detail. The results of present study illustrate the genetic heterogeneity of heart transplanted DCM as demonstrated by the fact that PM were found in >10 different genes. Not surprisingly, the large TTN gene was one of the principal genes in which mutations were found. Interestingly, disease-causing mutations were recurrently found in other less well-characterized genes e.g. BAG3 and FLNC. Previous studies in the field also raised the question of whether the adverse clinical course in DCM could be related to the effect of simultaneous multiple mutations in several DCM-causing genes. When systematic screening of DCM-associated genes has been applied to large DCM cohorts, possible or likely disease-causing genetic variants have been described in up to 27% of patients (20-22). Surprisingly, little information has been provided, up to now, in relation to the coexistence of ≥2 pathogenic mutations in multiple genes(20-22). Compound heterozygosity has been described in ARVC and HCM patients, in whom the presence of more than one mutation caused earlier onset and more severe disease(23,24). In our study, 11 patients (21.2%) showed a combination of PM with a second PM (5.8%) or with VUS (15.4%).
10 Excluding patients with the EMD - c.77T>C founder mutation increased the rate of patients with multiple genetic variants to a striking 23.1%. This is much higher than the rate of multiple mutations found among patients with other inherited cardiomyopathies and poor clinical course(24,25). Patients with multiple genetic variants did not show any specific clinical or histological finding in our cohort (Table 4S. Supplementary material 1), but all the patients included in our study showed a severe phenotype with end-stage heart failure. We hypothesize that the combination of multiple mutations could lead to a worse clinical course in patients with DCM. Further studies with a larger number of patients with diverse clinical courses, and robust genetic data, are needed to confirm if this genetic “multi-hit” damage determines prognosis in familial DCM. If confirmed, routine genetic testing with NGS could become of immense help in predicting the patients’ clinical course.
Yield of NGS analysis and importance of familial evaluation Over the last 2 decades, mutations in more than 50 genes have been associated with DCM(68). Despite these advances, the use of genetic screening in daily practice has been limited by the low yield of genetic testing strategies. In this report, we have used NGS technology to gain further insight into familial DCM genetics and to evaluate the yield of NGS studies in this setting. Applying NGS technology, we were able to analyze simultaneously 59 DCM-related genes, and we could determine the causal PM in 40% of our patients considering only the NGS data. Moreover, after performing detailed familial evaluation studies, the causal PM could be identified in 73% of patients, highlighting the crucial role of familial cosegregation studies in this disease. Patients in whom PM were identified showed less enlarged LV dimensions and higher prevalence of ventricular arrhythmias, SCD and family history of SCD, compared with patients in whom PM were not found (Table 5S. Supplementary material 1).
11 Previous genetic studies analyzing a mean of 6 to 12 genes by Sanger sequencing revealed a genetic testing yield ranging from 17% to 27% and around 25% when considering only familial DCM cases(19-22). Our results reflect a substantial increase in the yield obtained by modern NGS techniques. Nevertheless, NGS technologies also have the drawback that multiple genetic variants of unknown significance might be identified in genes both related and apparently unrelated to the investigated disease that would need further investigation(12-15). The landmark study of Hass and colleagues(16) reported a significant number of mutations in genes such as PKP2 and TTN that are known to have a high number of genetic variants that are difficult to classify(26,27). In the present study, we were extremely cautious in classifying the causal PM and VUS and used familial evaluation to confirm/discard their pathogenicity. Furthermore, we found that 25% of our patients exhibited relevant genetic variants in genes related to other cardiovascular diseases and some of them were indeed disease-causing mutations (Table 3S. Supplementary material 1). Expanded genetic studies with careful phenotypic and familial studies will help to classify genetic variants in the future, but we expect that this issue will remain one of the major challenges in the DCM field because the majority of mutations belong only to one or a few families, and functional data are often lacking.
Clinical implications The fact that approximately 20-25% of heart transplant patients have direct evidence of familial disease illustrates the importance of providing genetic counseling and prompt clinical evaluation to the relatives of patients with end-stage DCM. As shown in this study, new genetic testing techniques plus detailed familial evaluation have a strong potential to identify the causal PM in end-stage familial DCM. The genetic yield observed is higher than that reported in other genetically determined diseases such as HCM, in which genetic testing is advocated in guidelines with a class I recommendation(28). Incorporating genetic testing to
12 routine clinical practice requires a change in most heart failure/HTx units and will involve adopting a different approach to patients and families. In particular, genetic testing should always be offered to DCM patients undergoing HTx as it might have an impact on their relatives’ clinical management (possibility of providing genetic counselling). For those patients who are reluctant to undergo genetic testing, special emphasis should be made in favor of performing at least clinical screening in first-degree relatives. Identification of other relatives with overt DCM could facilitate genetic testing in that family. Regardless, genetic information should be carefully managed and we recommend that specific protocols should be adopted in advance in order to manage difficult situations (genetic testing in minors, intellectually disabled, post-mortem genetic studies, etc.). Finally, as some studies have shown differential clinical course in DCM based on the underlying genetic defect(29-31), additional genetic studies in DCM HTx recipients will help to define the clinical course and natural history of this complex disease.
Conclusions The genetic spectrum of familial DCM undergoing HTx is heterogeneous and involves multiple genes. Current NGS technology plus detailed familial studies allow the identification of causative mutations in the majority of familial DCM cases. Careful interpretation of genetic results plus detailed familial studies are critical to determine the pathogenicity of genetic variants. Genetic testing should always be offered to end-stage familial DCM patients.
Acknowledgments We gratefully acknowledge SECUGEN for help with the haplotype analysis and Kenneth McCreath for English editing.
13 Funding This work was supported by the Instituto de Salud Carlos III [grants PI11/0699, RD012/0042/0015, RD012/0042/0049, RD012/0042/0066 and RD12/0042/0069] and the Spanish Ministry of Science and Innovation [SAF2010-22153-C03-03]. Grants are supported by the Plan Nacional de I+D+I 2008-2011 and the Plan Estatal de I+D+I 2013-2016 – European Regional Development Fund (FEDER) “A way of making Europe”.
Disclosures IG-D is an employee of Health in Code. LM is shareholder of Health in Code. SC, LP-B, EL-P and PG-P have a patent on diagnostic testing. The other authors have no conflicts of interest to declare.
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Michels VV, Moll PP, Miller FA, et al. The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. N Engl J Med 1992;326:77-82.
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Burkett EL, Hershberger RE. Clinical and genetic issues in familial dilated cardiomyopathy. J Am Coll Cardiol 2005;45:969-81.
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Hershberger RE, Hedges DJ, Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol 2013;10:531-47.
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Kärkkäinen S, Reissell E, Heliö T, et al. Novel mutations in the lamin A/C gene in heart transplant recipients with end stage dilated cardiomyopathy. Heart 2006;92:524-6.
10. Garcia-Pavia P, Syrris P, Salas C, et al. Desmosomal protein gene mutations in patients with idiopathic dilated cardiomyopathy undergoing cardiac transplantation: A clinicopathological study. Heart 2011;97:1744-52. 11. Garcia-Pavia P, Cobo-Marcos M, Guzzo-Merello G, et al. Genetics in Dilated Cardiomyopathy. Biomark Med 2013;7:517-33. 12. George AL. Use of Contemporary Genetics in Cardiovascular Diagnosis. Circulation 2014;130:1971-80. 13. Sikkema-Raddatz B, Johansson LF, de Boer EN, et al. Targeted next-generation sequencing can
replace
Sanger sequencing in
clinical
diagnostics.
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15 16. Haas J, Frese KS, Peil B, et al. Atlas of the clinical genetics of human dilated cardiomyopathy. Eur Heart J 2015;36:1123-35. 17. Mestroni L, Maisch B, McKenna WJ, et al. Guidelines for the study of familial dilated cardiomyopathies. Collaborative Research Group of the European Human and Capital Mobility Project on Familial Dilated Cardiomyopathy. Eur Heart J 1999;20:93-102. 18. Richardson P, McKenna W, Bristow M, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 1996;93:841-2. 19. van Spaendonck-Zwarts KY, van Rijsingen IA, van den Berg MP, et al. Genetic analysis in 418 index patients with idiopathic dilated cardiomyopathy: overview of 10 years’ experience. Eur J Heart Fail 2013;15:628-36. 20. Hershberger RE, Norton N, Morales A, Li D, Siegfried JD, Gonzalez-Quintana J. Coding Sequence Rare Variants Identified in MYBPC3, MYH6, TPM1, TNNC1 and TNNI3 from 312 Patients with Familial or Idiopathic Dilated Cardiomyopathy. Circ Cardiovasc Genet 2010;3:155-61. 21. Hershberger RE, Parks SB, Kushner JD, et al. Coding sequence mutations identified in MYH7, TNNT2, SCN5A, CSRP3, LBD3, and TCAP from 313 patients with familial or idiopathic dilated cardiomyopathy. Clin Transl Sci 2008; 1:21-6. 22. Lakdawala NK, Funke BH, Baxter S, et al. Genetic testing for dilated cardiomyopathy in clinical practice. J Card Fail 2012;18:296-303. 23. Girolami F, Ho CY, Semsarian C, et al. Clinical Features and Outcome of Hypertrophic Cardiomyopathy Associated With Triple Sarcomere Protein Gene Mutations. J Am Coll Cardiol 2010;55:1444-53. 24. Quarta G, Muir A, Pantazis A, et al. Familial Evaluation in Arrhythmogenic Right Ventricular Cardiomyopathy / Clinical Perspective: Impact of Genetics and Revised Task Force Criteria. Circulation 2011;123:2701-9.
16 25. Garcia-Pavia P, Vázquez ME, Segovia J, et al. Genetic basis of end-stage Hypertrophic Cardiomyopathy. Eur J Heart Fail 2011;13:1193-201. 26. Kapplinger JD1, Landstrom AP, Salisbury BA, et al. Distinguishing arrhythmogenic right ventricular cardiomyopathy/dysplasia-associated mutations from background genetic noise. J Am Coll Cardiol 2011;57:2317-27.
27. Roberts AM, Ware JS, Herman DS, et al. Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease. Sci Transl Med 2015;7:270ra6. 28. Elliott PM, Anastasakis A, Borger MA, et al. 2014 ESC Guidelines on diagnosis and management of hypertrophic cardiomyopathy: The Task Force for the Diagnosis and Management of Hypertrophic Cardiomyopathy of the European Society of Cardiology (ESC). Eur Heart J 2014;14;35:2733-79. 29. Van Rijsingen IA, Arbustini E, Elliott PM, et al. Risk factors for malignant ventricular arrhythmias in lamin a/c mutation carriers a European cohort study. J Am Coll Cardiol 2012;59:493-500. 30. Diegoli M, Grasso M, Favalli V, et al. Diagnostic work-up and risk stratification in X-linked dilated cardiomyopathies caused by distrophin defects. J Am Coll Cardiol 2011;58:925-34. 31. Van Rijsingen IA, van der Zwaag PA, Groeneweg JA, et al. Outcome in phospholamban R14del carriers: results of a large multicentre cohort study. Circ Cardiovasc Genet 2014;7:455-65.
Tables Table 1. Genetic variants identified and initially classified as pathogenic mutations. Table 2. Genetic variants identified and initially classified as variants of unknown significance.
17
Figures Figure 1. Family trees from 3 selected families (Family 21, top left; Family 1, top right; Family 37, bottom). Family 21: Genetic variant was initially classified as a pathogenic mutation. Family evaluation confirmed its pathogenicity. Family 1: Genetic variant was initially classified as a variant of unknown significance. Family evaluation showed absence of cosegregation and the variant was reclassified as non-pathogenic. Family 37: Genetic variant was initially classified as a variant of unknown significance. Family evaluation confirmed cosegregation and the variant was reclassified as pathogenic. Squares and circles indicate male and female family members, respectively. Symbols with a single slash mark are deceased family members. Arrows indicate probands. Solid symbols are affected individuals. Symbols containing a dot are unaffected carriers. Symbols containing an “N” are unaffected noncarriers. The ages stated refer to age at the time of death for deceased family members and current age for living family members. HTx indicates heart transplant; y, years; CE, clinical evaluation (including EKG and echocardiogram); SCD, sudden cardiac death; ICD, implantable cardioverter-defibrillator; HF, heart failure; AMI, acute myocardial infarction; DCM, dilated cardiomyopathy; CVA, cerebrovascular accident.
Figure 2. Shared haplotype surrounding the EMD gene in c.77T>C mutation carrying patients. The table shows constructed haplotypes among 9 different heart transplanted individuals. The shared markers between different families are depicted in grey.
Figure 3. Final genetic results in 52 heart transplanted patients due to familial DCM.
18 Table 1. Genetic variants identified and initially classified as pathogenic mutations.
G en e
A BC C9
B A G 3
B A G 3
Mutation*
Coddin g effect
NM_005691:c.4570_457 2delTTAinsAAAT
Leu152 4Lysfs* 5
NM_004281.3:c.361C>T
Arg121 *
NM_004281.3:c.382_383 insAG
Ala128 Glufs* 84
Pa tie nt
7
11
12
Pre vio usly des crib ed
Yes
No
No
Des crib ed in con trol s
No
No
No
Cons erva tion
-
-
-
SIF T pre dict ion (sco re)
-
-
-
Pol yph en2 pre dict ion (sco re)
-
-
-
Mut atio n Tast er (sco re)
-
-
-
Cons eque nce
Trun catin g fram eshif t Nons ense muta tion Trun catin g fram eshif t
Cose grega tion
Final Class ificat ion
Not dem onstr ated
Path ogen ic muta tion
Not dem onstr ated
Path ogen ic muta tion
Yes
Path ogen ic muta tion
Yes
Path ogen ic muta tion
Yes
Path ogen ic muta tion
B A G 3
NM_004281.3:c.1316_13 17delTA
Val439 Glyfs* 4
13
No
No
-
-
-
Trun catin g fram eshif t
B A G 3
NM_004281.3:c.903delG
Arg301 Serfs* 6
15
No
No
-
-
-
D M D
NM_004006.2:c.4069G> T
Glu135 7*
6
No
No
-
-
-
DS P
NM_004415.2:c.5851C>T
Arg195 1*
41
No
No
-
-
-
FL N C FL N
NM_001458.4:c.6231del T
Ser207 7Argfs *50
10
No
No
NM_001458.4:c.4106_41 07insA
Asn13 69Lysf
27
No
No
-
-
-
-
-
-
-
Trun catin g fram eshif t Nons ense muta tion Nons ense muta tion Trun catin g fram eshif t Trun catin
Yes
Not dem onstr ated
Path ogen ic muta tion Path ogen ic muta tion
Yes†
Path ogen ic muta tion
Not dem
Path ogen
19 C
L M N A
L M N A
L M N A
s*36
NM_170707.3:c.569G>A
Arg190 Gln
9
Yes
No
High
Aff ects pro tein fun ctio n (0.0 0)
Pro bab ly Da ma gin g (0.9 98)
Pres uma bly dise ase caus ing (1.0 00)
NM_170707.3:c.961C>T
NM_170707.3:c.568C>T
Arg321 *
Arg190 Trp
23
52
Yes
Yes
T N N T2
TT N
TT N
TT N
TT N
NM_004572.3:c.223+1G >A
NM_001001430.1:c.422 G>A
NM_003319.4:c.39059C >A
NM_003319.4:c.13000A >T
16
Arg141 Gln
Ser130 20*
Arg433 4*
NM_003319.4:c.75042_7 5043delAG
Arg250 14Serf s*4
NM_003319.4:c.59900de lC
Pro199 67Leuf s*8
17
51
2
5
7
Yes
Yes
No
No
No
No
onstr ated
Miss ense muta tion
Not dem onstr ated
Path ogen ic muta tion
Yes
Path ogen ic muta tion
Nons ense muta tion
ic muta tion
No
-
-
-
No
Aff ects pro tein fun ctio n (0.0 0)
Pro bab ly Da ma gin g (0.9 58)
Pres uma bly dise ase caus ing (1.0 00)
Miss ense muta tion
Not dem onstr ated
Path ogen ic muta tion
Not dem onstr ated
Path ogen ic muta tion
Yes†
Path ogen ic muta tion
High
PK P2
g fram eshif t
No
No
No
No
No
No
High
-
-
-
-
-
-
-
Abno rmal splici ng
Tol erat ed (0.2 2)
Pro bab ly Da ma gin g (1.0 00)
Pres uma bly dise ase caus ing (1.0 00)
Miss ense muta tion
-
-
-
-
-
-
-
-
-
--
-
-
Nons ense muta tion Nons ense muta tion Trun catin g fram eshif t Trun catin g fram eshif t
Yes
Not dem onstr ated
Path ogen ic muta tion Path ogen ic muta tion
Yes
Path ogen ic muta tion
Not dem onstr ated
Path ogen ic muta tion
20
TT N
TT N
TT N
TT N
TT N
TT N
NM_003319.4:c.60310_6 0314delCAAGT
NM_003319.4:c.12052G >T
NM_003319.4:c.56232_5 6233insTTGTCGAAAAAC GATAAACTACA
NM_003319.4:c.48240G >A
NM_003319.4:c.53521C >T
NM_003319.4:c.23997_2 3998insT
Gln201 04Tyrf s*17
Glu401 8*
Arg187 45Leuf s*69
Trp160 80*
Arg178 41*
Lys800 0*
9
17
18
19
21
50
No
No
No
No
No
No
* Variants are referred to (GRCh37.p13). † Cosegregation along with other mutations
No
No
No
No
No
No
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Trun catin g fram eshif t Nons ense muta tion Trun catin g fram eshif t Nons ense muta tion Nons ense muta tion Trun catin g fram eshif t
Yes†
Path ogen ic muta tion
Yes†
Path ogen ic muta tion
Yes†
Path ogen ic muta tion
Yes
Yes
Not dem onstr ated
Path ogen ic muta tion Path ogen ic muta tion Path ogen ic muta tion
21 Table 2. Genetic variants identified and initially classified as variants of unknown significance.
Ge ne
AC TN 2
AC TN 2
Mutation*
NM_001103 .2:c.1040C> T
Codd ing effec t
Thr3 47M et
NM_001103 .2:c.26A>G
Gln9 Arg
BA G3
NM_004281 .3:c.343C>T
Pro1 15Se r
DM D
NM_004006 .2:c.2057C> T
Thr6 86Ile
DS C2
NM_024422 .3:c.2194T> G
Leu7 32Va l
DS C2
NM_024422 .3:c.13C>T
Arg5 Cys
Pa tie nt
11
20
43
52
24
33
Prev ious ly desc ribe d
No
Yes
Yes
No
Yes
No
DS G2
NM_001943 .3:c.1174G> A
Val3 92Ile
24
Yes
DS
NM_004415
Arg9
9
No
Des crib ed in cont rols
M AF (% )
No
-
Yes
0. 05 38
Yes
-
No
-
Yes
0. 10 76
No
-
Yes
0. 15 87
No
-
Poly phe n-2 pre dicti on (sco re) Pro babl y dam agin g (0.9 89)
Cons ervati on
SIFT pre dicti on (sco re)
High
Affe cts prot ein func tion (0.0 0)
High
Tole rate d (0.1 3)
Beni gn (0.0 00)
High
Tole rate d (1.0 0)
Beni gn (0.0 01)
High
Tole rate d (0.1 0)
Beni gn (0.1 47)
Medi um
Tole rate d (0.1 5)
Beni gn (0.0 05)
Weak
Tole rate d (0.1 3)
Poss ibly dam agin g (0.6 42)
Weak
Tole rate d (0.1 4)
Beni gn (0.0 01)
High
Affe
Beni
Mutat ion Taster (score ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (0.576 ) Polym orphis m (1.000 ) Presu mably diseas e causin g (1.000 ) Polym orphis m (0.989 ) Polym orphis m (1.000 ) Presu mably diseas e causin g (0.000 ) Presu
Cons eque nce
Coseg regati on
Final classi ficati on
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
No
Not patho genic
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Yes
Patho genic muta tion
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse
Yes†
VUS
22 P
.2:c.2720G> A
07Hi s
DS P
NM_004415 .2:c.6881C> G
Ala2 294G ly
DS P
NM_004415 .2:c.2186T> C
Met 729T hr
45
Val2 6Ala
14, 28, 31, 32, 35, 39, 40, 42, 43, 44, 45, 46, 47
EM D
FLN C
NM_000117 .2:c.77T>C
NM_001458 .4:c.14126C>G
-
FLN C
NM_001458 .4:c.6617G> A
Arg2 206G ln
MY BP C3
NM_000256 .3:c.594C>A
Asp1 98Gl u
MY BP C3
NM_000256 .3:c.3742G> A
Gly1 248A rg
10
1
18
34
29
Yes
No
No
No
No
No
Yes
Yes
No
No
No
No
0. 02 31
-
-
-
-
No
-
Yes
0. 02 45
High
Medi um
High
-
High
High
High
cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 1)
Tole rate d (0.0 7)
-
Affe cts prot ein func tion (0.0 4) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func
gn (0.0 08)
Pro babl y dam agin g (0.9 20)
mably diseas e causin g (0.999 ) Presu mably diseas e causin g (1.000 )
nse mutat ion
Misse nse mutat ion
Yes†
VUS
Not demo nstrat ed
VUS
Beni gn (0.0 08)
Polym orphis m (0.667 )
Misse nse mutat ion
Poss ibly dam agin g (0.5 95)
Polym orphis m (0.999 )
Misse nse mutat ion
Yes
Patho genic muta tion
-
Poten tial abnor mal splici ng
No
Not patho genic
Misse nse mutat ion
Yes†
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
No
Not patho genic
-
Beni gn (0.1 05)
Beni gn (0.1 94)
Poss ibly dam agin g
Presu mably diseas e causin g (0.995 ) Presu mably diseas e causin g (0.994 ) Presu mably diseas e causin
23
MY H6
NM_002471 .3:c.49C>T
Arg1 7Cys
MY H7
NM_000257 .2:c.1618T> C
Phe5 40Le u
MY PN
PK P2
PSE N2
PSE N2
NM_032578 .3:c.59A>G
NM_004572 .3:c.156G>C
NM_000447 .2:c.21+5G>C
NM_000447 .2:c.389C>T
Tyr2 0Cys
Lys5 2Asn
-
Ser1 30Le u
TN NC 1
NM_003280 .2:c.400G>A
Glu1 34Ly s
TN NT 2
NM_001001 430.1:c.586 C>T
Arg1 96Tr p
25
37
3
16
3
26
24
22
No
No
Yes
No
No
Yes
No
No
No
No
Yes
No
No
Yes
No
No
-
-
0. 09 23
-
-
0. 06 92
Medi um
High
High
Weak
-
Very high
-
High
-
Medi um
tion (0.0 0) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 0) Tole rate d (0.1 4)
-
Affe cts prot ein func tion (0.0 2) Affe cts prot ein func tion (0.0 3) Affe cts prot ein func tion
(0.5 63) Pro babl y dam agin g (0.9 55)
Beni gn (0.0 30)
Pro babl y dam agin g (0.9 98) Poss ibly dam agin g (0.7 80)
-
Poss ibly dam agin g (0.5 23) Pro babl y dam agin g (0.9 57) Pro babl y dam agin g
g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (0.613 )
-
Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Yes
Patho genic muta tion
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Poten tial abnor mal splici ng
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Misse nse mutat ion
Not demo nstrat ed
VUS
Yes
Patho genic muta tion
Misse nse mutat ion
24
TP M1
VCL
NM_001018 005.1:c.337 C>G
NM_014000 .2:c.1433A> G
Leu1 13Va l
Asn4 78Se r
48
33
No
No
No
No
-
-
* Variants are referred to (GRCh37.p13). † Cosegregation along with other mutations
Medi um
Very high
(0.0 1) Affe cts prot ein func tion (0.0 0) Affe cts prot ein func tion (0.0 4)
(0.9 78) Poss ibly dam agin g (0.6 53) Poss ibly dam agin g (0.7 55)
(0.999 ) Presu mably diseas e causin g (1.000 ) Presu mably diseas e causin g (1.000 )
Misse nse mutat ion
Yes
Patho genic muta tion
Misse nse mutat ion
Yes†
VUS
25