Heart failure and cardiomyopathies Original research Mutations in TRIM63 cause an autosomal-­recessive form of hypertrophic cardiomyopathy Joel Salazar-­Mendiguchía ‍ ‍,1,2,3 Juan Pablo Ochoa,1 Julian Palomino-­Doza,4,5 Fernando Domínguez,5,6 Carles Díez-­López,7 Mohammed Akhtar,8 Soraya Ramiro-­León,9 María M Clemente,10 Antonia Pérez-­Cejas,11 María Robledo,12 Iria Gómez-­Díaz,1 María Luisa Peña-­Peña,13 Vicente Climent,14 Francisco Salmerón-­Martínez,15 Celestino Hernández,16 Pablo E García-­Granja ‍ ‍,17 M Victoria Mogollón,18 Ivonne Cárdenas-­Reyes,1 Marcos Cicerchia,1 Diego García-­Giustiniani,1 Arsonval Lamounier Jr.,1 Belén Gil-­Fournier,9 Felícitas Díaz-­Flores,11 Rafael Salguero,4 Luis Santomé,1 Petros Syrris,19 Montse Olivé,20 Pablo García-­Pavía,5,6,21 Martín Ortiz-­Genga,1 Perry M. Elliott ‍ ‍,8,19 Lorenzo Monserrat,1 on behalf of GENESCOPIC Research Group ►► Additional material is Abstract estimated prevalence of at least 1:500.1 The under- published online only. To view Objective Up to 50% of patients with hypertrophic standing of the genetic architecture of HCM has please visit the journal online (http://​dx.​doi.​org/​10.​1136/​ cardiomyopathy (HCM) show no disease-­causing variants in greatly improved since the advent of next genera- heartjnl-​2020-​316913). genetic studies. TRIM63 has been suggested as a candidate tion sequencing, but approximately 50% of cases gene for the development of cardiomyopathies, although remain unresolved.2 3 For numbered affiliations see evidence for a causative role in HCM is limited. We sought TRIM63 encodes an E3 ubiquitin-­protein ligase end of article. known as muscle-­ specific RING-­ finger protein 1 to investigate the relationship between rare variants in TRIM63 and the development of HCM. (MuRF1), which belongs to a family of proteins Correspondence to Dr Joel Salazar-­Mendiguchía, Methods TRIM63 was sequenced by next generation with RING zinc-­finger motifs. It mainly localises Cardiovascular Genetics, Health sequencing in 4867 index cases with a clinical to the Z-­disk and M-­line of the sarcomere, where in Code, A Coruna 15006, diagnosis of HCM and in 3628 probands with other it interacts with several sarcomeric proteins.4 5 Spain; cardiomyopathies. Additionally, 3136 index cases MuRF1 participates in the regulation of sarcomeric ​joel.​salazar@​healthincode.​com protein degradation through a process known as with familial cardiovascular diseases other than Received 18 March 2020 cardiomyopathy (mainly channelopathies and aortic ubiquitination and plays an important role in linking Revised 3 April 2020 diseases) were used as controls. myofibril components with intermediate filaments Accepted 6 April 2020 Results Sixteen index cases with rare homozygous or and microtubules.6 TRIM63 expression is upregu- Published Online First compound heterozygous variants in TRIM63 (15 HCM lated by atrophic stimuli,4 and TRIM63 knockout 25 May 2020 and one restrictive cardiomyopathy) were included. No mice models exhibit an exaggerated hypertrophic homozygous or compound heterozygous were identified response to stress and an increase in hypertrophy in the control population. Familial evaluation showed biomarkers.7 8 that only homozygous and compound heterozygous Genetic variants in TRIM63 were first suggested had signs of disease, whereas all heterozygous family as a possible cause of HCM following the identifi- members were healthy. The mean age at diagnosis cation of two heterozygous missense variants and was 35 years (range 15–69). Fifty per cent of patients a heterozygous nonsense variant, p.(Gln247*), in had concentric left ventricular hypertrophy (LVH) and patients but not in controls.8 However, subsequent 45% were asymptomatic at the moment of the first data on p.(Gln247*) showed that this variant is examination. Significant degrees of late gadolinium relatively common in the European population, and enhancement were detected in 80% of affected the relationship between TRIM63 and the develop- ►► https://​doi.​org/​10.​1136/​ individuals, and 20% of patients had left ventricular (LV) ment of HCM has not been confirmed yet.9 10 Some heartjnl-​2020-​317043 systolic dysfunction. Fifty per cent had non-­sustained authors have suggested that variants in TRIM63 ventricular tachycardia. Twenty per cent of patients might act as modifiers of cardiac hypertrophy in suffered an adverse cerebrovascular event (20%). the presence of a sarcomere pathogenic variant.11 Conclusion TRIM63 appears to be an uncommon cause On the basis of its potential role in cardiovas- © Author(s) (or their employer(s)) 2020. Re-­use of HCM inherited in an autosomal-­recessive manner and cular diseases, we sought to determine the role of permitted under CC BY-­NC. No associated with concentric LVH and a high rate of LV TRIM63 as a candidate gene for the development commercial re-­use. See rights dysfunction. of cardiomyopathies in a large cohort of patients and permissions. Published referred for clinical genetic testing. by BMJ. To cite: Salazar-­ Mendiguchía J, Ochoa JP, Introduction Methods Palomino-­Doza J, et al. Heart Hypertrophic cardiomyopathy (HCM) is the From March 2013 to January 2019, TRIM63 was 2020;106:1342–1348. most common inherited cardiac disorder, with an sequenced as a candidate gene using next-­generation 1342   Salazar-­Mendiguchía J, et al. Heart 2020;106:1342–1348. doi:10.1136/heartjnl-2020-316913 Heart failure and cardiomyopathies sequencing in 4867 index cases with a clinical diagnosis of HCM Statistical analysis and in 3628 probands with other cardiomyopathies referred to Continuous variables were expressed as mean±SD, and compar- our centre for molecular genetic diagnosis. Additionally, 3136 ison between groups was performed using the Student’s t test index cases with familial cardiovascular diseases other than or the Mann-­Whitney U test according to values distribution. cardiomyopathies (mainly channelopathies and aortic diseases) Non-­continuous variables were expressed as an integer number were used as controls. The phenotypes had been established by (per cent of total) and compared using the χ² test or Fisher’s the referring centres prior to the genetic study. Patient samples exact test, as appropriate. A two-­sided p<0.05 was considered were mainly received from hospitals in Spain and the UK, and to indicate statistical significance. the predominant ethnicity was European (>90%). The frequen- Analysis was performed using the R V.3.4.3 (The R Founda- cies of variants in the general population were determined by tion for Statistical Computing Platform). using the gnomAD database (http://​gnomad.​broadinstitute.​org; V.2.1.1, December 2019). According to gnomAD, the constraint LOD score (Logarithms of Odds) calculation score (observed/expected number of variants) for missense and We calculated two-­point LOD score for informative families by loss-­of-­function (LoF) variants in TRIM63 suggests that this gene using the PARAMLINK package for R software. The model was is tolerant to the aforementioned genetic changes. However, set for a recessive inheritance pattern, with θ=0, phenocopy the variants reported in gnomAD are mainly found in hetero- rate=0.005 and a penetrance of 0.95 for homozygous carriers. zygosity. To investigate the role of non-­synonymous TRIM63 An indeterminate status was assigned to family members with a (NM_032588.3) variants in homozygosity or compound hetero- confounding cardiac diagnosis, as well as to individuals younger zygosity, probands harbouring rare variants (defined as those than 40 years who did not meet clinical criteria for HCM and with minor allele frequency (MAF)<0.001) in homozygous could develop the disease afterwards. or compound heterozygous state were analysed and invited to participate in segregation studies. Clinical and genetic familial cascade screening was performed when possible. In cases where Results a second pathogenic variant in a sarcomeric gene was identified, A total of 20 (0.39%) index cases harbouring homozygous or the latter variant was also included in family screening to deter- compound heterozygous rare variants in TRIM63 were identi- mine the role of each of the identified variants in the familial fied, all of them of European descent (non-­Finnish). Nineteen phenotype. Finally, the clinical characteristics and outcomes of had a clinical diagnosis of HCM and one was diagnosed with homozygous and compound heterozygous individuals (including restrictive cardiomyopathy (RCM). No individuals with biallelic probands and relatives) were assessed. Our study was conducted variants were identified in the disease controls. Likewise, in the in accordance with the Declaration of Helsinki. Written consent gnomAD database, no homozygous individuals harbouring rare was obtained from all participants. variants (MAF<0.001) are identified. Fifteen of the homozygous/compound heterozygous patients with a clinical diagnosis of HCM and the patient Patient involvement with restrictive cardiomyopathy accepted to participate in this Patients were not involved in the design of this study. The index study. Five missense and three protein-­truncating variants in cases actively participated in the recruitment of first-­ degree TRIM63 (two frameshifts and one nonsense) were identified relatives. They were central to the dissemination of the baseline in these families. All the identified variants are present in the information and our hypothesis, which helped to motivate the gnomAD database, with a low frequency (MAF<0.001) and involvement of their families. All index cases received informa- only in heterozygosity, except for the p.(Leu37Val) and p.(Lys- tion on the basis of genetic inheritance in order to adequately 146Thrfs*24) variants, which are absent in this database. The inform their relatives prior to contacting the research team. identified variants, their frequency in gnomAD, the result of Once our study is published, we aim to disseminate the main the bioinformatic predictors, their classification in ClinVar results to all trial participants. (whenever present) and their significance according to the ACMG recommendations can be found in online supplemen- Genetic studies, variant filtering and variant classification tary table 2. Only one HCM individual harbouring a homozy- Coding exons and intronic boundaries of 219 genes related gous truncating variant in TRIM63 also carried a pathogenic to inherited cardiovascular diseases and sudden cardiac death truncating variant in a sarcomeric gene (MYBPC3), whereas in (online supplementary table 1) were captured using a custom the rest of the included individuals, no other variants associ- probe library (SureSelect Target Enrichment Kit for Illumina ated with the phenotype were identified. paired-­end multiplexed sequencing method; Agilent Technolo- gies, Santa Clara, California, USA) and sequenced on the HiSeq Familial cosegregation 1500 platform (Illumina, San Diego, California, USA) following Twelve of the included families accepted further clinical and Illumina protocols. The read depth (number of times that a base genetic screening as part of the evaluation. Three other fami- was sequenced by independent reads) of every nucleotide of lies accepted to undergo only clinical evaluation, and in one genes related to the referred phenotype (including TRIM63) was homozygous index case, family study was not possible. Clinical >30 (mean 250–400). Exons that did not fulfil this standard and genetic data on all evaluated family members as well as the were complementary sequenced using the Sanger method. Only family pedigrees are available online (online supplementary table likely protein-­altering variants (missense, in-­ frame insertions/ S3 and figure S1). deletions, frameshifts, nonsense and consensus splice-­site vari- After familial evaluation, three more homozygous and one ants) in the TRIM63 gene were analysed. Bioinformatic analysis compound heterozygous individuals were identified. Two of was performed by means of a custom pipeline including software these met current diagnostic criteria for HCM,13 whereas the for variant calling, genotyping and annotation. To determine the remaining two were considered likely to be affected, manifesting pathogenicity of the identified variants, the American College of early signs of the disease given the grossly abnormal ECG, even Medical Genetics and Genomics recommendations were used.12 though the left ventricular (LV) wall thickness did not fulfil the Salazar-­Mendiguchía J, et al. Heart 2020;106:1342–1348. doi:10.1136/heartjnl-2020-316913 1343 Heart failure and cardiomyopathies Figure 1 Left ventricular maximal wall thickness according to zygosity. Left ventricular maximal wall thickness is higher in homozygous/compound heterozygous patients than in their heterozygous (p<0.001) or wild-­type (p<0.001) family members. No statistically significant differences are observed between wild-­type and heterozygous individuals. classical criteria for HCM (ECGs available as online supplemen- at the first examination, with abnormal ECG being the most tary figure 2). None of the heterozygous or wild-­type family frequent cause of diagnosis (25%). Fifty per cent of patients members showed signs of the disease. Figure 1 shows the differ- with available information had concentric left ventricular ence in LV maximal wall thickness in homozygous/compound hypertrophy (LVH), and none had LV outflow tract obstruc- heterozygous individuals, compared with their heterozygous or tion. The mean maximal LV wall thickness was 19 mm (±5). wild-­type family members. After segregation analysis in the fami- At the moment of inclusion, no patient had extreme LV hyper- lies, a combined LOD score of 4.25 was obtained, which is indic- trophy (ie, >30 mm); however, one patient with a maximal LV ative of strong cosegregation (online supplementary table 4). wall thickness of 17 mm showed significant wall thinning, with a previously reported septal wall thickness of 33 mm (without Clinical characteristics of homozygous and compound septal reduction therapies). Significant degrees of late gado- heterozygous individuals linium enhancement (LGE) (ie, transmural and involving more The baseline characteristics of all homozygous and compound than one myocardial segment) were observed in almost 80% of heterozygous individuals after family screening and their homozygous/compound heterozygous and 20% of our popula- comparison with wild-­type and heterozygous family members tion progressed to LV systolic dysfunction (defined as LV ejec- are shown in table 1. tion fraction (LVEF) <50%). All patients with LV dysfunction More detailed clinical, echocardiographic and electro- showed LGE on MRI studies. Figure 3 shows two examples cardiographic data on each of the homozygous/compound of patients’ cardiac MRIs. Fifty per cent of the patients had heterozygous are shown in online supplementary table 5. non-­sustained ventricular tachycardia on Holter monitoring. The most frequently identified variant in our cohort was One patient had hemodynamically stable sustained ventric- p.(Gln247*), followed by the p.(Cys23Tyr) variant. The distri- ular tachycardia, which prompted the implantation of an bution of the identified variants throughout the different func- implantable cardioverter defibrillator for secondary preven- tional domains of the protein is shown in figure 2. HCM was tion. Twenty per cent of the included individuals underwent a the most frequent diagnosis (95%), with only one individual formal neurological examination, including muscular imaging with biallelic variants in TRIM63 with a different phenotype techniques, and none of them showed evident neuromuscular (restrictive cardiomyopathy). The mean age at diagnosis was alterations. Only one individual underwent muscle biopsy, 35 years (range 15–69). Forty-­five per cent were asymptomatic which was normal. Creatine-­ kinase leves (CK) levels were 1344 Salazar-­Mendiguchía J, et al. Heart 2020;106:1342–1348. doi:10.1136/heartjnl-2020-316913 Heart failure and cardiomyopathies disorders were sequenced, and rare homozygous and compound Table 1 Baseline characteristics of homozygous/compound heterozygous variants in TRIM63 were identified only in subjects heterozygous individuals with HCM (and one case with RCM), whereas they were absent Homozygous/compound Heterozygous/ in our control population. Likewise, homozygous individuals heterozygous (n=20) wild-­type (n=44) P value harbouring rare variants in TRIM63 are absent in the gnomAD Age (range) 33 (15–69) 48 (12–80) 0.002 database. Moreover, familial evaluation confirmed these find- Hypertension (%) 4/20 (20) 7/44 (16) 0.4 ings, as heterozygous carriers were healthy and only homozygous HCM (%) 19/20 (95) 0/44 <0.001 and compound heterozygous individuals showed manifestations  ASH 8/16 (50) of the disease. Even though two family members (homozygous)  Concentric 8/16 (50) did not fulfil the classical criteria for HCM, we classified them Restrictive 1/20 (5) 0/44 0.15 as possibly affected on the basis of an abnormal ECG (online cardiomyopathy (%) supplementary figure 2). It has been described that ECG changes Reason for diagnosis (%) in the absence of diagnostic morphological abnormalities (in  Syncope 2/20 (10%) N/A N/A the context of a familial disease) may represent an early or mild  Angina 2/20 (10%) form of HCM.13  Dyspnoea 3/20 (15%) Sarcomeric proteins undergo constant turnover in order to  ECG disturbance 5/20 (25%) adapt to the physiological needs as well as to repair damaged  Palpitations 3/20 (15%) proteins.4 Central to this turnover is the ubiquitin proteasome  Family screening 4/20 (20%) system (UPS). E3 ubiquitin ligases direct the addition of ubiq-  Others 1/20 (5%) uitin to specific target proteins, marking them for degradation.15 NYHA functional class 19/20 (95) N/A N/A Substrates targeted by MuRF1 include several members of the I–II (%) contractile apparatus, such as myosin heavy chain, troponins, ECG LVH (%) 13/16 (81) 0/44 <0.001 tropomyosin and titin.6 16 17 Online supplementary figure 3 LVMWT (mm) 19.2 (±5.6) 9.8 (±1.9) <0.001 shows a schematic representation of the role of ubiquitin ligases LVMWT>25 mm (%) 3/20 (15) 0/44 <0.001 in the UPS. Due to its interactions with sarcomeric proteins, PW (mm) 13.6 (±3) 8.8 (±2) 0.01 TRIM63, encoding MuRF1, has been proposed as a candidate LVEDD (mm) 49.4 (±7.5) 45.4 (±4.8) 0.02 gene for the development of cardiomyopathies. However, most LVEF (%) 60 (±12) 63.4 (±3.7) <0.001 descriptions focused on heterozygous carriers or isolated homo- LV dysfunction 4/20 (20) 0/44 0.003 zygous individuals without familial cosegregation studies,8 18 and (EF<50%) (%) its true relevance as a cardiomyopathy-­causing gene has not yet LA diameter (mm) 43 (±5) 32 (±5) 0.7 been elucidated. LVOTO 0/20 N/A N/A One previous study19 reported a family in which a male CMR LGE (%) 10/12 (83) N/A N/A harbouring the p.(Gln247*) variant in TRIM63 in homozygosity Atrial fibrillation (%) 3/20 (15) 0/40 0.012 and the heterozygous missense TRIM54 variant p.(Asp106Asn) NSVT on Holter (%) 9/18 (50) N/A N/A had HCM and skeletal myopathy. His siblings heterozygous for ICD implant (%) 7/20 (35) N/A  N/A the TRIM63 variant were asymptomatic and healthy. One of his Betablockers (%) 12/20 (60) N/A N/A sisters was also a homozygous for the p.(Gln247*) variant, but Oral anticoagulants (%) 5/20 (25) 0/44 <0.001 not a carrier of the TRIM54 variant, and was diagnosed with Events LV hypertrophy thought to be secondary to hypertension. This  Cardiovascular death 0/20 group reported that the presence of digenic mutations (TRIM63  CVE 4/20 1/44 and TRIM54) would be necessary for the development of the  Heart failure death/ 0/20 phenotype and that the homozygous TRIM63 variant would heart transplant deserve further investigation in order to establish its contribu-  SCD 0/20 tion to the development of LV hypertrophy. ASH, asymmetric septal hypertrophy; CMR, cardiac magnetic resonance; CVE, cerebrovascular event; ECG, electrocardiogram; HCM, hypertrophic cardiomyopathy; ICD, The presence of concentric hypertrophy is not a frequent implantable cardioverter defibrillator; LA, left atrium; LVEDD, left ventricular end-­diastolic presentation in sarcomeric HCM and may be more common diameter; LVEF, left ventricular ejection fraction; LVH, left ventricular hypertrophy; LVMWT, in metabolic and infiltrative disorders.13 However, 50% of the left ventricular maximal wall thickness; LVOTO, left ventricular outflow tract obstruction; NSVT, non-­sustained ventricular tachycardia; NYHA, New York Heart Association; PW, patients with HCM included in our study showed this distribu- posterior wall; SCD, sudden cardiac death. tion. The finding of concentric HCM together with extensive LGE distribution prompted the search for an infiltrative disease in three of our patients (15%), and was ruled out in all of them. elevated in 3 out of 11 evaluated patients. None of the evalu- The clinical suspicion of infiltrative disorder together with the ated cases had previous episodes of sudden cardiac death. Four presence of extensive LGE and LV systolic dysfunction in our patients (20%) had a previous cerebrovascular event, none of population matches the description of the cardiac phenotype in them disabling. the proband reported by Olivé et al.19 End-­stage HCM (defined as EF <50%) has been identified in Discussion up to 10% of individuals with HCM in modern cohorts, and it Our study demonstrates a relationship between homozygous has been suggested that the progression to this phenotype may and compound heterozygous rare variants in TRIM63 and the be lower in individuals with sarcomeric HCM when compared development of HCM. Autosomal-­ recessive (AR) inheritance with phenocopies.1 20–22 Nevertheless, in our study, 20% of indi- has been considered the least common form of inheritance viduals showed progression to LV dysfunction. Moreover, in one in inherited cardiac muscle diseases and is mainly associated family, there was also a history of heart transplant due to end-­ with metabolic or complex multi-­system disorders.13 14 More stage HCM in an individual with unknown genotype. Animal than 11 600 probands with different cardiovascular inherited studies have shown an increase in myocardial fibrosis in mice Salazar-­Mendiguchía J, et al. Heart 2020;106:1342–1348. doi:10.1136/heartjnl-2020-316913 1345 Heart failure and cardiomyopathies Figure 2 Schematic representation of the protein’s domains. Location of the identified homozygous/compound heterozygous variants in TRIM63 at protein level. harbouring mutations in TRIM63, and myocardial fibrosis has of cysteine (a neutral polar side chain amino acid) for tyro- been shown to be a key determinant of LV adverse remodelling sine (an aromatic hydrophobic side chain amino acid), which in other cardiomyopathies.23–25 causes very significant changes in the physicochemical prop- Interestingly, the previously described variant p.(Gln247*) erties and may alter the structure and function of the protein. was the most frequently identified variant in our cohort. Func- Although our findings will affect a small subset of patients tional studies have shown that this variant reduces the localisa- with HCM, they are important in clinical practice because they tion of the protein to the Z-­disk and impairs the ubiquitination have a relevant impact on the genetic counselling of these fami- of MuRF1 substrates in cardiac myocytes.8 RT-­PCR analysis lies, as all the available evidence suggests that TRIM63 is a cause suggested that the transcripts undergo nonsense-­ mediated of HCM with AR inheritance. Moreover, the clinical profile of decay (a mechanism responsible for haploinsufficiency); more- these patients (moderate to severe hypertrophy, high incidence over, transient expression of wild-­type MuRF1 rescued the of ventricular arrhythmias, extensive fibrosis and frequent LV phenotype in adult human myocytes.19 Sixty per cent of the systolic dysfunction) suggests that individuals homozygous and identified missense variants in our cohort are located in the compound heterozygous for variants in TRIM63 are at high RING domain, a highly conserved region (see figure 2 and risk of adverse events. The absence of arrhythmic events in our online supplementary figure 4) which mediates ubiquitination cohort may be due to a lack of long-­term follow-­up of the iden- events.26 Other groups8 have shown that missense variants tified carriers. located in the RING domain can significantly decrease the enzyme’s autoubiquitination function and the ubiquitination of the substrates, as well as producing an activation in the Key messages mTOR pathway signalling (important for the development of ventricular hypertrophy). Furthermore, three out of five (60%) What is already known on this subject? of all the missense variants identified involve the substitution ►► Up to 50% of patients with hypertrophic cardiomyopathy (HCM) show no disease-­causing variants in genetic studies. TRIM63 has been suggested as a candidate gene for the development of cardiomyopathies, although evidence for a causative role in HCM is limited. What might this study add? ►► This study adds to the genetic architecture of HCM by showing that TRIM63 is a cause of a non-­classical form of HCM, with an autosomal-­recessive inheritance, presenting as concentric HCM with a high rate of left ventricular (LV) dysfunction. How might this impact on clinical practice? ►► Our findings have a relevant impact on the genetic counselling of these families, as all the available evidence Figure 3 Cardiac MRI. Panels A and B show patient II.3 from family suggests that TRIM63 is a cause of HCM only with 6897. Severe concentric LV hypertrophy (20 mm IVS) and severe autosomal-­recessive inheritance. Moreover, the clinical profile transmural LGE were observed. Panels C and D show patient II.2 from of these patients (moderate to severe hypertrophy, high family 2523, who developed LV systolic dysfunction and dilatation. incidence of ventricular arrhythmias, extensive fibrosis and Severe transmural LGE is observed in the apical segments and near- frequent LV systolic dysfunction) suggests that homozygous transmural LGE in the interventricular septum. Hypertrabeculation of the and compound heterozygous individuals for variants in lateroapical segments was found. LGE, late gadolinium enhancement; TRIM63 are at high risk of adverse events. LV, left ventricular. 1346 Salazar-­Mendiguchía J, et al. Heart 2020;106:1342–1348. doi:10.1136/heartjnl-2020-316913 Heart failure and cardiomyopathies Study limitations Funding St. Bartholomew’s Hospital (Drs P M Elliot and M Akhtar) and Hospital The fact that no other TRIM protein family members have Universitario Puerta de Hierro Majadahonda (Drs P Garcia-­Pavia and F Domínguez) are members of the European Reference Network on Rare and Complex Diseases of been studied in our cohort made it impossible to study whether the Heart (Guard-­Heart; http://​guard-​heart.​ernnet.​eu). digenic variants could explain the development of clinical myop- Competing interests JS-­M, JPO, IG-­D, IJC-­R, MNC, DG-­G, AL, LS and MO-­G are athy or elevated CK observed in some patients. employees of Health in Code SL. LM is a stakeholder and CEO of Health in Code SL. Patient and public involvement Patients and/or the public were not involved in Conclusion the design, or conduct, or reporting, or dissemination plans of this research. TRIM63 is a cause of HCM with an AR inheritance pattern, Patient consent for publication Not required. which may be associated with concentric LVH and a high rate of LV dysfunction. It accounts for approximately 0.4% of HCM Provenance and peer review Not commissioned; externally peer reviewed. cases, although its importance in sporadic cases could be higher. Data availability statement All data relevant to the study are included in the If these findings are validated by other groups, it is possible that article or uploaded as supplementary information. TRIM63 is the most frequent cause of HCM with a recessive Open access This is an open access article distributed in accordance with the inheritance pattern. Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-­commercially, and license their derivative works on different terms, provided the original work is Author affiliations properly cited, appropriate credit is given, any changes made indicated, and the use 1 Cardiovascular Genetics, Health in Code, A Coruna, Spain is non-­commercial. See: http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/. 2 Genetics Department, Universitat Autonoma de Barcelona, Barcelona, Spain 3 Clinical Genetics Department, Hospital Universitario de Bellvitge, Barcelona, Spain ORCID iDs 4 Inherited Cardiac Diseases Unit. Cardiology Department, Hospital Universitario 12 Joel Salazar-­Mendiguchía http://​orcid.​org/​0000-​0003-​2171-​8508 de Octubre, Madrid, Spain Pablo E García-­Granja http://​orcid.​org/​0000-​0002-​0750-​1567 5 Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares Perry M. Elliott http://​orcid.​org/​0000-​0003-​3383-​3984 (CIBERCV), Instituto Carlos III, Madrid, Spain 6 Inherited Cardiac Diseases Unit. Cardiology Department, Hospital Universitario Puerta de Hierro, Madrid, Spain 7 Heart Failure and Cardiomyopathy Unit. Cardiology Department, Hospital References Universitario de Bellvitge, Barcelona, Spain 1 Maron BJ. Clinical course and management of hypertrophic cardiomyopathy. N Engl J 8 Centre for Inherited Cardiac Diseases. Barts Heart Centre, Saint Bartholomew’s Med 2018;379:655–68. Hospital, London, United Kingdom 2 Van Driest SL, Ellsworth EG, Ommen SR, et al. Prevalence and spectrum of 9 Genetics Department, Hospital Universitario de Getafe, Getafe, Spain thin filament mutations in an outpatient referral population with hypertrophic 10 Cardiology Department, Hospital Virgen del Puerto, Plasencia, Spain cardiomyopathy. 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IDIBELL, Hospital Universitario human hypertrophic cardiomyopathy. Circ Res 2012;111:907–19. de Bellvitge, Barcelona, Spain 9 Płoski R, Pollak A, Müller S, et al. Does p.Q247X in TRIM63 cause human hypertrophic 21 University Francisco de Vitoria (UFV), Madrid, Spain cardiomyopathy? Circ Res 2014;114:2–5. 10 Walsh R, Buchan R, Wilk A, et al. Defining the genetic architecture of hypertrophic cardiomyopathy: re-­evaluating the role of non-­sarcomeric genes. Eur Heart J Twitter Joel Salazar-­Mendiguchía @JoelSMG 2017;38:ehw603–8. Acknowledgements The authors would like to thank Health in Code’s laboratory, 11 Su M, Wang J, Kang L, et al. Rare variants in genes encoding MuRF1 and MuRF2 are bioinformatics and administrative teams for their everyday effort. We thank Mr modifiers of hypertrophic cardiomyopathy. Int J Mol Sci 2014;15:9302–13. Radek Suchac and Mrs Alicia Pallas for their support in English editing. 12 Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Collaborators GENESCOPIC Research group: Health in Code: Xusto Fernández; medical genetics and genomics and the association for molecular pathology. Genet Elena Veira; David de Uña; Pablo Iglesias; Noël Brögger; Germán Fernández; Juan Med 2015;17:405–23. Pablo Trujillo-­Quintero Hospital Universitario Puerta de Hierro: Marta Cobo-­Marcos; 13 , Elliott PM, Anastasakis A, et al, Authors/Task Force members. 2014 ESC guidelines María A. Restrepo-­Córdoba; Esther González-­LópezSaint Bartholomew’s Hospital: on diagnosis and management of hypertrophic cardiomyopathy: the task force for the Luis R. Lopes Hospital Universitario de Getafe: Jesús Saavedra Hospital Clínico diagnosis and management of hypertrophic cardiomyopathy of the European Society of cardiology (ESC). Eur Heart J 2014;35:2733–79. Universitario de Valladolid: María Sandín-­Fuentes; Iria Duro-­Aguado Hospital General 14 Rapezzi C, Arbustini E, Caforio ALP, et al. Diagnostic work-­up in cardiomyopathies: Universitario de Alicante: María García Álvarez. bridging the gap between clinical phenotypes and final diagnosis. A position Contributors JS-­M is the principal investigator, wrote the proposal, coordinated statement from the ESC Working group on myocardial and pericardial diseases. Eur the study, supervised the data and prepared the article. JPO had a central role in Heart J 2013;34:1448–58. statistical analysis and critical revision. JP-­D, FD, CD-­L, MA, SR, AP-­C, MMC, MR, 15 Willis MS, Schisler JC, Portbury AL, et al. Build it up-­Tear it down: protein quality MLPP, VC-­P, FMS, CH, PEG-­G, MVM, IC-­R, MC, DG-­G, AL, BG-­F, FD-­F and RS helped control in the cardiac sarcomere. Cardiovasc Res 2009;81:439–48. in data acquisition and coordinated the family studies. IG-­D, LS and PS did a critical 16 McElhinny AS, Kakinuma K, Sorimachi H, et al. Muscle-­specific RING finger-1 interacts review on all the bioinformatic aspects of the identified genetic variants. MO critically with titin to regulate sarcomeric M-­line and thick filament structure and may have revised the manuscript and led the neuromuscular investigations. PG-­P, MO-­G and nuclear functions via its interaction with glucocorticoid modulatory element binding PME made substantial contributions to the design of the work and added important protein-1. J Cell Biol 2002;157:125–36. intellectual content to the final manuscript. LM conceived the initial idea and acted 17 Henderson CA, Gomez CG, Novak SM, et al. Overview of the muscle cytoskeleton. as co-­coordinator. JS-­M and LM act as guarantors. Compr Physiol 2017;7:891–944. Salazar-­Mendiguchía J, et al. 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