Significance of Sarcomere Gene Mutations Analysis in the End-Stage Phase of Hypertrophic Cardiomyopathy
Short title: Genetics of end-stage HC
Elena Biagini, MD, PhDa, Iacopo Olivotto, MDb, Maria Iascone, BSc, PhDc, Maria I. Parodi, BScd, Francesca Girolami, BSce, Giulia Frisso, MD, PhDf, Camillo Autore, MDg, Giuseppe Limongelli, MDh, Massimiliano Cecconi, Bsc, PhDd, Barry J. Maron, MDi, Martin S. Maron, MDj, Stefania Rosmini, MDa, Francesco Formisano, MDK, Beatrice Musumeci, MDg, Franco Cecchi, MDb, Attilio Iacovoni, MDl, Tammy S. Haas, RNi, Maria L. Bacchi Reggiani, MSc, MStata, Paolo Ferrazzi, MDl, Francesco Salvatore, MD, PhDf, Paolo Spirito, MDK, Claudio Rapezzi, MDa
aDipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Alma Mater Studiorum, Universita di
Bologna, Italy; bCentro di Riferimento per le Cardiomiopatie, Azienda Ospedaliero-Universitaria Careggi,
Firenze Italy; cUSSD Laboratorio Genetica Medica, Ospedali Riuniti, Bergamo, Italy; dS.C. Laboratorio di
Genetica Umana, E.O. Ospedali Galliera, Genova, Italy; eSOD Diagnostica Genetica, Azienda
Ospedaliero-Universitaria Careggi, Firenze,Italy, fCEINGE-Biotecnologie Avanzate s.c.ar.l. Napoli e Dipartimento di
Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli “Federico II”, IRCCS-Fondazione SDN, Napoli, Italy; gDivisione di Cardiologia, Dipartimento di Medicina Clinica e Molecolare,
Università La Sapienza, Roma, Italy; hDipartimento di Cardiologia, Seconda Università degli Studi, Napoli,
Italy; iHypertrophic Cardiomyopathy Center, Minneapolis Heart Institute Foundation, Minneapolis, MN;jTufts
Medical Center, Hypertrophic Cardiomyopathy Center, Boston, Massachusetts; KS.C. Cardiologia, E. O.
Ospedali Galliera, Genova, Italy; l Policlinico di Monza, Monza, Italy. Corresponding Author:
Elena Biagini
Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Alma Mater Studiorum, Università di Bologna, Via Massarenti n 9, 40138 Bologna, Italy.
Tel: +39 3471016933, Fax: +39 051 344859. E-mail: elena.biagini73@gmail.com
Funding Sources
Dr Stefania Rosmini was supported by a grant from “Fondazione del Monte di Bologna e Ravenna”, Italy.
Galliera Genetic Bank - “Telethon Network of Genetic Biobanks” was supported by Italian Telethon grants (project no. GTB07001)
ABSTRACT
End-stage hypertrophic cardiomyopathy (ES-HC) has an ominous prognosis. Whether genotype can influence ES-HC occurrence is unresolved. We assessed the spectrum and clinical correlates of HC associated mutations in a large multicenter cohort with end-stage ES-HC. Sequencing analysis of 8 sarcomere genes (MYH7, MYBPC3, TNNI3, TNNT2, TPM1, MYL2, MYL3, ACTC1) and 2 metabolic genes (PRKAG2, LAMP2) was performed in 156 ES-HC patients with left ventricular (LV) ejection fraction (EF) <50%. A comparison among mutated and negative ES-HC patients and a reference cohort of 181 HC patients with preserved LVEF was performed. Overall, 131 mutations (36 novel) were identified in 104 ES-HC patients (67%) predominantly affecting MYH7 and MYBPC3 (80%). Complex genotypes with double or triple mutations were present in 13% compared to 5% of the reference cohort (p=0.013). The distribution of mutations was otherwise indistinguishable in the 2 groups. Among ES-HC patients, those presenting at first evaluation before the age of 20 had a 30% prevalence of complex genotypes compared to 19% and 21% in the subgroups aged 20-59 and ≥60 years (p=0.003). MYBPC3 mutation carriers with ES-HC were older than patients with MYH7, other single mutations or multiple mutations (median 41 vs. 16, 26 and 28 years, p=<0.001). Outcome of ES-HC patients was severe irrespective of genotype. In conclusion, the ES phase of HC is associated with a variable genetic substrate, not distinguishable from that of patients with HC and preserved EF, except for a higher frequency of complex genotypes with double or triple mutations of sarcomere genes.
The end-stage (ES) phase of hypertrophic cardiomyopathy (HC) carries an ominous prognosis due to high rates of refractory heart failure and sudden arrhythmic death and represents the sole indication for heart transplantation in HC (1-6). Several different mechanisms have been proposed to explain the evolution toward ES, such as progressive cardiomyocyte energy depletion,
microvascular ischemia and replacement fibrosis although it remains largely unresolved (7-11). It has also been hypothesized that development of ES may be heavily influenced by the specific genetic background. Indeed multiple sarcomere gene mutations are associated with early onset of HC disease and particularly severe phenotypes, including evolution toward the ES (12-17). To date, however, systematic studies on the genetic background of ES-HC are limited, probably due to the relative rarity of this condition in most HC cohorts (18, 19). Therefore, to adequately
investigate this issue, we assessed the prevalence and spectrum of sarcomere gene mutations in the largest multicenter cohort of patients with ES-HC reported to date, with respect to phenotypic expression, clinical course and outcome. Moreover, a comparison of the genetic substrate in ES-HC with a subset of ES-HC patients with preserved left ventricular (LV) ejection fraction (EF) was assessed.
METHODS
In this multicenter cross-sectional and longitudinal study, we retrospectively identified 156 patients diagnosed with ES-HC (130 index cases and 26 family members) from January 1981 to June 2010 and genetically screened between January 2007 and June 2010 at 6 referral centers in Italy and 2 in the US. Five of these centers (Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Alma Mater Studiorum, Università di Bologna, Italy; Dipartimento Cardiovascolare, Ospedali Riuniti, Bergamo, Italy; Dipartimento di Cardiologia, Seconda Università degli Studi, Napoli, Italy; Hypertrophic Cardiomyopathy Center, Minneapolis Heart Institute Foundation,
Minneapolis; Tufts Medical Center, Hypertrophic Cardiomyopathy Center, Boston, Massachusetts) have dedicated heart failure management units including heart transplantation programs.
Diagnosis of HC was based on echocardiographic and/or cardiovascular magnetic resonance imaging documentation of a hypertrophied, non-dilated LV, in the absence of other cardiac or systemic diseases that could produce the magnitude of LV hypertrophy evident (20). ES-HC was defined as 2D echocardiographic LV EF <50% at rest, reflecting global systolic dysfunction, at study entry or during follow-up (3, 4). Patients with prior surgical myectomy or alcohol septal ablation (N= 16), known atherosclerotic coronary artery disease (N= 9) or severe valvular heart disease (N= 8) were excluded.
A subset of 181 patients with HC and normal LV systolic function consecutively genotyped for 10 genes between January 2007 and June 2010 at Centro di Riferimento per le Cardiomiopatie, Azienda Ospedaliero-Universitaria Careggi, Firenze, Italy were examined as a reference cohort (14). Median age at first evaluation was 52 years (IQR 36-63); 64 (35%) were female, median maximum LV wall thickness was 22 mm (IQR 19-27), left atrium diameter 45 mm (IQR 40-51); 34 (19%) had LV outflow tract obstruction in resting conditions; 164 out of 181 patients (91%) were in New York Heart Association (NYHA) class I-II; 25 (19%) were implanted with an ICD for primary or secondary protection from sudden death.
We considered echocardiographic studies performed at first evaluation at the referral center and first documentation of ES. Maximal wall thickness, LV end-diastolic cavity dimension, left atrial dimension, LV outflow tract obstruction and LVEF were evaluated. LV hypertrophy was assessed with 2-dimensional echocardiography, and the site of maximum wall thickness was identified.
All patients underwent genetic testing for the 8 most frequently mutated sarcomeric genes associated with HC: cardiac beta-myosin heavy chain (MYH7, NM_000257.2); cardiac myosin binding protein C (MYBPC3, NM_000256.3); troponin I (TNNI3, NM_000363.4); troponin T (TNNT2, NM_001001430.1); alpha-tropomyosin (TPM1, NM_000366.5); regulatory myosin light chain (MYL2, NM_000432.3); essential myosin light chain (MYL3, NM_000258.2) and alpha-cardiac actin (ACTC1, NM_005159.4), as well for lysosome-associated membrane protein 2 (LAMP2, NM_002294.2) and AMP-activated protein kinase (PRKAG2, NM_016203.3) as possible
cause of HC phenocopies. Written informed consent was obtained in each case. Conventional DNA sequencing was performed using standard method (14).
Each identified variant was confirmed by direct sequencing from an independent amplifica-tion product, and, whenever possible, by restricamplifica-tion enzyme digesamplifica-tion. The significance of each variation was defined by the following criteria, though not always all of them present for each muta-tion: (i) absence in 300 adult control chromosomes from ethnically matched individuals, tested by sequencing; (ii) minor allele frequency (MAF) <1%, collected from ESP (Exome Sequencing Project, http://evs.gs.washington.edu/EVS/, data release ESP6500SI-V2) and dbSNP137 (http://www.ncbi.nlm.nih.gov/SNP/), that includes 1000Genomes Project data; (iii) evolutionary conservation of the nucleotide or amino acid, calculated by multiple alignments of 46 vertebrate species and measured by phyloP score; and (iv) in silico functional prediction of effect by in silico tools: PolyPhen (http://genetics.bwh.harvard.edu/pph/), SIFT (http://sift.jcvi.org/) and Mutation-Taster (http://neurocore.charite.de/MutationMutation-Taster) for coding variants; a specific software Alamut 2.0 was used to search for splicing prediction . Moreover the presence in the literature of identified variant was recorded. When family members were available, cosegregation of the mutation with disease was tested, especially for complex genotype.
Cardiovascular mortality was defined as death due to cardiovascular causes, including sudden cardiac death, heart-failure related death and stroke-related death. Deaths due to cardiac complications following heart transplantation (such as acute rejection) were also included.
HC-related death or equivalents: cumulative end-point consisting of cardiovascular mortality (as previously defined) as well as cardiac death equivalents including appropriate implantable cardioverter–defibrillator (ICD) intervention for ventricular fibrillation or ventricular tachycardia with a heart rate ≥200bpm and cardiac transplantation (21).
Continuous variables were expressed as median and interquartile range (IQR) and were compared with Mann-Whitney rank sum test or Kruskall-Wallis test. Non-continuous variables were expressed as proportions and were compared with Pearson χ2 test. Probability values were
considered significant when ≤ 0.05. Univariate and multivariate logistic regression analysis were used to identify baseline clinical predictors of cardiovascular mortality and HC-related death or equivalents. Probability of death-free survival was calculated using the Kaplan-Meier method, and survival curves were compared using the log-rank test.
RESULTS
A total of 156 patients was identified with ES-HC (Table 1). Ninety-two patients (59%) had only mild or no symptoms (NYHA functional class I-II) at the time of ES diagnosis. Sequencing analysis identified a total of 131 mutations in 104 ES HC patients (overall prevalence 67%) including 101 missense, 13 splicing, 11 frame-shift, 4 nonsense, 2 in-frame deletion mutations. Of these, 95 (73%) mutations have been previously reported as associated to HC phenotype, whereas 36 (27%) were novel variants. The majority occurred in MYBPC3 (n=71, 5%), MYH7 (n=34, 26%) and TNNI3 (n=12, 9%), while mutations in TPM1, TNNT2, ACTC1, MYL3, MYL2 and LAMP2 were uncommon and collectively accounted for 11% of all identified variants. Eighty-three patients (53%) had single mutations (38 in MYBPC3, 26 in MYH7, 9 in TNNI3, 3 in TPM1, 3 in TNNT2, 2 in MYL2, and 1 in MYL3 and LAMP2 each), whereas 21 patients (13%) had a complex genotype characterized by double-gene mutation heterozygosis (n=6; 4%), compound heterozygosis (n=9; 6%), triple mutations (n=4; 3%) and homozygosis (n=2; 1%) (Figure 1). The gene most frequently involved in complex genotype was MYBPC3 (19 out of 21 complex genotypes, 90%). A novel LAMP2 mutation probably altering the splicing was identified in one patient. No mutations were identified in PRKAG2.
The minor allele frequency value (MAF) was collected from ESP and dbSNP137, which also includes the 1000Genome Project data. Based on this analysis, no mutation (novel or previously reported) had a MAF >1%. In particular, none of our novel mutations is present in any publicly available database (MAF = 0), while among the previously reported mutations, 16 have been reported as very rare, showing a MAF between 0.0077% and 0.0392% but depicted as probably or possibly damaging. Furthermore, we had found also c.83C>T and c.833G>A in TNNT2
and MYBPC3 gene, respectively, previously classified as mutations. These two variants are now reported in ESP database as benign; therefore we decided to eliminate them.
Compared to the 181 patients with HC phenotype and preserved LVEF, patients with ES-HC showed a greater prevalence of complex genotypes (21/156 or 13% vs. 9/181 or 5%; p=0.013); otherwise, the overall prevalence and gene distribution of mutations was not different in the 2 groups; specifically, the location of mutations and the mutated residues were comparable (Figure 2).
Clinical and echocardiographic features did not differ significantly between genotype-positive and genotype-negative ES-HC patients, except for prevalence of HC family history (Table 1). Although median age at first evaluation was 5 years younger, patients with complex genotypes were comparable to those with single or no mutations regarding clinical or demographic features. With regard to specific genes involved, patients with single mutations in MYBPC3 gene were older at HC diagnosis, first evaluation and ES diagnosis compared to those with single mutations in MYH7, single mutations in other genes or with multiple mutations (Table 2). Patients with single mutations in genes different from MYBPC3 or MYH7 (mostly TNNI3) showed a higher LVEF at first detection of ES compared to patients with single mutations in MYBPC3, MYH7 or complex genotypes [47% (IQR 43-49%) vs. 45% (IQR 35-49%), 40% (IQR 35-45%), and 42% (IQR 33-45%) respectively, p=0.018] (Table 2). Of note, there was a 30% prevalence of patients with complex genotypes among those presenting at first evaluation before the age of 20 compared to 19% and 21% in the subgroups aged 20-59 years and ≥ 60 years, respectively (p=0.003). Conversely, single MYBPC3 mutations were by far the most prevalent genotype among patients ≥60 years at first evaluation (79% vs. 20% and 31% in the other age subgroups; p=0.003).
In 95 out of 104 patients with positive genotype a time course from the diagnosis of HC with preserved systolic function to ES evolution was identifiable. In these 95 patients median LVEF at first evaluation was 60% (IQR 58-66), maximal wall thickness 22 mm (IQR 18-26), LV end-diastolic diameter was 45 mm (IQR 40-50), LV outflow obstruction was present in 14 (15%) and massive LV
hypertrophy (maximal wall thickness ≥30 mm) in 10 (10%). Progression to ES was characterized by a 25% reduction in EF (from a median value of 60% to 45%), a 13% increase in LV end-diastolic diameter (from a median value of 45 mm to 51 mm) and a 23% reduction in LV thickness (from a median value of 22 mm to 17 mm) during a median interval of 16 years.
Among the 95 ES-HC patients with normal systolic function at initial presentation, median age at presentation was 16 years (IQR 10-32) for patients with single mutations in MYH7 gene, compared to 26 years (IQR 11-45) in patients with single mutations in other genes including other thick filament genes. Conversely, patients with single MYBPC3 mutations were older both at HC diagnosis and at ES diagnosis compared to patients with single MHY7 mutations, other single gene mutations and multiple mutations (p= 0.0002 and p=0.0008, respectively; Figure 3).
Median duration of follow-up from first detection of ES was 3.6 years (IQR 1.18–8.02). During follow-up, 103 patients (66%) progressed to NHYA functional class III-IV. Overall, 26 patients died (17%): 4 experienced sudden death, 14 due to heart failure, 2 due to stroke, 2 of non cardiac causes and 4 following cardiac transplantation. Of note, 7 of these 26 patients had had 1 or more appropriate ICD interventions before death. Therefore, 130 patients (83%) were alive at the end of follow-up. By comparison, 74 patients (47%) reached the end-point HC-related death or equivalents. In addition to the 26 patients who died, 44 underwent heart transplantation (including 3 with prior ICD interventions), and 4 patients (who were alive at the end of follow-up) experienced appropriate ICD discharges. The total number of patients who experienced appropriate ICD interventions during follow-up was 14 (9%, or 2.5%/year). In the light of these findings, only 53% of ES-HC patients would have plausibly been alive at the end of follow-up in the absence of aggressive management. Of note, cardiac transplantation was associated with favourable outcome in 44/48 cases (92%), who were alive after a median of 7.4 years (IQR 4.3-12.1). Of the 4 patients who died in this subset, 1 died of acute rejection and 3 of non-cardiac causes respectively at 1 month, 6 years and 18 years after transplantation.
Outcome was similar both in terms of cardiovascular death or HC-related death in genotype-positive and genotype-negative patients (16% vs. 8%, p=0.213, and 46% vs. 48%; p=0.253, respectively). Similarly, no significant differences were present between patients with single mutations in MYH7, MYBPC3, other single mutations or complex genotypes (p=0.65). At multivariate analysis, age at initial evaluation (HR 1.03; CI 1.01-1.07; p=0.03) and NYHA III-IV at
initial evaluation (HR 3.04; CI 1.23-7.51; p=0.02) were independently associated with cardiovascular death. Furthermore, age at ES diagnosis, family history of ES-HC, LVEF at initial
evaluation and NYHA III-IV at initial evaluation were independently associated with HC-related death or equivalents (HR 0.97; CI 0.95-0.99 p=0.002, HR 1.79, CI 1.03-3.10; p=0.036, HR 0.97; CI
0.95-0.99; p=0.018 and HR 2.53; CI 1.45-4.41; p=0.001 respectively). Kaplan–Meier estimates of the proportion of patients without heart failure-related death and cardiac transplantation according
to the structural characteristics of mutated proteins are shown in Figure 4. DISCUSSION
The present study assessed the genetic and clinical profile of the largest cohort of ES-HC patients reported to date. In over 150 HC patients with a LVEF <50% a comprehensive sarcomere gene screening showed that: 1) the rate and spectrum of mutations were similar to those of other patients with HC and preserved systolic function; 2) a higher frequency of multiple mutations was present compared to HC with preserved EF, particularly among younger patients; 3) the clinical course and outcome of ES-HC patients was confirmed to be extremely severe but independent of genotype.
The 67% yield for 1 or more sarcomere mutations in our ES cohort is comparable with the prevalence in cohorts with HC and preserved systolic function (14, 22). The majority of mutations were identified in MYBPC3, MYH7 and TNNI3 accounting for almost 90% of genotype-positive patients. Conversely, mutations in TPM1, TNNT2, ACTC1, MYL3, MYL2 and LAMP2 were uncommon, and none of the patients had mutations in PRKAG2. Of note, the prevalence of complex genotypes, characterized by 2 or even 3 mutations coexisting in the same patient, was
13% in our cohort of ES-HC, i.e. about 3-fold times higher than the values reported in literature for HC with preserved EF (12-14) as well as in our reference cohort group. Notably 30% of patients presenting at first evaluation before the age of 20 were found to have complex genotypes.
These findings strengthen the hypothesis that the mutational load may play an important role in the phenotypic expression of HC contributing to disease severity not only regarding malignant arrhythmias, sudden death and massive hypertrophy but also evolution to the ES phase (12-16). On the other hand, while the 13% prevalence observed in our cohort is indeed high compared to classic HC with preserved systolic function, the vast majority of end-stage patients were not accounted for by double or triple sarcomere gene mutations. Thus, complex genotypes may be considered predictors of end-stage progression, but the negative predictive value remains low.
With the exception of the overrepresentation of complex genotypes, however, there was no significant difference in the distribution of mutations and in the mutated residues in ES-HC patients compared to our reference cohort with HC and preserved EF, or to the case series reported in literature. Specifically, the overall prevalence of variants for each of 8 genes and the location of the variants within the various exons was similar in HC patients with and without systolic dysfunction.
Among ES-HC patients, genotype-positive patients showed a greater evidence of familial disease. Within the mutated group, the early age at presentation of thick filament patients was a myosin heavy chain effect; in fact patients with single mutations in MYH7 gene were younger at presentation compared to patients with single mutations in MYBPC3 or other genes. Differently, patients with single MYBPC3 mutations were older at HC diagnosis, first evaluation and onset of ES, compared to those with single MYH7 mutations, single mutations in other genes or multiple mutations (23). Consistently, Kubo et al. have reported a frequent, late onset of systolic dysfunction in HC patients with a founder MYPBC3-mutation in Japan (24), whereas Fujino et al. have found an independent relation between age and development of systolic dysfunction (19). To date, MYBPC3 remains a largely unknown entity within the sarcomere; recent data based on
mouse model of HC suggest that sarcomere dysfunction in MYBCP3-HC could be related to failure of nonsense mRNA-mediated decay or ubiquitine-proteasome system in effectively removing the mutant mRNA and protein from the cardiomyocyte, adding a quota of poison effect to the pre-existing haploinsufficiency (25). An age-related impairment in these molecular systems may therefore account for the delayed onset of ES in MYBPC3-positive patients. Moreover, MYBPC3 is the gene most frequently involved in complex genotypes further supporting the dosage-effect of mutations.
Within the ES cohort, long term prognosis does not seem to be influenced by the presence, type and number of mutations. In particular, mutation-negative patients share essentially the same poor natural history as mutation-positive ones. Overall, these findings emphasize the complexity of HC pathophysiology, in which the pathways leading to the fully expressed phenotype, including evolution of LV hypertrophy, subsequent adverse remodeling and systolic dysfunction, cannot be ascribed to a simple cause-effect relationship with a specific gene or mutation. On the other hand, findings such as the excess number of complex genotypes and the delayed onset of ES in single MYBPC3 mutations suggest that the genotype may play a role in certain subgroups. A number of genetic and epigenetic modifiers could influence the ultimate, individual nature and course of the disease, thus amplifying or diluting the specific effects of disease-causing mutations. These modifiers are likely to include an array of genetic, metabolic, hormonal and environmental factors, which might explain the large heterogeneity of HC phenotypes even within the same pedigree, although this still remains speculative. The opportunities offered by new technologies, such as next generation sequencing, will deepen our understanding of the genetic and epigenetic mechanisms of ES pathogenesis, although the increasing number of variants of undetermined significance identified will determine subsequent difficulties in interpretation of clinical significance (26, 27).
The large number of ES-HC patients assembled in this study also allowed us to evaluate the disease course and outcome over a relatively long follow-up period. Consistent with previous reports (2-4, 14), 50% of our ES patients would have died over a relatively short follow-up in the
absence of aggressive management (i.e. ICD implantation and heart transplantation). This confirms the necessity of an early detection of the ES phase and the importance of referring these patients to tertiary centres where early consideration for heart transplantation and ICD will occur.
One of the limitations of this study is that the segregation of mutations in complex genotypes was not always possible due to the small number of family members available for genetic screening. This is particularly important considering that it is very common in HC to encounter “private” mutations that are novel and therefore of uncertain clinical significance (many of the mutations reported in previous studies, when lacking appropriate validation, cannot be considered certainly pathogenic) and that the majority of double mutations involved a single gene (MYBPC3). Moreover the used definition of mutation or genotype-negative implies only the absence of mutations in the analyzed genes, although mutations in other known disease genes could not be excluded.
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FIGURE LEGENDS
Figure 1. Pedigrees of 4 families with ES-HC and multiple mutations.
Figure 2. Distribution of mutations in MHY7, MYBPC3, TPM1 and TNNT2 functional domains: a comparison between patients with ES-HC and patients with HC and normal LVEF.
Figure 3. Time-line with patient ages describing evolution towards the ES phase in the 95 patients with positive genotype according to the structural characteristics of mutated proteins.
Figure 4. Kaplan-Meier estimates of heart failure-related death/cardiac transplantation free survival of patients with ES-HC according to the structural characteristics of mutated proteins.
