Novel α‐Actin Gene Mutation p.(Ala21Val) Causing Familial Hypertrophic Cardiomyopathy, Myocardial Noncompaction, and Transmural Crypts. Clinical‐Pathologic Correlation
Background Mutations of α‐actin gene (ACTC1) have been phenotypically related to various cardiac anomalies, including hypertrophic cardiomyopathy and dilated cardiomyopathy and left ventricular (LV) myocardial noncompaction. A novel ACTC mutation is reported as cosegregating for familial hypertrophic cardiomyopathy and LV myocardial noncompaction with transmural crypts.
Methods and Results In an Italian family of 7 subjects, 4 aged 10 (II‐1), 14 (II‐2), 43 (I‐4) and 46 years (I‐5), presenting abnormal ECG changes, dyspnea and palpitation (II‐2, I‐4, and I‐5), and recurrent cerebral ischemic attack (I‐5), underwent 2‐dimensional echo, cardiac magnetic resonance, Holter monitoring, and next‐generation sequencing gene analysis. Patients II‐2 and I‐5 with ventricular tachycardia underwent a cardiac invasive study, including coronary with LV angiography and endomyocardial biopsy. In all the affected members, ECG showed right bundle branch block and left anterior hemiblock with age‐related prolongation of QRS duration. Two‐dimensional echo and cardiac magnetic resonance documented LV myocardial noncompaction in all and in I‐4, I‐5, and II‐2 a progressive LV hypertrophy up to 22‐mm maximal wall thickness. Coronary arteries were normal. LV angiography showed transmural crypts progressing to spongeous myocardial transformation with LV dilatation and dysfunction in the oldest subject. At histology and electron microscopy detachment of myocardiocytes were associated with cell and myofibrillar disarray and degradation of intercalated discs causing disanchorage of myofilaments to cell membrane. Next‐generation sequencing showed in affected members an unreported p.(Ala21Val) mutation of ACTC.
Conclusions Novel p.(Ala21Val) mutation of ACTC1 causes myofibrillar and intercalated disc alteration leading to familial hypertrophic cardiomyopathy and LV myocardial noncompaction with transmural crypts.
What Is New?
The identification of a novel p.(Ala21Val) ACTC1 gene mutation cosegregating for familial hypertrophic cardiomyopathy and left ventricular myocardial noncompaction with transmural crypts is reported.
This single‐point mutation of ACTC1 causes a composite structural change involving sarcomeres and intercalated discs.
What Are the Clinical Implications?
Dysfunction of cytoplasmic α‐actin causes a disanchorage of myofibrils to sarcolemmal membrane followed by myofibryllolysis and possibly cell death.
Intercalated discs seem to be particularly involved by this mutation given that they appear irregular, and fragmented, favoring cell disconnection.
HCM is characterized morphologically and defined by a hypertrophied, nondilated left ventricle in the absence of another systemic or cardiac disease that is capable of producing the magnitude of wall thickening evident.2 More than 90% of HCM is inherited as an autosomal‐dominant disease with variable expressivity and age‐related penetrance. HCM occurs mainly because of mutations in genes encoding for the cardiac sarcomere. Around 70% of these mutations are in the sarcomere genes encoding for cardiac β‐myosin heavy chain (MYH7; MIM *160760) and cardiac myosin binding protein C (MYBPC3; MIM *600958). Other sarcomeric genes involved in HCM are regulatory myosin light chain (MYL2; *160781), cardiac troponin T (TNNT2; MIM *191045), cardiac troponin I (TNNI3; MIM *191044), and actin (ACTC1; MIM *102540). Nonsarcomeric genes, such as genes encoding plasma membrane and mitochondrial proteins, as well as sarcomere adjacent Z‐disc encoding genes, account for the other HCM cases.
Left ventricular (LV) noncompaction (LVNC), the most recently classified form of cardiomyopathy, is characterized by abnormal trabeculations in the left ventricle, most frequently at the apex. LVNC can be associated with LV dilation or hypertrophy, systolic or diastolic dysfunction, or both or various forms of congenital heart disease. Genetic inheritance arises in at least 30% to 50% of patients, and several genes that cause LVNC have been identified.3, 4 These genes seem generally to encode sarcomeric (contractile apparatus) or cytoskeletal proteins. Disrupted mitochondrial function and metabolic abnormalities were demonstrated to have a causal role, too.5
Recent literature has documented genetic evidence to prove LVNC and HCM as having causative genes, which are overlapping. Two separate autosomal‐dominant LVNC families with mutations in the sarcomeric MYH7 gene, known to be associated with HCM, restricted cardiomyopathy, and dilated cardiomyopathy, were described.6, 7 Monserrat et al8 described another 5 families, 1 with LVNC and 4 with HCM with mutations in the ACTC1 gene.
In the following study, a novel p.(Ala21Val) ACTC1 mutation manifesting with familial hypertrophic cardiomyopathy and myocardial noncompaction with transmural crypts, is reported. Combination of myocyte and myofibrillar disarray with degradation of intercalated discs because of abnormal anchorage of mutated ACTC1 protein to sarcolemmal membrane are shown to be at the base of the ultrastructural abnormality.
The data, analytical methods, and study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedure.
In an Italian family of 9 subjects (Figure 1), 1 dead at 50 years of age for cardiac arrest (I‐1), 4 presented abnormal ECG changes, and 3 were symptomatic for palpitations and/or dyspnea. The oldest affected patient (II:5) presented also recurrent episodes of cerebrovascular transient ischemic attacks.
The 4 patients with ECG abnormalities (10 [III:1], 14 [III:2], 43 [II:4], and 46 [II:5] year‐old male) had a 24 hs Holter monitoring that showed in the 14‐ (III:2) and 46‐year‐old subjects (II:5) runs of nonsustained ventricular tachycardia. The 4 subjects with ECG abnormalities had additionally an echocardiogram. The 2 patients with electrical instability had an implemental cardiac magnetic resonance and invasive cardiac study, including coronary angiography, LV angiography, and endomyocardial biopsy. All 7 subjects underwent a next‐generation sequencing (NGS) gene analysis (Table).
Institutional review board approval was obtained, according to the guidelines.
Cardiac magnetic resonance exams have been perfomed on a 1.5 Tesla scanner (Avanto; Siemens Medical Solutions, Erlangen, Germany). Standard cardiac magnetic resonance protocol included: (1) Cine magnetic resonance images acquired during breath‐holds in the short‐axis, 2‐chamber, and 4‐chamber; (2) black blood T2‐weighted short tau inversion recovery images on short‐axis planes covering the entire left ventricle during 6 to 8 consecutive breath‐holds for myocardial edema detection; (3) late gadolinium‐enhanced imaging performed 15 minutes after injection of 0.2 nmol/kg of gadoteratemeglumine (gadolinium‐DOTA, Dotarem; Guerbet, Paris, France), and signal intensity value 2 SDs above the mean signal intensity of the remote normal myocardium was considered suggestive for myocardial fibrosis.
Cardiac catheterization, selective coronary with LV angiography, and endomyocardial biopsy followed patients’ written informed consent. Biopsies (5 samples each patient) were performed in the septal‐apical region of the left ventricle.
Histology and Electron Microscopy
Endomyocardial biopsies were fixed in 10% buffered formalin and paraffin embedded. Sections (5 μm) were stained with hematoxylin and eosin, Masson thrichrome, and Miller's Elastic Van Gieson. For transmission electron microscopy, endomyocardial biopsies were fixed by 2.5% glutaraldehyde solution in phosphate buffer (0.1 mol/L, pH 7.3 at cold), then postfixed in osmium tetroxide and processed following a standard schedule for embedding in Epon resin. Semithin plastic sections were stained at warm with AzurII and basic fucsin. Ultrathin sections were stained with uranyl acetate and lead hydroxide. A Philips (CM‐10; Philips, Best, The Netherlands) transmission electron microscope was used for observation and photographic analysis.
NGS and Structural Interpretation of Variants
Genomic DNA was isolated from peripheral blood using Macherey‐Nagel NucleoSpin blood extraction kit (Macherey‐Nagel, Duren, Germany). Targeted enrichment was performed using TruSight Cardio Sequencing Kit (Illumina, San Diego, CA). This target panel includes 174 genes with known associations to 17 different inherited cardiac conditions (see Table S1 for the list of genes included in the gene panel; Pua et al).14 Captured libraries were loaded onto a MiSeq sequencing platform (Illumina, San Diego, CA).
Sequences (aka reads) were analyzed using a custom software pipeline. In brief, paired‐end reads were aligned to the GRCh37/hg19 reference genome by Bowtie 2 (version 2.3.0). BAM files were sorted by SAM tools (version 1.3.2) and purged from candidate PCR duplicates using Mark Duplicates from the Picard suite (version 2.9.0). The alignment process was optimized by the local realignment and base‐quality‐score recalibration functions, as implemented in the Genome Analysis Toolkit (GATK 3.5). To confer higher confidence, both reads and alignments underwent an extensive evaluation of the base‐per‐position and mapping‐per‐read quality scores. Reads with mapping quality scores lower than 20 or with more than one half nucleotides with quality scores less than 30 were filtered out. The GATK's Haplotype Caller was used to identify single‐nucleotide polymorphisms and insertions/deletions. Genetic variants were annotated by ANNOVAR and subjected to filtering. In particular, only novel or rare exonic and splicing variants were considered. Thus, these variants exhibited an allele frequency lower than 1% in the general population, as from dbSNP 149 (https://www.ncbi.nlm.nih.gov/projects/SNP/), GO‐ESP (http://evs.gs.washington.edu/EVS/) and ExAC (http://exac.broadinstitute.org/). The pathogenicity of these variants was assessed in silico using 13 pathogenicity predictors (eg, SIFT, PolyPhen 2, Mutation Assessor and CADD, and thermodynamically). In particular, the thermodynamic stability of the wild‐type or mutant proteins was investigated using the FoldX algorithm. It computed the total energy of proteins, as a proxy of their overall stability, and the Van der Waals inter‐residue clashes, as energy penalization factors. Thus, the 3‐dimensional structures of both proteins were minimized, namely all the side chains were slightly moved in order to reduce the Van der Waals’ clashes. It was run with standard parameters on a system modeled on a template of the rabbit skeletal muscle actomyosin rigor complex (Protein Data Bank ID: 5h53). A stand‐alone version of FoldX is available from http://foldx.crg.es (Schymkowitz et al).15 Potentially harmful variants were finally prioritized considering familial segregation. Variants identified using NGS were validated by Sanger sequencing and segregation analysis using the ABI BigDye Terminator Sequencing Kit v.3.1 (Life Technologies, Carlsbad, CA) and an ABI 3130XL automated sequencer.
ECG in the 4 affected members showed similar changes consisting in right bundle branch block and left anterior hemiblock with increased QRS duration in the oldest patient, associated to negative T waves in the anterolateral leads. Ventricular ectopic beats were documented in patients II‐4, II‐5, and III‐2. In patients III‐2 and II‐5 runs of nonsustained ventricular tachycardia were also registered.
Two‐dimensional echocardiogram showed prominent trabeculations with evidence of irregular endocardial surface in all 4 affected family members. Particularly in II‐4 and II‐5 patients, deep endocardial recesses were documented with color Doppler, meeting the echocardiographic diagnostic criteria for LVNC (ie, noncompaction/compaction ratio ≥2).16 A remarkable LV hypertrophy with an LV maximal wall thickness of 12‐, 17‐, and 22‐mm thickness was observed in patients II‐4, II‐5, and III‐2, respectively. The youngest 10‐year‐old boy (II‐1) had normal cardiac wall thickness, dimensions, and function. The II‐5 had also a dilated (end‐diastolic diameter, 67 mm) and severely hypokinetic (ejection fraction, 30%) left ventricle. Cardiac magnetic resonance showed in the III‐2 symmetric mild LV hypertrophy diffusely involving all myocardial segments with a maximal wall thickness of 12 mm (Figure 2). In particular, cine steady‐state free precession images acquired on midventricular short‐axis (Figure 2A and 2B) and vertical long‐axis (Figure 2C and 2D) views showed trabeculated noncompacted myocardium involving anterior and lateral walls, and deep transmural crypts located at the basal inferior LV wall. No areas of gadolinium enhancement have been detected on late gadolinium‐enhanced inversion recovery imaging.
In the II‐5 patient, cine steady‐state free precession images showed deep myocardial crypts penetrating almost the entire thickness of the myocardium, involving almost all segments, hyperthophy of basal septum (maximal wall thickness, 22 mm), and highly trabeculated midapical anterolateral wall as commonly observed in noncompaction myocardium (Figure 3A through 3C). Late gadolinium‐enhanced inversion recovery short‐axis image showed diffuse enhancement of anterolateral wall consistent with diffuse interstitial myocardial fibrosis (Figure 3D).
Coronary angiography obtained in the III‐2 and II‐5 affected members was normal. In these patients, LV angiography confirmed the presence of transmural crypts (Figures 2 and 3E, 3F) particularly prominent in the oldest patient where they assumed a spongeous conformation (Figure 3E and 3F) becoming the likely source of cerebral thromboembolism.
Histology and Electronmicroscopy
At histology, cardiomyocytes were hypertrophied up to 60 μm in diameter at nuclear level and often in total disarray (Figures 2 and 3G). Myocytes were diffusely separated by unendotheliolized spaces and channels of various width. These changes were more prominent in the I‐5 patient where a remarkable interstitial and replacement fibrosis was also documented. At ultrastructural examination diffuse myofibrillar disarray was present. The intercalated discs were diffusely irregular, fragmented (Figure 2H), and often detached (Figure 3H), being connected with areas of myofibrillolysis, likely attributed to disanchorage of mutated cytoplasmic alpha‐actin.
The affected family members having LVNC associated with HCM (I:5 and II:2) were mutation scanned using the targeted NGS TruSight Cardio Kit panel including 174 genes involved in inherited cardiac conditions.14 Among a total of 32 000 variants identified by NGS analysis, after selection and prioritization filtering, 2 variants, both confirmed by Sanger sequencing, were found to be shared by both individuals, 1 in the ACTC1 gene [NM_005159.4:c.62C>T;p.(Ala21Val)], encoding for the skeletal muscle alpha‐actin, and the other in the TTN gene [NM_001267550:c.28127C>G;p.(Tyr9376Arg)], encoding for Titin, a giant muscle protein expressed in the cardiac and skeletal muscles. ACTC1 p.(Ala21Val) variant alters a highly conserved amino acid, up to Baker's yeast, located within the actin‐like domain of the protein. It is not reported by public single‐nucleotide polymorphism databases (1000 genomes, ExAC) and is deemed deleterious by various in silico prediction programs (SIFT, score=0.03; PolyPhen2, score=0.85; MutationTaster, P=1; CADDphred=24.2). p.(Tyr9376Arg) variant is a rare TTN1 allele reported with a frequency of C=0.00005/6 in the ExAC population database (rs749875409). No protein conservation data are available for this variant, which is also reported as a variant with uncertain significance in the ClinVar database, a public archive of reports of clinical significance of variants (https://www.ncbi.nlm.nih.gov/clinvar/). To further prioritize the 2 variants, their patterns of segregation were analyzed by Sanger sequencing in available family members (II:1, II:2, II:3, II:4, and III:2). Sequence analysis confirmed segregation of the ACTC1 p.(Ala21Val) variant with the disease in 2 further affected family members (II:4, III:1), whereas TTN variant p.(Tyr9376Arg) was found not to segregate with the disease phenotype. Therefore, we propose that ACTC1 p.(Ala21Val) is the disease‐causing mutation of this family. To further confirm the pathological effects of ACTC1 p.(Ala21Val) missense variant, its thermodynamic impact on the stability of the overall 3‐dimensional structure of ACTC1 was assessed by the FoldX algorithm (Figure 4). Following free energy calculations, ΔGmut=213.85 kcal/mol and ΔGwt=211.49 kcal/mol, we estimated a ΔΔG increase, after mutation, of 2.36 kcal/mol (±0.005 kcal/mol), which is compatible with the highly destabilizing character hypothesized for the variant under examination.
Treatment and Follow‐up
The three patients with preserved cardiac dimensions and function were treated with bisoprolol (2.5 mg daily for the III:1 and III:2; 5 mg daily for I:4). The II:5 with cardiac dilatation and severe dysfunction received carvedilol 25 mg tid in addition to amiodarone 200 mg daily, diuretics and angiotensin‐converting enzyme inhibitors. Warfarin was introduced because of recurrent cerebrovascular transient ischemic attacks. He also underwent an implantable cardioverter defibrillator implantation and was suggested for cardiac transplantation.
The first 3 patients were followed for a 5‐year time. Clinical conditions were stable; no major arrhythmias were recorded at repeated (every 3 months) Holter monitoring; LV wall thickness, dimension, and function remained unchanged.
A novel α‐actin gene mutation p.(Ala21Val) causing familial HCM, myocardial noncompaction, and transmural crypts is reported on. This ACTC1 mutation is exceedingly rare and was not detected in over 126 000 individuals in the ExAC database. Moreover, a range of mutation analysis approaches (SIFT, PolyPhen2, MutationTaster, and CADDphred), homology studies, and protein modeling all suggest that the p.(Ala21Val) mutation is pathogenic. Furthermore, the p.(Ala21Val) mutation strongly cosegregates with the disease, providing additional support for its pathogenicity. ACTC1 gene mutations have been phenotypically related with diverse cardiac anomalies, including dilated cardiomyopathy,17 hypertrophic cardiomyopathy,18, 19, 20 myocardial noncompaction,8, 21 and congenital heart defects, in particular atrial septal defect.22
To our knowledge, this is the first report in which a sarcomeric gene mutation is found to cosegregate with both HCM and LVNC in multiple family members, suggesting that ACTC1 p.(Ala21Val) mutation causes a high penetrance for both HCM and LVNC.
Genotype‐phenotype correlations within the ACTC1 mutation spectrum are not unexpected, for example, ACTC1 mutations resulting in congenital heart defects (essentially atrial septal defects) are restricted to the first half of the protein (from residue Met84 to residue Met178),23 whereas beyond residue Met178, all reported ACTC1 mutations are found to result in cardiomyopathies17, 18, 19, 20 with an unusual prevalence of noncompaction or hypertrophied apex.
In 2 patients with ACTC1 mutation and pronounced ventricular arrhythmias, an additional mutation of TTN gene has been found, suggesting that it might have contributed to the arrhythmic phenotype. On the other hand, TTN mutation has been associated with arrhythmic cardiomyopathies.24
Diagnosis of HCM has been supported by an age‐related progressive thickening of LV walls associated to severe hypertrophy with disarray of myocardiocytes at histology. Myocytes were also found frequently separated by unendotheliolized spaces of various width, suggesting the coexistence of myocardial noncompaction. Crypts, defined as narrow, deep blood‐filled endocardial invaginations within the myocardium, were transmural and diffusely distributed along the left ventricle if compared with isolated HCM and healthy volunteers where they are of limited number and depth.25, 26 It may be that these architectural abnormalities, first described at postmortem with HCM27, 28, 29 and recently recognized by cardiac magnetic resonance in 5% of normal, 4% of HCM, and 61% of genotype‐positive phenotype‐negative (prehypertrophic) HCM,21, 22 may contribute to disease progression when diffusely associated to myocardial noncompaction.
What appears from gene analysis by NGS of affected and nonaffected members of our family is that the previously unreported p.(Ala21Val) missense mutation of ACTC1 is associated with multiple structural changes involving sarcomeres and their anchorage to sarcolemmal membrane. Indeed, this variant alters a highly conserved amino acid located within the actin‐like domain of the protein. In addition, the thermodynamic studies of mutated protein obtained in our cases suggest that this mutation is highly destabilizing conferring to the protein a pathogenic impact.
Structural consequences of the mutated protein are delineated by histological and ultrastructural examination of LV endomyocardial biopsies obtained in 2 affected members. Specifically, mutation of sarcomeric α‐actin is followed by fibrils disarray and hypertrophy with disarray of myocardiocytes. Dysfunction of cytoplasmic α‐actin causes a disanchorage of myofibrils to the sarcolemmal membrane followed by myofibryllolysis and, possibly, cell death. Intercalated discs seem to be particularly involved by this mutation given that they appear irregular, and fragmented, favoring cell disconnection.
Clinical implications have been reported as arrhythmic early in life, hypertrophic with diastolic dysfunction, and dyspnea up to middle age evolving toward cardiac dilatation and dysfunction in the fifth decade of life. Spongeous transformation of LV myocardium and heart failure eventually predispose to cardiac thrombus formation and systemic embolization.
As far as therapy is concerned, early recognition of this entity and treatment with beta‐blockers may control cardiac arrhythmias reducing cellular stress and perhaps limit the disease progression. Anticoagulation may be required in the occurrence of heart failure and/or thromboembolic events.
In conclusion, p.(Ala21Val) mutation of ACTC1 is a novel pathogenic variant giving rise to a complex structural change in human myocardium, including HCM, myocardial noncompaction, and transmural crypts. This results in electrical instability, cardiac dilatation, and failure requiring heart transplantation in the fifth decade of life.
Sources of Funding
The study was supported by the “Ateneo La Sapienza” grant C26H15H9LP “Differential mutated gene expression causing cardiomyocyte phenotype variation in human hypertrophic cardiomyopathy.”
Table S1. Alignment of Amino Acid Residues Adjacent to ACTC1 p.(Ala21Val) Sequence Variant Showing Level of Conservation Among Different Species
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