Background
Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death (SCD) in the young as well as in the trained athletes, and is also a critical substrate for heart failure disability at any age. HCM is a clinical heterogeneous but relatively common form of genetic heart defect transmitted as autosomal dominant trait (affecting 1 in 500 people). It is characterized by unexplained cardiac hypertrophy, myocyte disarray, and fibrosis [
1]. The yield of genetic testing among HCM cases is as high as ~70%, and comprehensive or targeted (
MYH7, MYBPC3, TNNI3, TNNT2, TPM1) HCM genetic testing is recommended for patients with HCM and first-degree relatives based on clinical history, family history, and electrocardiographic/echocardiographic phenotype [
2,
3]. HCM patients with J point and ST segment elevation are related to higher risk of ventricular arrhythmias and SCD [
4]. However, the genetic background of this situation has not been deeply investigated before.
Early repolarization (ER) pattern is defined as J wave (>0.1 mV) with or without ST segment elevation in at least two continuous leads in standard 12-lead ECGs [
5]. It has been related with malignant ventricular tachycardia and SCD [
6]. ER pattern also has a tendency of heritability [
7]. Several ion channel mutations have been linked to ER syndrome, such as genes in I
K,ATP and I
Na [
8‐
10]. It is worth mentioning that, cardiac calcium channel mutations are related to ER with shortened QTc interval because of its influence on the action potential duration (APD). These patients had higher tendencies for cardiac event and SCD [
8].
Here we report a Chinese family of HCM with early repolarization pattern, and borderline short QTc interval. The clinical phenotypes included severe cardiac hypertrophy and left ventricular outflow tract obstruction, left ventricular dysfunction, atrial fibrillation, and sustained ventricular tachycardia. Novel trigenic mutations in DES/MYPN/CACNA1C are detected in the proband and his family. The clinical characteristics and genetic background of the family members are investigated in detail.
Discussion
Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric hypertrophy of interventricular septum, disorganization of cardiomyocytes, as well as myocardial interstitial fibrosis. The basis of HCM has been ascribed to multiple etiologies; however, in 1989 researchers first mapped a genetic mutation for HCM to chromosome [
12]. Subsequently, hundreds mutations have been found in HCM patients. Most mutations involve the myofilaments of the cardiac sarcomere (
MYH7,
MYBPC3, et al.); however, there is increasing awareness of non sarcomeric mutations as well, such as Z-disk or intracellular calcium modulators.
Comprehensive testing of five HCM genes is strongly recommended to assess patients with HCM by clinical guidelines [
2]. Whereas Nextgen sequencing approaches have facilitated much broader testing panels to be widely available, such as the one we employed in this study (Table
2). In both the proband and his daughter, we simultaneously identified trigenic mutations of p.E234K in the
DES, p.R989H in the
MYPN, and p.R1973P in
CACNA1C.
There was few reported trigenic mutation related with cardiomyopathy. One study described a case of HCM with trigenic mutations (LAMA4, PKP2 and TTN) [
13], but the ECG characteristics of the patient was not available. On the relationship between gene mutation and QT interval, one study showed that patients with HCM-related gene mutation frequently exhibited QT interval disruption, which was related with increased occurrence of ventricular arrhythmias [
14]. Although CACNA1C mutation very likely underlined the shortened QT intervals in our case, it was hard to determine the role of each individual mutation in the genesis of the clinical phenotypes, due to incomplete family history as well as lacking of previous references.
It is well known that QT intervals are slightly prolonged in most HCM patients due to the increased late sodium current and fibrosis [
15]. However, the QTc of this proband is significantly short. The intervals from the J points to the peak of T waves were approximately 120 ms. Combined with typical ER in the inferioral-lateral leads, the characteristics of short QT interval might be attributed to the mutation p.R1973P in
CACNA1C.
CACNA1C lie in chromosome 12, coding for α subunit of the L-type calcium channels. The genetic defects in I
Ca,L have been linked with LQTS [
16], Brugada syndrome [
17], ERS [
8,
9] and SQTS [
18]. Of the reported defects, loss-of-function mutations are extremely rare. In this case, the mutated site was located in the C-terminal of the channel protein (Fig.
4a), which is a “hot region” for loss-of-function mutations. It plays an important role in the Ca
2+ influx and kinetics procedure, as well as intracellular signal transductions [
19,
20]. In order to illuminate the disease-causing ability of the
CACNA1C-R1973P mutation, heterologous expressions and patch clamping techniques were conducted. We found out that the R1937P mutant dramatically decreased peak current density by ~68%, while exerting no significant influences on the channel kinetics, which is consistent with the previously reported V2014I mutations in the vicinity area [
8]. We speculated that the channel current reductions might be due to the trafficking deficiencies of the channel protein. Decreased I
Ca,L results in increased net outward currents and shortened cardiomyocyte repolarization period. Due to the transmural discrepancies of outward potassium currents (I
to, I
K, etc.), the increase in net outward current result in partial or complete loss of the action potential dome, leading to a transmural voltage gradient that manifests as ER waves. Meanwhile, accelerated repolarization caused by the mutation could lead to subsequent short QT interval in the surface ECG. The SQT4-6 are linked to mutations in different subunits of calcium channel [
8,
18]. There is a high prevalence of ER in SQTs [
21], suggesting the genetic background of the two diseases may share common fields.
The
CACNA1C mutation also linked with the slowed AV conduction and bundle branch block, because I
Ca,L is the main depolarization current in cardiac conduction cells. Decreased I
Ca,L hampers the depolarization of these cells and causes delayed conductions. However, it is also noteworthy that the
DES mutations could cause atrioventricular blocks [
22]. So the conduction delay is probably the outcome of compound genetic defects of both.
The daughter of the patient carries the
CACNA1C mutation while her ECG presented far less prominent ER, and normal QTc interval. The incomplete penetrance of the
CACNA1C mutation is probably caused by genetic background or certain factors, such as the gender protection. It is well known that sex hormones have significant influence on the ion channel functions as well as on the cardiac repolarization procedure. Testosterone can shorten cardiomyocyte APDs in guinea pigs, through inhibiting the I
Ca and enhancing I
Ks [
23]. Thus, testosterone’s inhibiting effect on the I
Ca,L may worsen the effect of the
CACNA1C mutation and cause the more severe clinical symptoms of the father. As a matter of fact, ER pattern are more common in male population [
24]. Another possible explanation is the matter of age. Experiment on the animals show that the expression of the I
Ca,L is decreased with aging [
25]. Aging has also been related to decreased expressions of potassium and sodium channels [
26,
27]. Collectively, the net inward currents during cardiac repolarization decreased with age. The symptoms of ventricular tachycardia appeared when the patient was in his 50’s, indicating the importance of aging in the pathogenesis. Last but not the least, the degree of the myocardial hypertrophy is also a critical factor. The more severe myocardial hypertrophy of the father may increase the transmural dispersions in the cardiac repolarization and enhance the ST segment elevations, as well as the onset of ventricular tachycardia. Previous studies indicate that variants in sarcomere genes may individually or collectively affect cardiac morphology and function, even without causing overt HCM. HCM is transmitted as a dominant trait. However, penetrance is incomplete and lowest at young ages [
11]. Identical HCM mutations in the same pedigree can produce distinct LVH morphologies, varying amounts of myocardial fibrosis, and differing susceptibility to arrhythmias. Genetic modifiers, epigenetic differences, and unique environmental factors are likely to influence these variables. But without long term follow-up and deeper translational research, especially more information form the pedigree, it is hard for us to deduce which assumption is the major cause of ventricular arrhythmia. The proband suffered from severe obstructive HCM and his daughter also showed mild myocardial hypertrophy. The
CACNA1C mutations have been related to HCM in previous reports. However, there were two other mutations detected in this family, which might be part of the pathogenesis of the HCM. The
DES gene is located in chromosome 2, coding for desmin protein. Desmin is the type III intermediate filament protein with the molecular weight of 53.5 kD of 470 residues, which is expressed in various types of muscle cells. In the heart, it integrates the Z disc, the sarcolemma and the myocyte nucleus. Mutations in the
DES gene are the causes of the desmin-related myopathies (DRM) [
28]. Recent reports linked
DES mutations with arrhymogenic right ventricular cardiomyopathy (ARVC) [
29] and dilated cardiomyopathy (DCM) [
30].
DES mutations are rarely associated with HCM except in few sporadic cases [
31]. The
MYPN gene is a 145.2 kDa protein of 1320 residue for encoding myopalladin, which is located in chromosome 10. Myopalladin is the component of the myofilament, connecting the Z disc, the sarcolemma and the myocyte nucleus. The myopalladin participate in the regulations of the contraction and adhension of the cardiomyocytes, as well as the gene expressions. Also, the mutations in
MYPN were linked with DCM, HCM and restrictive cardiomyopathy (RCM) [
32]. Both desmin and myopalladin were important components of the cytoskeleton. The dual mutations cause instability of the sarcomeres and decreased contraction efficiencies. On the other hand, the Ca
2+ cycling exert significant impact on the sarcomere contractions. Intracellular Ca
2+ level is crucial for the normal contraction of cardiomyocytes [
33]. The impaired function of I
Ca,L lead to decreased influx of Ca
2+ ions, influencing the intracellular Ca
2+ cycling and reduce the efficiency of sarcomere contraction. The impaired cytoskeletons caused by digenic
DES/MYPN mutations may also hamper the
CACNA1C expressions. Thus, the three mutations work together in the impairment of the sarcomere functions and subsequent onset of cardiomyopathy.
The managements of HCM include optimized drug therapy and septal reduction therapy, when the LVOT gradients exceed 50 mmHg with clinical symptoms like dyspnea or chest pain. Either surgical myectomy or septal ablation is generally effective in reducing LOVT obstructions and relieving clinical symptoms. The patient developed refractory LV hypertrophy and severe LVOT obstruction 3 years after the septal ablation, due to those mutations. Interestingly, the CRT significantly reduces the outflow tract gradient. The CRT was not considered as the standard therapy for HCM patients [
2], but there are some reports on the effectiveness of resynchronization therapy on reducing the outflow tract gradients in HCM patients [
34‐
36]. Although the mechanism is not clear yet, some proposes that the biventricular pacing changed the sequence of the ventricular excitation, which helps to reduce the outflow tract gradient and to reverse the remodeling of the left ventricles [
35]. Our report adds clinical support to the effectiveness of the CRT on reducing the outflow tract gradients, which might be a promising strategy for the treatment of HCM patients.
The daughter gave birth to a boy recently. Unfortunately, she refused genetic screening for the baby boy. To observing the progressive nature of the disease, we plan to follow the daughter as well as her son continuously. Meanwhile, we plan to build human induced pluripotent stem cells (hiPSC) from skin tissue of the proband and family members, to replicating the situation and perform further research on stem cell level with all the genetic features in this pedigree.
In this study, we reported a family with ER, SQTSs and HCM. Novel CACNA1C mutation is the pathogenic substrate of the electrophysiological as well as structural abnormities. Meanwhile the rare trigenic mutations make the clinical manifestation complicated and aggravated in this HCM family. At last, the study also suggests the effectiveness of CRT-D on reducing the LVOTG of septal ablation refractory hypertrophic cardiomyopathy.
Authors’ contributions
DH and WXY designed the study. CYH, ZDX, WXH, CCH, ZRJ, SJJ, WXM, TYJ, JWD, WXY, and DH performed clinical evaluations. CYH, HBM and DH coordinated the pathological phenotyping of study subjects. DH supervised and coordinated the laboratory work. CYH, HBM and DH constructed plasmids, and DH and HBM performed the electrophysiology laboratory work. CYH, DH and HBM analyzed, organized and summarized the data. DH and WXY developed the conceptual approaches to data analysis. CYH, DH, HBM and WXY wrote the manuscript. All co-authors contributed to editing of manuscript. All authors read and approved the final manuscript.