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Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy
IJCA-28186; No of Pages 7 International Journal of Cardiology xxx (xxxx) xxx
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Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy Shenghua Liu a, Yuanyuan Xie a, Hongliang Zhang a,b, Zongqi Feng a,c, Jian Huang a, Jie Huang a, Shengshou Hu a,⁎, Yingjie Wei a,⁎ a State key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China b Department of Cardiology, First Affiliated Hospital of Jiamusi University, Jiamusi, Heilongjiang 154002, China c Inner Mongolia People's Hospital, 010017 Hohhot, China
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Article history: Received 6 July 2019 Received in revised form 21 November 2019 Accepted 2 December 2019 Available online xxxx Keywords: Left ventricular noncompaction cardiomyopathy Exome sequencing Genetics
a b s t r a c t Background: Left ventricular noncompaction cardiomyopathy (LVNC) is a primary cardiomyopathy with an unclear aetiology. The clinical symptoms range from asymptomatic to heart failure, arrhythmias and sudden cardiac death. This study aimed to characterize the genetic features and clinical outcomes of LVNC who underwent heart transplantation (HTx) to reveal the potential genetic pathogenesis. Methods and results: We recruited 16 cases who underwent HTx in our hospital. Exome-sequencing was performed to reveal genetic background. Clinical information and histopathology features of patients were investigated. Gene expression profiling of tissue fibrosis were evaluated by quantitative PCR. The median age of patients was 21 years. Of the 16 patients, 14 harboured multiple gene variants involved in LVNC. Ten of the patients harboured biallelic variants and/or truncating variants. Young patients (b18) with biallelic variants and/or truncating variants and lower LVEF (b45%) at initial symptom deteriorated quickly. Except for noncompaction myocardium, myocardial fibrosis was a remarkable pathological feature, and gene profiles related to immune inflammation and extracellular matrix remodelling were upregulated. Conclusions: This study showed that multiple pathologic variants were underlie genetic mechanism of LVNC who in high risks, suggesting that genetic screening should be applied to the diagnosis of LVNC. LVNC patient with multiple variants should be considered carefully follow-up. Genetics involved in the phenotype and cardiac fibrosis, and is the major causing for LVNC. © 2019 Published by Elsevier B.V.
1. Introduction Left ventricular noncompaction cardiomyopathy (LVNC) is a primary cardiomyopathy with numerous prominent trabeculae and multiple deep intertrabecular recesses in the left ventricle. Patients are diagnosed with LVNC when the ratio of the noncompacted layer to compacted myocardium is N2.0 (NC/C N 2.0) as measured by echocardiography (ECG) or cardiac magnetic resonance imaging (MRI) examination. The clinical outcomes of patients range from asymptomatic to heart failure, malignant arrhythmias, and sudden cardiac death [1,2]. For adolescents and adults with isolated LVNC, 31% of symptomatic patients died or underwent heart transplantation (HTx) during a median follow-up of 2.7 years [3]. Herein, distinguishing LVNC patient who is in high risk is benefit for clinician to make therapy strategy. ⁎ Corresponding authors at: Fuwai Hospital, No.167 North Lishi Road, Xicheng District, Beijing 100037, China. E-mail addresses: [email protected] (S. Hu), [email protected] (Y. Wei).
Genetics may play an important role in LVNC population. Nextgeneration sequencing, such as exome sequencing and genomic sequencing, has conveniently been used to screen potential pathologic variants associated with cardiomyopathy, and an increasing number of potential genes have been correlated with LVNC. The yield of genetic testing ranges from 17% to 42% depending on the panel of incorporated genes and sequencing methods [4–6]. And the risk of cardiac events occurrence was higher for genetic patients than sporadic cases [4]. In addition, LVNC is also presumed to be a developmental defect of the ventricular myocardium during foetal embryogenesis. One retrospective study reported that approximately a quarter of LVNC patients were accompanied by congenital heart diseases [7], which suggested that LVNC has related with cardiac development defects. However, this hypothesis of heart development defects has been questioned. The study of comparison between normal and noncompaction foetal human hearts showed that excessive trabeculation is probably not the result of failed compaction [8]. For the precise pathogenesis remains poorly understood, we speculated that genetics might influence or modulate the development of noncompacted myocardium. Identifying
https://doi.org/10.1016/j.ijcard.2019.12.001 0167-5273/© 2019 Published by Elsevier B.V.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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the underlying genetics can be essential for genetic counseling of LVNC families. Now, we describe a cohort of unrelated LVNC patients who undergoing HTx and review their clinical outcomes, genetic background and histological features. We focused on the relationship between the genotype and phenotype and tried to reveal the aetiology of cardiomyopathy. 2. Methods
variants, we applied an MAF threshold of 0.001 as a conservative threshold to exclude more frequent variants that would not be the potential pathogenic variants. For gene variant verification, total RNA was extracted from the frozen heart tissues by using TRIzol method and quantified using Nanodrop2000 and electrophoresis. The cDNA was generated from 2 μg total RNA using an M-MLV Reverse Transcriptase Kit (Promega), and then amplified the target area to sequence. The primers were designed to flank the variants. Then, the PCR products were sequenced after TA cloning.
2.1. Patient clinical information 2.4. Fibrosis gene expression profiling Unrelated LVNC patients were recruited who underwent heart transplantation (HTx) in our hospital who fulfilled a defined diagnostic criterion as determined by pathologists, the layer of noncompaction: compaction ≥2:1(NC:C ≥ 2:1), were included. Clinical data were retrieved retrospectively from the medical records, including patient basic information, first symptoms, familial disease history, electrocardiogram (ECG), echocardiography, and cardiac magnetic resonance imaging-late gadolinium enhancement (LGE). Disease history and pedigree were completed according to records of clinician counseling and recording. Patients were classified as sporadic if they had no family history of cardiomyopathy.
Gene expression profiling of heart samples was analysed with the Fibrosis Pathway RT2 Profiler™ PCR Array Kits (Qiagen, USA), according to the manufacturer's protocol. For the cDNA synthesis, total RNA (1 μg) was reversed by RT2 First Strand Kit. Next, the cDNA was mixed with RT2SYBR Green Mastermix and performed real time RT-PCR was performed on the ABI ViiA 7. The relative expression of the signal pathway genes was determined from raw data using the ΔΔCt method. The positive result was defined as a 2-fold change. A heatmap was produced by OmicShare (http://www.omicshare.com/). 2.5. Histological studies
2.2. Ethical approval This study was approved by the Ethics Committees of Fuwai Hospital. All patients and their family relative who took part in the study, provided written informed consent for research of the recipient heart and blood samples before HTx. The investigation also followed the principles outlined in the Declaration of Helsinki.
For histopathological analysis, paraffin-embedded heart tissues of the left ventricle were cut into 5-μm-thick tissue sections. Masson staining was used to assess the morphologic features and fibrosis of the heart samples. 3. Results
2.3. Exome sequencing and analysis
3.1. Clinical features of LVNC patients
Total DNA was extracted from frozen myocardium specimens or blood samples according to the handbook of the Genomic DNA Purification kit (Promega) as previously reported [9]. Exomes were captured with Agilent SureSelect Human All Exon V6 Kit and sequenced with one lane per sample with Illumina HiSeq X. Reads passed instrument quality control were aligned to the human genome (GRCh37/hg19) and variants were identified by BWA, VarScan and SAMtools. All variants were annotated by Annovar software. The variant frequency in the population was annotated in dbSNP147, 1000 Genomics and ExAC. Variants in candidate genes were considered to pathogenic or likely pathogenic variants based on the following the criteria, which are based on ACMG guidelines [10]: 1) variants in candidate genes that have been previously reported to be associated with LVNC phenotype were considered pathogenic.1 2) New variant locations in candidate genes that have been confirmed to damage or disrupt the function of protein, such as loss-of-function variants (i.e., frameshift, nonsense, canonical splice site and start loss), and truncating variants and nonframeshift insertion–deletion variants were considered pathogenic. Furthermore, new variants in candidate genes and new potential pathological genes were predicted to be pathological variants based on the following: 1) Refer to Online Medline Database, HGMD, and ClinVar databases, candidate genes were reported to associated with cardiomyopathy phenotype. 2) expressed enrichment in heart tissues according to the HPA database (https://www.proteinatlas.org/) and UniPort database (https://www.uniprot.org/), and having a role in heart development and/or heart structure;2 3) The deleterious effect of variants was assessed by the algorithm methods, Polyphen-2, SIFT and Variant Taster et al. Potential pathological variants were predicted to be damaging by at least two of algorithms. All results of prediction algorithms were listed in Table S1 by VarCards (http://varcards.biols.ac.cn/). For new
A total of 16 unrelated LVNC patients were included: 12 males and 4 females (median age at diagnosis, 21 years; age range, 12 to 47 years) (Table 1). All probands underwent HTx for heart failure and cardiac arrythmias. Three of the patients have family history of cardiomyopathy (18%, 1 DCM and 2 HCM) (Fig. 1A). Patient #3 was diagnosed with LVNC at 14 years old, and his father was diagnosed with DCM and presented initial symptoms at 40 years old (Fig. 1B). In the family of patient #5, the
1 2
(Table supplementary 1) (Table Supplementary 2)
Table 1 Comparison of clinical information of LVNC patients. Clinical features Basic information Age* Gender (M/F) Genetic consulting for family HCM DCM Clinical feature Combine CHD Syncope Abnormal conduction LBBB Ventricular tachycardia ICD implantation MRI (LGE positive) LVEF(b45%, initial onset) Surgery in one year NC/C
Patients b18 y (N = 8)
Patients N18 y (N = 8)
21 y 12:4 1 1
1 0
2 3 1 2 0 4 6 7 N2:1
0 2 0 7 1 7 1 0
Age*: time of initial symptom. CHD: congenital heart disease. LBBB: left bundle branch block. ICD: implantable cardioverter defibrillator. LVEF: left ventricular ejection fraction. MRI: magnetic resonance imaging. LGE: late gadolinium enhancement. NC/C: ratio of non-compaction/compaction.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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Fig. 1. Clinical information about LVNC patients. A. Age of patients. Onset-age, the age of patients showed initial symptoms; HTx-age, the age of patients accepted HTx surgery. * marks patients with family disease history. B–D. Pedigree of patients. Two patients had HCM-related sudden cardiac death family disease history.
heart of his father and son have remained normal situation according to echocardiogram examination for now, and his cousin-sister was diagnosed with HCM. Two cases in this family were suspected of cardiac sudden death (I-1 and III-4) (Fig. 1C). Patient #10 was diagnosed with cardiomyopathy at 22 years old, and his father with HCM was suspected to have died from sudden cardiac death (Table 1, Fig. 1D). Two LVNC patients combined with congenital heart defects (CHD, #1 with ASD and #2 with Ebstein's anomaly). Five patients had initial symptoms of syncope. One patient combined with LBBB (left ventricular block). Echocardiography and cardiac magnetic resonance (CMR) findings of these patients were notable for myocardium noncompaction. According to the CMR, LGE was present in 10 of 15 patients in the left ventricular walls (one patient had no CMR information for ICD implantation). LGE-positive signals involved in left ventricular walls and/or interventricular septum were calculated. Of 8 young patients 7 was underwent surgery in one year, which suggested that the heart situation
of young patients (at initial symptomb18) seemed to be deteriorated quickly (Table 1). 3.2. Genetics in LVNC Of these 16 patients, 14 patients had multiple mutant genes which associated with cardiomyopathy (87.5%). In total, 44 variants in 22 cardiomyopathy genes were considered likely pathogenic and contributed to the pathogenesis of cardiomyopathy, 12 premature stop variants, one in-frameshift variant and 31 missense variants. Ten patients harboured truncating variants and/or biallelic variants. All of variants were heterozygous. Sarcomere variants were the most common variants in this population. Nine patients had pathological variants in the TNNT2, MYH7 and MYBPC3 genes (Table S1). In total, eight of patients harboured premature stop variants in genes (MYH7, MYBPC3, RBM20, DMD, ALPK3, TNNC1, LAMP2,
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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ANKRD1, SMYD1, FLNC and LRRC39), which were predicted to induce haploinsufficiency of protein by nonsense-mediated decay. And, six of patients harboured biallelic variants in genes (MYH7, RBM20, MYBPC3, ALPK3 and LRRC39). MYH7, MYBPC3 and LRRC39 were component of the sarcomeric, RBM20 and ALPK3 were involved in cardiomyocyte differentiation. Detail mutant loci information were listed in Table S1. In order to assess the effect of gene variants on cardiac function, mutant genes have function in heart development and regulated cardiomyocyte differentiation were listed. Sarcomere genes were the most popular mutant genes in this group. Except sarcomere genes, five variants in ion-channel genes (SCN5A, HCN4, KCNE2 and KCNE1) were identified in these patients. Gene function with heart development, RBM20, ALPK3, SMYD1 and ANKRD1, which play an essential role in the early differentiation of cardiomyocytes and the modulation of cardiomyocyte morphology were categorized (Table S1). 3.3. Histological features of LVNC Except non-compaction myocardium, three types of fibrosis were observed in these patient specimens: endocardial fibrosis, interstitial fibrosis and perivascular fibrosis. In this cohort, endocardial fibrosis was observed in three specimens (Fig. 2A.#1,3,6). Perivascular fibrosis was noticed in one patient specimen (Fig. 2A.#4). Obvious myocardial interstitial fibrosis was observed in the myocardium of the remaining patients. Patient #1 who had endocardia fibrosis, showed left bundle branch block (LBBB), suggesting that normal electrical activity in the His-Purkinje system might be interrupted by endocardia fibrosis. Moreover, we investigated the expression of fibrosis genes in left ventricular samples. The expression level of fibrosis pathway genes was evaluated by qPCR-based array. Remarkably, matrix metalloproteinase (MMP9) and inflammatory factor (CCL3) were expressed at high levels, and cell adhesion molecule (ITGB8) expression was decreased in the fibrotic process of LVNC (Fig. 2B). 4. Discussion The genetics and clinical features of a cohort of LVNC patients with severe symptom who underwent HTx were described. Complex and heterogenous genetics were observed in these LVNC patients, and were play an important role on phenotype of LVNC. 4.1. Clinical phenotype of LVNC Patients were recruited after heart transplantation surgery, who presented the severe cases of LVNC population. Average age of operation was 21 years, and clinical outcome include malignant arrhythmia and heart failure. Three out of 16 probands have family history of subtype cardiomyopathy (DCM, HCM), which suggested LVNC has the common genetic pathogenesis with other cardiomyopathies. Two probands combined with CHD have serious clinical outcomes in adolescence, suggested heat defect exacerbates the phenotype of LVNC. 4.2. Genetic features of LVNC Multiple gene variants were more popular in this group, which suggested multiple gene variants synergistically function in the phenotype of LVNC who was in high cardiac risk. Multiple genetic defects may also explain the situation that a specific mutation can result in a different myocardial phenotype (e.g., LVNC, HCM or DCM) in a LVNC family. For instance, Patient #5 harboured biallelic MYBPC3 (p.R943X and p. R502Q) and MYH7 (p.A1777T) variants. MYBPC3 (p.R943X) was shared in the family and did not co-segregate with the phenotype of cardiomyopathy (Fig. 1C). And recently, complex genetic variants were proved to be underlie mechanism of a LVNC family by CRISPR-Cas9 editing mice. Compound heterozygosity for all three variants (MKL2, MYH7, and
NKX2-5) recapitulated the LVNC phenotype [11]. In addition, only a few of researches had mentioned that multiple gene variants associated with LVNC, depending on the panel of incorporated genes and sequencing methods [4–6]. Therefore, multiple gene variants should be pay more attention in LVNC genetic screening in the future study. And the pathogenesis of two patients (#13 and #14) who harboured single genetic defect, and have not family cardiomyopathy history, need to be further investigated. Biallelic variants and truncating variants were popular in this cohort. Biallelic variants in MYH7, MYBPC3, LRRP3, ALPK3 and RBM20 were showed in six patients. MYH7, LRRP3 and MYBPC3 were essential components of sarcomere, which produce force during cardiomyocyte contraction. Premature stop variants in these genes were predicted to induce haploinsufficiency of product and reduced force-generating capacity of cardiomyocytes and myofibril density, induced to cardiomyocyte hypertrophy or dilatation. Referring to the literature on patients with biallelic MYBPC3 and MYH7 variants, patients with heterozygous truncating variants usually presented with gentle and late-onset clinical symptoms (N40 years) [12]. And, the truncating variant combined with another missense variant (especially another truncating variant) seemed to induce a more serious phenotype and severe onset symptom of LVNC [13]. A similar situation occurred in patients with biallelic ALPK3 variants. ALPK3 plays a role in cardiomyocyte differentiation and regulates the expression of numerous genes involved in heart development. Probands harboured biallelic variants present with pediatric cardiomyopathy, and their family members with heterozygous truncating ALPK3 variant showed a milder phenotype of hypertrophic cardiomyopathy and could survive to adulthood [14]. And two patients combine with CHD also harboured biallelic variants in MYH7, supporting the previously reported association of heart development defect and MYH7 mutations. Therefore, LVNC patients who carried multiple pathological variants, especially biallelic variants with truncating variant should be followed-up carefully. These young patients showed clinical outcome of heart failure and arrhythmia. Except sarcomere gene variants, ion channel gene variants were also identified, SCN5A (c.3584CNT, p.R1195H), SCN5A (c.3578GNA, p.R1193Q, polymorphism), HCN4 (c.1740CNG, p.E580D), KCNE2 (c.80GNA, p.R27C), and KCNE1(c.253CNT, p.D85N, polymorphism), which are well-established as being associated with cardiac arrythmias. Ion-channel gene variants might to be one of reasons for malignant arrhythmia and the phenotype of LVNC [15], yet we do not have an approach to find a solid evidence to illustrate this mechanism. We remain highly skeptical of missense variants of these ion channel genes and assistant ion-channel genes, such as CMYA1 and PKP2. HCN4 contributes to pacemaker currents (If) in the sinoatrial node and regulates the rhythm of the heart and has been observed in LVNC patients [16,17]. SCN5A encodes a sodium channel of the heart and is involved in the arrhythmia of LVNC [18]. KCNE1 and KCNE2, which are well-associated with cardiac arrythmias and long QT syndrome (LQTS) [19]. CMYA1 encodes Xin actin-binding repeat-containing protein and interacts with K+ channel-interacting protein 2, which might influence the ion channel function of cardiomyocytes [20]. PKP2 encodes an essential armadillo repeat protein of the cardiac desmosome, which is one of disease-causing genes of arrhythmogenic right ventricular cardiomyopathy (ARVC). Overlapping in genetic causes with subtype cardiomyopathies (HCM, DCM, ARVC, Cardiac ion channelopathies) is common in this LVNC population [21], which implies subtype cardiomyopathies have common molecular pathologies and explains the overlap phenotypes of cardiomyopathy. 4.3. Histological features of LVNC In this cohort, all of patients fulfilled the diagnosed criteria of LVNC, and most of patients showed initial symptom before 45 years, yet there was no obvious correlation between the ratio of noncompaction part and the severity of clinical outcome. Although the extensive LV
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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Fig. 2. Cardiac fibrosis of specimens in LVNC. A. Masson staining showed fibrosis (blue) in the myocardium of LVNC patients. Endocardial fibrosis in #1, #3 and #6; remarkable interstitial fibrosis in myocardium #5 and #10; Perivascular fibrosis showed in patient #4. B. Comparison of the expression level of the fibrosis gene profile of patient specimens. Gene expression analysis of the fibrosis gene profile in LVNC revealed increasing ECM gene expression in LVNC. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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trabeculation is the remarkable histological feature for LVNC, whether it is due to cardiomyopathy or is just a form of morphology with potential contributions from cardiac remodelling processes is still under debate. On the one hand, trabeculae are important to maintain LV stiffness in normal situations [22]. On the other hand, stimuli actions, like exercise or pregnancy, result in an increased cardiac preload in individuals with sarcomere variants, might leading to pathological hyper-trabeculae adaptive growth. In LVNC patients, noncompaction myocardium (massive hyper-trabeculae) was reckoned to affected the contraction of ventricular wall. But, the previously report showed that the ratio of noncompaction/compaction part not seemed to exactly predict a poor prognosis of LVNC [23]. Comprehensively consideration of genetics and disease progress, we predicted that genetic background combined with other nongenetic factors might defined the phenotype and outcomes of our LVNC patients. Apart from remarkable hyper-trabeculae, endocardial fibrosis or fibroelastosis was the most frequently reported abnormality in LVNC [24]. Endocardial fibrosis or fibroelastosis in LVNC was considered a symbol pathophysiology feature, with uncertain clinical effect [25]. In this cohort, LGE positivity is consistent with cardiac interstitial fibrosis and should be applied to predict poor prognosis in LVNC. In normal situations, the cardiac fibrosis network was originally the normal schedule of cardiac muscle that provided stress during heart contractile. Abnormal sarcomeric structures and alterations in calcium signalling would destroy the cardiac homeostasis and can result in cardiomyocyte remodelling and systolic and diastolic dysfunction. In response to extrastress, cardiac fibroblasts convert into activated cardiac myofibroblasts, proliferate and enhance cardiac fibrosis deposition. In this study, we compared the gene expression profiles of fibrosis between patients
and normal control myocardium. These genes related to fibrosis were extremely highly expressed and irregularly expressed in the myocardium of LVNC. In particularly, gene function with inflammatory and extracellular matrix proteolysis, suggested that immune inflammation was also involved in the fibrosis pathway in LVNC. We suspected that variants in sarcomere and/or other functional cardiomyocyte structure induced to abnormal contraction of cardiac myocytes, which lead to disturbance of myocardium development and myocardium remodelling (Fig. 3). This may help to answer the question whether LVNC can be a failure of trabecular compaction during foetal development or a distinct pathologic phenotype of cardiomyopathy caused by genetics.
4.4. Limitation No doubt, this is a small cohort and all of patients came from HTx Databank. All of patients have severe and complex clinical manifestations, which hampered the establishment of genotype and phenotype. Exome sequencing provided us an opportunity to investigate more genetic information about this cardiomyopathy, but only those reported genes and proteins associated with LVNC and cardiomyopathy were priority to list in this study, which would induce to neglection of potential pathological variants. In addition, the high-throughput sequencing method has technique limitation, some potential genetic defects contributed to LVNC phenotype would be missed, such as big deletion and inversion of genes. We have plans to dig pathological variants or mutation by other genetic screening methods, which would help for expand the genetic panel of disease.
Fig. 3. Summary of inference about genetics in LVNC. Pathologic variants in genes have function with heart development. Physiological activities synergistically contribute to the phenotype of LVNC.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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5. Conclusions Multiple gene variants synergistically function in the phenotype of LVNC who underwent HTx. Biallelic variants and truncating variants involved in the pathogenesis of LVNC who was in a high risk and should be considered carefully follow-up. Young patients with multiple pathologic variants and lower LVEF (b45%) at initial symptom seemed to be deteriorated quickly. Pathological variants in cardiac genes induced to abnormal contraction of cardiac myocytes involved in the pathological progress of LVNC. These results support that genetic screening should be applied to the diagnosis and prognosis of LVNC patients. CRediT authorship contribution statement Shenghua Liu: Data curation, Formal analysis and Writing-original draft. Yuanyuan Xie: Data curation, Formal analysis and Writingoriginal draft. Hongliang Zhang: Data curation, Formal analysis and Writing-original draft. Zongqi Feng: Data curation, Formal analysis and Writing-original draft. Jian Huang: Data curation, Formal analysis and Writing-original draft. Jie Huang: Data curation, Formal analysis and Writing-original draft. Shengshou Hu: Data curation, Formal analysis and Writing-original draft. Yingjie Wei: Data curation, Formal analysis and Writing-original draft. Acknowledgements We thank the contributors to the primary cardiomyopathy cohort, and we are grateful to our patient and his family for the support and collaboration in this study. Funding This work was supported by CAMS Initiative for Innovative Medicine (CAMS-I2M, 2016-I2M-1-015 to Y.J.W.) and PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (Grant 33320140167 to S.H.L). Contributors Conceived and designed the experiment: S.H.L and Y.J.W; Clinical support: Doctor H.L.Z, J.H. and S.H.H.; Collected the samples and conducted the experiments: S.H.L, Y.Y.X, and Z.Q.F; Analysed exome data: S.H.L and Y.Y.X.; Wrote the manuscript: S.H.L, and Y.J.W; S.H.L and Y.Y.X. contributed equally to this work. All the authors have reviewed the manuscript. Declaration of competing interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijcard.2019.12.001. References [1] F. Negri, A. De Luca, E. Fabris, R. Korcova, C. Cernetti, C. Grigoratos, et al., Left ventricular noncompaction, morphological, and clinical features for an integrated diagnosis, Heart Fail. Rev. 24 (2019) 315–323.
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[2] G. Habib, P. Charron, J.-C. Eicher, R. Giorgi, E. Donal, T. Laperche, et al., Isolated left ventricular non-compaction in adults: clinical and echocardiographic features in 105 patients. Results from a French registry, Eur. J. Heart Fail. 13 (2011) 177–185. [3] M. Greutmann, M.L. Mah, C.K. Silversides, S. Klaassen, C.H.A. Jost, R. Jenni, et al., Predictors of adverse outcome in adolescents and adults with isolated left ventricular noncompaction, Am. J. Cardiol. 109 (2012) 276–281. [4] J.I. van Waning, K. Caliskan, Y.M. Hoedemaekers, K.Y. van Spaendonck-Zwarts, A.F. Baas, S.M. Boekholdt, et al., Genetics, clinical features, and long-term outcome of noncompaction cardiomyopathy, J. Am. Coll. Cardiol. 71 (2018) 711–722. [5] P. Richard, F. Ader, M. Roux, E. Donal, J.C. Eicher, N. Aoutil, et al., Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity, Clin. Genet. 95 (2019) 356–367. [6] S. Klaassen, S. Probst, E. Oechslin, B. Gerull, G. Krings, P. Schuler, et al., Mutations in sarcomere protein genes in left ventricular noncompaction, Circulation 117 (2008) 2893–2901. [7] B.E. Staehli, C. Gebhard, P. Biaggi, S. Klaassen, E.V. Buechel, C.H.A. Jost, et al., Left ventricular non-compaction: prevalence in congenital heart disease, Int. J. Cardiol. 167 (2013) 2477–2481. [8] B. Jensen, P. Agger, B.A. de Boer, R.-J. Oostra, M. Pedersen, A.C. van der Wal, et al., The hypertrabeculated (noncompacted) left ventricle is different from the ventricle of embryos and ectothermic vertebrates, Biochim. Biophys. Acta, Mol. Cell Res. 1863 (2016) 1696–1706. [9] S. Liu, Y. Bai, J. Huang, H. Zhao, X. Zhang, S. Hu, et al., Do mitochondria contribute to left ventricular non-compaction cardiomyopathy? New findings from myocardium of patients with left ventricular non-compaction cardiomyopathy, Mol. Genet. Metab. 109 (2013) 100–106. [10] S. Richards, N. Aziz, S. Bale, D. Bick, S. Das, J. Gastier-Foster, et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet. Med. 17 (2015) 405–424. [11] C.A. Gifford, S.S. Ranade, R. Samarakoon, H.T. Salunga, T.Y. de Soysa, Y. Huang, et al., Oligogenic inheritance of a human heart disease involving a genetic modifier, Science 364 (2019) 865–870. [12] H.G. van Velzen, A.F.L. Schinkel, R.A. Oldenburg, M.A. van Slegtenhorst, I.M.E. FrohnMulder, J. van der Velden, et al., Clinical characteristics and long-term outcome of hypertrophic cardiomyopathy in individuals with a MYBPC3 (myosin-binding protein C) founder mutation, Circ. Cardiovasc. Genet. 10 (2017), e001660. [13] K. Kolokotronis, J. Kuhnisch, E. Klopocki, J. Dartsch, S. Rost, C. Huculak, et al., Biallelic mutation in MYH7 and MYBPC3 leads to severe cardiomyopathy with left ventricular non-compaction phenotype, Hum. Mutat. (2019). [14] R. Almomani, J.M. Verhagen, J.C. Herkert, E. Brosens, K.Y. van Spaendonck-Zwarts, A. Asimaki, et al., Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy, J. Am. Coll. Cardiol. 67 (2016) 515–525. [15] L. Shan, N. Makita, Y. Xing, S. Watanabe, T. Futatani, F. Ye, et al., SCN5A variants in Japanese patients with left ventricular noncompaction and arrhythmia, Mol. Genet. Metab. 93 (2008) 468–474. [16] P.A. Schweizer, J. Schroeter, S. Greiner, J. Haas, P. Yampolsky, D. Mereles, et al., The symptom complex of familial sinus node dysfunction and myocardial noncompaction is associated with mutations in the HCN4 channel, J. Am. Coll. Cardiol. 64 (2014) 757–767. [17] A. Milano, A.M.C. Vermeer, E.M. Lodder, J. Barc, A.O. Verkerk, A.V. Postma, et al., HCN4 mutations in multiple families with bradycardia and left ventricular noncompaction cardiomyopathy, J. Am. Coll. Cardiol. 64 (2014) 745–756. [18] N. Makita, K. Sasaki, H. Yokoshiki, S. Yamada, H. Tsutsui, Left ventricular noncompaction associated with mutations in cardiac Na channel gene SCN5A, Circulation 114 (2006) 126. [19] Y. Nishio, T. Makiyama, H. Itoh, T. Sakaguchi, S. Ohno, Y.Z. Gong, et al., D85N, a KCNE1 polymorphism, is a disease-causing gene variant in long QT syndrome, J. Am. Coll. Cardiol. 54 (2009) 812–819. [20] Q. Wang, K.-H. Wu, P.-H. Jiang, C.-I. Lin, J.L. Lin, E.A. Adams, et al., Loss of intercalated disc protein mXin beta leads to atrioventricular conduction defects and channel remodeling, Circulation 128 (2013). [21] J.I. van Waning, K. Caliskan, M. Michels, A.F.L. Schinkel, A. Hirsch, M. Dalinghaus, et al., Cardiac phenotypes, genetics, and risks in familial noncompaction cardiomyopathy, J. Am. Coll. Cardiol. 73 (2019) 1601–1611. [22] D.L. Halaney, A. Sanyal, N.A. Nafissi, D. Escobedo, M. Goros, J. Michalek, et al., The effect of trabeculae carneae on left ventricular diastolic compliance: improvement in compliance with trabecular cutting, J. Biomech. Eng. 139 (2017). [23] F. Zemrak, M.A. Ahlman, G. Captur, S.A. Mohiddin, N. Kawel-Boehm, M.R. Prince, et al., The relationship of left ventricular trabeculation to ventricular function and structure over a 9.5-year follow-up the MESA study, J. Am. Coll. Cardiol. 64 (2014) 1971–1980. [24] G. Nucifora, G.D. Aquaro, A. Pingitore, P.G. Masci, M. Lombardi, Myocardial fibrosis in isolated left ventricular non-compaction and its relation to disease severity, Eur. J. Heart Fail. 13 (2011) 170–176. [25] J.A. Towbin, A. Lorts, J.L. Jefferies, Left ventricular non-compaction cardiomyopathy, Lancet. 386 (2015) 813–825.
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Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy Shenghua Liu a, Yuanyuan Xie a, Hongliang Zhang a,b, Zongqi Feng a,c, Jian Huang a, Jie Huang a, Shengshou Hu a,⁎, Yingjie Wei a,⁎ a State key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China b Department of Cardiology, First Affiliated Hospital of Jiamusi University, Jiamusi, Heilongjiang 154002, China c Inner Mongolia People's Hospital, 010017 Hohhot, China
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Article history: Received 6 July 2019 Received in revised form 21 November 2019 Accepted 2 December 2019 Available online xxxx Keywords: Left ventricular noncompaction cardiomyopathy Exome sequencing Genetics
a b s t r a c t Background: Left ventricular noncompaction cardiomyopathy (LVNC) is a primary cardiomyopathy with an unclear aetiology. The clinical symptoms range from asymptomatic to heart failure, arrhythmias and sudden cardiac death. This study aimed to characterize the genetic features and clinical outcomes of LVNC who underwent heart transplantation (HTx) to reveal the potential genetic pathogenesis. Methods and results: We recruited 16 cases who underwent HTx in our hospital. Exome-sequencing was performed to reveal genetic background. Clinical information and histopathology features of patients were investigated. Gene expression profiling of tissue fibrosis were evaluated by quantitative PCR. The median age of patients was 21 years. Of the 16 patients, 14 harboured multiple gene variants involved in LVNC. Ten of the patients harboured biallelic variants and/or truncating variants. Young patients (b18) with biallelic variants and/or truncating variants and lower LVEF (b45%) at initial symptom deteriorated quickly. Except for noncompaction myocardium, myocardial fibrosis was a remarkable pathological feature, and gene profiles related to immune inflammation and extracellular matrix remodelling were upregulated. Conclusions: This study showed that multiple pathologic variants were underlie genetic mechanism of LVNC who in high risks, suggesting that genetic screening should be applied to the diagnosis of LVNC. LVNC patient with multiple variants should be considered carefully follow-up. Genetics involved in the phenotype and cardiac fibrosis, and is the major causing for LVNC. © 2019 Published by Elsevier B.V.
1. Introduction Left ventricular noncompaction cardiomyopathy (LVNC) is a primary cardiomyopathy with numerous prominent trabeculae and multiple deep intertrabecular recesses in the left ventricle. Patients are diagnosed with LVNC when the ratio of the noncompacted layer to compacted myocardium is N2.0 (NC/C N 2.0) as measured by echocardiography (ECG) or cardiac magnetic resonance imaging (MRI) examination. The clinical outcomes of patients range from asymptomatic to heart failure, malignant arrhythmias, and sudden cardiac death [1,2]. For adolescents and adults with isolated LVNC, 31% of symptomatic patients died or underwent heart transplantation (HTx) during a median follow-up of 2.7 years [3]. Herein, distinguishing LVNC patient who is in high risk is benefit for clinician to make therapy strategy. ⁎ Corresponding authors at: Fuwai Hospital, No.167 North Lishi Road, Xicheng District, Beijing 100037, China. E-mail addresses: [email protected] (S. Hu), [email protected] (Y. Wei).
Genetics may play an important role in LVNC population. Nextgeneration sequencing, such as exome sequencing and genomic sequencing, has conveniently been used to screen potential pathologic variants associated with cardiomyopathy, and an increasing number of potential genes have been correlated with LVNC. The yield of genetic testing ranges from 17% to 42% depending on the panel of incorporated genes and sequencing methods [4–6]. And the risk of cardiac events occurrence was higher for genetic patients than sporadic cases [4]. In addition, LVNC is also presumed to be a developmental defect of the ventricular myocardium during foetal embryogenesis. One retrospective study reported that approximately a quarter of LVNC patients were accompanied by congenital heart diseases [7], which suggested that LVNC has related with cardiac development defects. However, this hypothesis of heart development defects has been questioned. The study of comparison between normal and noncompaction foetal human hearts showed that excessive trabeculation is probably not the result of failed compaction [8]. For the precise pathogenesis remains poorly understood, we speculated that genetics might influence or modulate the development of noncompacted myocardium. Identifying
https://doi.org/10.1016/j.ijcard.2019.12.001 0167-5273/© 2019 Published by Elsevier B.V.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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the underlying genetics can be essential for genetic counseling of LVNC families. Now, we describe a cohort of unrelated LVNC patients who undergoing HTx and review their clinical outcomes, genetic background and histological features. We focused on the relationship between the genotype and phenotype and tried to reveal the aetiology of cardiomyopathy. 2. Methods
variants, we applied an MAF threshold of 0.001 as a conservative threshold to exclude more frequent variants that would not be the potential pathogenic variants. For gene variant verification, total RNA was extracted from the frozen heart tissues by using TRIzol method and quantified using Nanodrop2000 and electrophoresis. The cDNA was generated from 2 μg total RNA using an M-MLV Reverse Transcriptase Kit (Promega), and then amplified the target area to sequence. The primers were designed to flank the variants. Then, the PCR products were sequenced after TA cloning.
2.1. Patient clinical information 2.4. Fibrosis gene expression profiling Unrelated LVNC patients were recruited who underwent heart transplantation (HTx) in our hospital who fulfilled a defined diagnostic criterion as determined by pathologists, the layer of noncompaction: compaction ≥2:1(NC:C ≥ 2:1), were included. Clinical data were retrieved retrospectively from the medical records, including patient basic information, first symptoms, familial disease history, electrocardiogram (ECG), echocardiography, and cardiac magnetic resonance imaging-late gadolinium enhancement (LGE). Disease history and pedigree were completed according to records of clinician counseling and recording. Patients were classified as sporadic if they had no family history of cardiomyopathy.
Gene expression profiling of heart samples was analysed with the Fibrosis Pathway RT2 Profiler™ PCR Array Kits (Qiagen, USA), according to the manufacturer's protocol. For the cDNA synthesis, total RNA (1 μg) was reversed by RT2 First Strand Kit. Next, the cDNA was mixed with RT2SYBR Green Mastermix and performed real time RT-PCR was performed on the ABI ViiA 7. The relative expression of the signal pathway genes was determined from raw data using the ΔΔCt method. The positive result was defined as a 2-fold change. A heatmap was produced by OmicShare (http://www.omicshare.com/). 2.5. Histological studies
2.2. Ethical approval This study was approved by the Ethics Committees of Fuwai Hospital. All patients and their family relative who took part in the study, provided written informed consent for research of the recipient heart and blood samples before HTx. The investigation also followed the principles outlined in the Declaration of Helsinki.
For histopathological analysis, paraffin-embedded heart tissues of the left ventricle were cut into 5-μm-thick tissue sections. Masson staining was used to assess the morphologic features and fibrosis of the heart samples. 3. Results
2.3. Exome sequencing and analysis
3.1. Clinical features of LVNC patients
Total DNA was extracted from frozen myocardium specimens or blood samples according to the handbook of the Genomic DNA Purification kit (Promega) as previously reported [9]. Exomes were captured with Agilent SureSelect Human All Exon V6 Kit and sequenced with one lane per sample with Illumina HiSeq X. Reads passed instrument quality control were aligned to the human genome (GRCh37/hg19) and variants were identified by BWA, VarScan and SAMtools. All variants were annotated by Annovar software. The variant frequency in the population was annotated in dbSNP147, 1000 Genomics and ExAC. Variants in candidate genes were considered to pathogenic or likely pathogenic variants based on the following the criteria, which are based on ACMG guidelines [10]: 1) variants in candidate genes that have been previously reported to be associated with LVNC phenotype were considered pathogenic.1 2) New variant locations in candidate genes that have been confirmed to damage or disrupt the function of protein, such as loss-of-function variants (i.e., frameshift, nonsense, canonical splice site and start loss), and truncating variants and nonframeshift insertion–deletion variants were considered pathogenic. Furthermore, new variants in candidate genes and new potential pathological genes were predicted to be pathological variants based on the following: 1) Refer to Online Medline Database, HGMD, and ClinVar databases, candidate genes were reported to associated with cardiomyopathy phenotype. 2) expressed enrichment in heart tissues according to the HPA database (https://www.proteinatlas.org/) and UniPort database (https://www.uniprot.org/), and having a role in heart development and/or heart structure;2 3) The deleterious effect of variants was assessed by the algorithm methods, Polyphen-2, SIFT and Variant Taster et al. Potential pathological variants were predicted to be damaging by at least two of algorithms. All results of prediction algorithms were listed in Table S1 by VarCards (http://varcards.biols.ac.cn/). For new
A total of 16 unrelated LVNC patients were included: 12 males and 4 females (median age at diagnosis, 21 years; age range, 12 to 47 years) (Table 1). All probands underwent HTx for heart failure and cardiac arrythmias. Three of the patients have family history of cardiomyopathy (18%, 1 DCM and 2 HCM) (Fig. 1A). Patient #3 was diagnosed with LVNC at 14 years old, and his father was diagnosed with DCM and presented initial symptoms at 40 years old (Fig. 1B). In the family of patient #5, the
1 2
(Table supplementary 1) (Table Supplementary 2)
Table 1 Comparison of clinical information of LVNC patients. Clinical features Basic information Age* Gender (M/F) Genetic consulting for family HCM DCM Clinical feature Combine CHD Syncope Abnormal conduction LBBB Ventricular tachycardia ICD implantation MRI (LGE positive) LVEF(b45%, initial onset) Surgery in one year NC/C
Patients b18 y (N = 8)
Patients N18 y (N = 8)
21 y 12:4 1 1
1 0
2 3 1 2 0 4 6 7 N2:1
0 2 0 7 1 7 1 0
Age*: time of initial symptom. CHD: congenital heart disease. LBBB: left bundle branch block. ICD: implantable cardioverter defibrillator. LVEF: left ventricular ejection fraction. MRI: magnetic resonance imaging. LGE: late gadolinium enhancement. NC/C: ratio of non-compaction/compaction.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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Fig. 1. Clinical information about LVNC patients. A. Age of patients. Onset-age, the age of patients showed initial symptoms; HTx-age, the age of patients accepted HTx surgery. * marks patients with family disease history. B–D. Pedigree of patients. Two patients had HCM-related sudden cardiac death family disease history.
heart of his father and son have remained normal situation according to echocardiogram examination for now, and his cousin-sister was diagnosed with HCM. Two cases in this family were suspected of cardiac sudden death (I-1 and III-4) (Fig. 1C). Patient #10 was diagnosed with cardiomyopathy at 22 years old, and his father with HCM was suspected to have died from sudden cardiac death (Table 1, Fig. 1D). Two LVNC patients combined with congenital heart defects (CHD, #1 with ASD and #2 with Ebstein's anomaly). Five patients had initial symptoms of syncope. One patient combined with LBBB (left ventricular block). Echocardiography and cardiac magnetic resonance (CMR) findings of these patients were notable for myocardium noncompaction. According to the CMR, LGE was present in 10 of 15 patients in the left ventricular walls (one patient had no CMR information for ICD implantation). LGE-positive signals involved in left ventricular walls and/or interventricular septum were calculated. Of 8 young patients 7 was underwent surgery in one year, which suggested that the heart situation
of young patients (at initial symptomb18) seemed to be deteriorated quickly (Table 1). 3.2. Genetics in LVNC Of these 16 patients, 14 patients had multiple mutant genes which associated with cardiomyopathy (87.5%). In total, 44 variants in 22 cardiomyopathy genes were considered likely pathogenic and contributed to the pathogenesis of cardiomyopathy, 12 premature stop variants, one in-frameshift variant and 31 missense variants. Ten patients harboured truncating variants and/or biallelic variants. All of variants were heterozygous. Sarcomere variants were the most common variants in this population. Nine patients had pathological variants in the TNNT2, MYH7 and MYBPC3 genes (Table S1). In total, eight of patients harboured premature stop variants in genes (MYH7, MYBPC3, RBM20, DMD, ALPK3, TNNC1, LAMP2,
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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ANKRD1, SMYD1, FLNC and LRRC39), which were predicted to induce haploinsufficiency of protein by nonsense-mediated decay. And, six of patients harboured biallelic variants in genes (MYH7, RBM20, MYBPC3, ALPK3 and LRRC39). MYH7, MYBPC3 and LRRC39 were component of the sarcomeric, RBM20 and ALPK3 were involved in cardiomyocyte differentiation. Detail mutant loci information were listed in Table S1. In order to assess the effect of gene variants on cardiac function, mutant genes have function in heart development and regulated cardiomyocyte differentiation were listed. Sarcomere genes were the most popular mutant genes in this group. Except sarcomere genes, five variants in ion-channel genes (SCN5A, HCN4, KCNE2 and KCNE1) were identified in these patients. Gene function with heart development, RBM20, ALPK3, SMYD1 and ANKRD1, which play an essential role in the early differentiation of cardiomyocytes and the modulation of cardiomyocyte morphology were categorized (Table S1). 3.3. Histological features of LVNC Except non-compaction myocardium, three types of fibrosis were observed in these patient specimens: endocardial fibrosis, interstitial fibrosis and perivascular fibrosis. In this cohort, endocardial fibrosis was observed in three specimens (Fig. 2A.#1,3,6). Perivascular fibrosis was noticed in one patient specimen (Fig. 2A.#4). Obvious myocardial interstitial fibrosis was observed in the myocardium of the remaining patients. Patient #1 who had endocardia fibrosis, showed left bundle branch block (LBBB), suggesting that normal electrical activity in the His-Purkinje system might be interrupted by endocardia fibrosis. Moreover, we investigated the expression of fibrosis genes in left ventricular samples. The expression level of fibrosis pathway genes was evaluated by qPCR-based array. Remarkably, matrix metalloproteinase (MMP9) and inflammatory factor (CCL3) were expressed at high levels, and cell adhesion molecule (ITGB8) expression was decreased in the fibrotic process of LVNC (Fig. 2B). 4. Discussion The genetics and clinical features of a cohort of LVNC patients with severe symptom who underwent HTx were described. Complex and heterogenous genetics were observed in these LVNC patients, and were play an important role on phenotype of LVNC. 4.1. Clinical phenotype of LVNC Patients were recruited after heart transplantation surgery, who presented the severe cases of LVNC population. Average age of operation was 21 years, and clinical outcome include malignant arrhythmia and heart failure. Three out of 16 probands have family history of subtype cardiomyopathy (DCM, HCM), which suggested LVNC has the common genetic pathogenesis with other cardiomyopathies. Two probands combined with CHD have serious clinical outcomes in adolescence, suggested heat defect exacerbates the phenotype of LVNC. 4.2. Genetic features of LVNC Multiple gene variants were more popular in this group, which suggested multiple gene variants synergistically function in the phenotype of LVNC who was in high cardiac risk. Multiple genetic defects may also explain the situation that a specific mutation can result in a different myocardial phenotype (e.g., LVNC, HCM or DCM) in a LVNC family. For instance, Patient #5 harboured biallelic MYBPC3 (p.R943X and p. R502Q) and MYH7 (p.A1777T) variants. MYBPC3 (p.R943X) was shared in the family and did not co-segregate with the phenotype of cardiomyopathy (Fig. 1C). And recently, complex genetic variants were proved to be underlie mechanism of a LVNC family by CRISPR-Cas9 editing mice. Compound heterozygosity for all three variants (MKL2, MYH7, and
NKX2-5) recapitulated the LVNC phenotype [11]. In addition, only a few of researches had mentioned that multiple gene variants associated with LVNC, depending on the panel of incorporated genes and sequencing methods [4–6]. Therefore, multiple gene variants should be pay more attention in LVNC genetic screening in the future study. And the pathogenesis of two patients (#13 and #14) who harboured single genetic defect, and have not family cardiomyopathy history, need to be further investigated. Biallelic variants and truncating variants were popular in this cohort. Biallelic variants in MYH7, MYBPC3, LRRP3, ALPK3 and RBM20 were showed in six patients. MYH7, LRRP3 and MYBPC3 were essential components of sarcomere, which produce force during cardiomyocyte contraction. Premature stop variants in these genes were predicted to induce haploinsufficiency of product and reduced force-generating capacity of cardiomyocytes and myofibril density, induced to cardiomyocyte hypertrophy or dilatation. Referring to the literature on patients with biallelic MYBPC3 and MYH7 variants, patients with heterozygous truncating variants usually presented with gentle and late-onset clinical symptoms (N40 years) [12]. And, the truncating variant combined with another missense variant (especially another truncating variant) seemed to induce a more serious phenotype and severe onset symptom of LVNC [13]. A similar situation occurred in patients with biallelic ALPK3 variants. ALPK3 plays a role in cardiomyocyte differentiation and regulates the expression of numerous genes involved in heart development. Probands harboured biallelic variants present with pediatric cardiomyopathy, and their family members with heterozygous truncating ALPK3 variant showed a milder phenotype of hypertrophic cardiomyopathy and could survive to adulthood [14]. And two patients combine with CHD also harboured biallelic variants in MYH7, supporting the previously reported association of heart development defect and MYH7 mutations. Therefore, LVNC patients who carried multiple pathological variants, especially biallelic variants with truncating variant should be followed-up carefully. These young patients showed clinical outcome of heart failure and arrhythmia. Except sarcomere gene variants, ion channel gene variants were also identified, SCN5A (c.3584CNT, p.R1195H), SCN5A (c.3578GNA, p.R1193Q, polymorphism), HCN4 (c.1740CNG, p.E580D), KCNE2 (c.80GNA, p.R27C), and KCNE1(c.253CNT, p.D85N, polymorphism), which are well-established as being associated with cardiac arrythmias. Ion-channel gene variants might to be one of reasons for malignant arrhythmia and the phenotype of LVNC [15], yet we do not have an approach to find a solid evidence to illustrate this mechanism. We remain highly skeptical of missense variants of these ion channel genes and assistant ion-channel genes, such as CMYA1 and PKP2. HCN4 contributes to pacemaker currents (If) in the sinoatrial node and regulates the rhythm of the heart and has been observed in LVNC patients [16,17]. SCN5A encodes a sodium channel of the heart and is involved in the arrhythmia of LVNC [18]. KCNE1 and KCNE2, which are well-associated with cardiac arrythmias and long QT syndrome (LQTS) [19]. CMYA1 encodes Xin actin-binding repeat-containing protein and interacts with K+ channel-interacting protein 2, which might influence the ion channel function of cardiomyocytes [20]. PKP2 encodes an essential armadillo repeat protein of the cardiac desmosome, which is one of disease-causing genes of arrhythmogenic right ventricular cardiomyopathy (ARVC). Overlapping in genetic causes with subtype cardiomyopathies (HCM, DCM, ARVC, Cardiac ion channelopathies) is common in this LVNC population [21], which implies subtype cardiomyopathies have common molecular pathologies and explains the overlap phenotypes of cardiomyopathy. 4.3. Histological features of LVNC In this cohort, all of patients fulfilled the diagnosed criteria of LVNC, and most of patients showed initial symptom before 45 years, yet there was no obvious correlation between the ratio of noncompaction part and the severity of clinical outcome. Although the extensive LV
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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Fig. 2. Cardiac fibrosis of specimens in LVNC. A. Masson staining showed fibrosis (blue) in the myocardium of LVNC patients. Endocardial fibrosis in #1, #3 and #6; remarkable interstitial fibrosis in myocardium #5 and #10; Perivascular fibrosis showed in patient #4. B. Comparison of the expression level of the fibrosis gene profile of patient specimens. Gene expression analysis of the fibrosis gene profile in LVNC revealed increasing ECM gene expression in LVNC. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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trabeculation is the remarkable histological feature for LVNC, whether it is due to cardiomyopathy or is just a form of morphology with potential contributions from cardiac remodelling processes is still under debate. On the one hand, trabeculae are important to maintain LV stiffness in normal situations [22]. On the other hand, stimuli actions, like exercise or pregnancy, result in an increased cardiac preload in individuals with sarcomere variants, might leading to pathological hyper-trabeculae adaptive growth. In LVNC patients, noncompaction myocardium (massive hyper-trabeculae) was reckoned to affected the contraction of ventricular wall. But, the previously report showed that the ratio of noncompaction/compaction part not seemed to exactly predict a poor prognosis of LVNC [23]. Comprehensively consideration of genetics and disease progress, we predicted that genetic background combined with other nongenetic factors might defined the phenotype and outcomes of our LVNC patients. Apart from remarkable hyper-trabeculae, endocardial fibrosis or fibroelastosis was the most frequently reported abnormality in LVNC [24]. Endocardial fibrosis or fibroelastosis in LVNC was considered a symbol pathophysiology feature, with uncertain clinical effect [25]. In this cohort, LGE positivity is consistent with cardiac interstitial fibrosis and should be applied to predict poor prognosis in LVNC. In normal situations, the cardiac fibrosis network was originally the normal schedule of cardiac muscle that provided stress during heart contractile. Abnormal sarcomeric structures and alterations in calcium signalling would destroy the cardiac homeostasis and can result in cardiomyocyte remodelling and systolic and diastolic dysfunction. In response to extrastress, cardiac fibroblasts convert into activated cardiac myofibroblasts, proliferate and enhance cardiac fibrosis deposition. In this study, we compared the gene expression profiles of fibrosis between patients
and normal control myocardium. These genes related to fibrosis were extremely highly expressed and irregularly expressed in the myocardium of LVNC. In particularly, gene function with inflammatory and extracellular matrix proteolysis, suggested that immune inflammation was also involved in the fibrosis pathway in LVNC. We suspected that variants in sarcomere and/or other functional cardiomyocyte structure induced to abnormal contraction of cardiac myocytes, which lead to disturbance of myocardium development and myocardium remodelling (Fig. 3). This may help to answer the question whether LVNC can be a failure of trabecular compaction during foetal development or a distinct pathologic phenotype of cardiomyopathy caused by genetics.
4.4. Limitation No doubt, this is a small cohort and all of patients came from HTx Databank. All of patients have severe and complex clinical manifestations, which hampered the establishment of genotype and phenotype. Exome sequencing provided us an opportunity to investigate more genetic information about this cardiomyopathy, but only those reported genes and proteins associated with LVNC and cardiomyopathy were priority to list in this study, which would induce to neglection of potential pathological variants. In addition, the high-throughput sequencing method has technique limitation, some potential genetic defects contributed to LVNC phenotype would be missed, such as big deletion and inversion of genes. We have plans to dig pathological variants or mutation by other genetic screening methods, which would help for expand the genetic panel of disease.
Fig. 3. Summary of inference about genetics in LVNC. Pathologic variants in genes have function with heart development. Physiological activities synergistically contribute to the phenotype of LVNC.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001
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5. Conclusions Multiple gene variants synergistically function in the phenotype of LVNC who underwent HTx. Biallelic variants and truncating variants involved in the pathogenesis of LVNC who was in a high risk and should be considered carefully follow-up. Young patients with multiple pathologic variants and lower LVEF (b45%) at initial symptom seemed to be deteriorated quickly. Pathological variants in cardiac genes induced to abnormal contraction of cardiac myocytes involved in the pathological progress of LVNC. These results support that genetic screening should be applied to the diagnosis and prognosis of LVNC patients. CRediT authorship contribution statement Shenghua Liu: Data curation, Formal analysis and Writing-original draft. Yuanyuan Xie: Data curation, Formal analysis and Writingoriginal draft. Hongliang Zhang: Data curation, Formal analysis and Writing-original draft. Zongqi Feng: Data curation, Formal analysis and Writing-original draft. Jian Huang: Data curation, Formal analysis and Writing-original draft. Jie Huang: Data curation, Formal analysis and Writing-original draft. Shengshou Hu: Data curation, Formal analysis and Writing-original draft. Yingjie Wei: Data curation, Formal analysis and Writing-original draft. Acknowledgements We thank the contributors to the primary cardiomyopathy cohort, and we are grateful to our patient and his family for the support and collaboration in this study. Funding This work was supported by CAMS Initiative for Innovative Medicine (CAMS-I2M, 2016-I2M-1-015 to Y.J.W.) and PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (Grant 33320140167 to S.H.L). Contributors Conceived and designed the experiment: S.H.L and Y.J.W; Clinical support: Doctor H.L.Z, J.H. and S.H.H.; Collected the samples and conducted the experiments: S.H.L, Y.Y.X, and Z.Q.F; Analysed exome data: S.H.L and Y.Y.X.; Wrote the manuscript: S.H.L, and Y.J.W; S.H.L and Y.Y.X. contributed equally to this work. All the authors have reviewed the manuscript. Declaration of competing interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijcard.2019.12.001. References [1] F. Negri, A. De Luca, E. Fabris, R. Korcova, C. Cernetti, C. Grigoratos, et al., Left ventricular noncompaction, morphological, and clinical features for an integrated diagnosis, Heart Fail. Rev. 24 (2019) 315–323.
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[2] G. Habib, P. Charron, J.-C. Eicher, R. Giorgi, E. Donal, T. Laperche, et al., Isolated left ventricular non-compaction in adults: clinical and echocardiographic features in 105 patients. Results from a French registry, Eur. J. Heart Fail. 13 (2011) 177–185. [3] M. Greutmann, M.L. Mah, C.K. Silversides, S. Klaassen, C.H.A. Jost, R. Jenni, et al., Predictors of adverse outcome in adolescents and adults with isolated left ventricular noncompaction, Am. J. Cardiol. 109 (2012) 276–281. [4] J.I. van Waning, K. Caliskan, Y.M. Hoedemaekers, K.Y. van Spaendonck-Zwarts, A.F. Baas, S.M. Boekholdt, et al., Genetics, clinical features, and long-term outcome of noncompaction cardiomyopathy, J. Am. Coll. Cardiol. 71 (2018) 711–722. [5] P. Richard, F. Ader, M. Roux, E. Donal, J.C. Eicher, N. Aoutil, et al., Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity, Clin. Genet. 95 (2019) 356–367. [6] S. Klaassen, S. Probst, E. Oechslin, B. Gerull, G. Krings, P. Schuler, et al., Mutations in sarcomere protein genes in left ventricular noncompaction, Circulation 117 (2008) 2893–2901. [7] B.E. Staehli, C. Gebhard, P. Biaggi, S. Klaassen, E.V. Buechel, C.H.A. Jost, et al., Left ventricular non-compaction: prevalence in congenital heart disease, Int. J. Cardiol. 167 (2013) 2477–2481. [8] B. Jensen, P. Agger, B.A. de Boer, R.-J. Oostra, M. Pedersen, A.C. van der Wal, et al., The hypertrabeculated (noncompacted) left ventricle is different from the ventricle of embryos and ectothermic vertebrates, Biochim. Biophys. Acta, Mol. Cell Res. 1863 (2016) 1696–1706. [9] S. Liu, Y. Bai, J. Huang, H. Zhao, X. Zhang, S. Hu, et al., Do mitochondria contribute to left ventricular non-compaction cardiomyopathy? New findings from myocardium of patients with left ventricular non-compaction cardiomyopathy, Mol. Genet. Metab. 109 (2013) 100–106. [10] S. Richards, N. Aziz, S. Bale, D. Bick, S. Das, J. Gastier-Foster, et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet. Med. 17 (2015) 405–424. [11] C.A. Gifford, S.S. Ranade, R. Samarakoon, H.T. Salunga, T.Y. de Soysa, Y. Huang, et al., Oligogenic inheritance of a human heart disease involving a genetic modifier, Science 364 (2019) 865–870. [12] H.G. van Velzen, A.F.L. Schinkel, R.A. Oldenburg, M.A. van Slegtenhorst, I.M.E. FrohnMulder, J. van der Velden, et al., Clinical characteristics and long-term outcome of hypertrophic cardiomyopathy in individuals with a MYBPC3 (myosin-binding protein C) founder mutation, Circ. Cardiovasc. Genet. 10 (2017), e001660. [13] K. Kolokotronis, J. Kuhnisch, E. Klopocki, J. Dartsch, S. Rost, C. Huculak, et al., Biallelic mutation in MYH7 and MYBPC3 leads to severe cardiomyopathy with left ventricular non-compaction phenotype, Hum. Mutat. (2019). [14] R. Almomani, J.M. Verhagen, J.C. Herkert, E. Brosens, K.Y. van Spaendonck-Zwarts, A. Asimaki, et al., Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy, J. Am. Coll. Cardiol. 67 (2016) 515–525. [15] L. Shan, N. Makita, Y. Xing, S. Watanabe, T. Futatani, F. Ye, et al., SCN5A variants in Japanese patients with left ventricular noncompaction and arrhythmia, Mol. Genet. Metab. 93 (2008) 468–474. [16] P.A. Schweizer, J. Schroeter, S. Greiner, J. Haas, P. Yampolsky, D. Mereles, et al., The symptom complex of familial sinus node dysfunction and myocardial noncompaction is associated with mutations in the HCN4 channel, J. Am. Coll. Cardiol. 64 (2014) 757–767. [17] A. Milano, A.M.C. Vermeer, E.M. Lodder, J. Barc, A.O. Verkerk, A.V. Postma, et al., HCN4 mutations in multiple families with bradycardia and left ventricular noncompaction cardiomyopathy, J. Am. Coll. Cardiol. 64 (2014) 745–756. [18] N. Makita, K. Sasaki, H. Yokoshiki, S. Yamada, H. Tsutsui, Left ventricular noncompaction associated with mutations in cardiac Na channel gene SCN5A, Circulation 114 (2006) 126. [19] Y. Nishio, T. Makiyama, H. Itoh, T. Sakaguchi, S. Ohno, Y.Z. Gong, et al., D85N, a KCNE1 polymorphism, is a disease-causing gene variant in long QT syndrome, J. Am. Coll. Cardiol. 54 (2009) 812–819. [20] Q. Wang, K.-H. Wu, P.-H. Jiang, C.-I. Lin, J.L. Lin, E.A. Adams, et al., Loss of intercalated disc protein mXin beta leads to atrioventricular conduction defects and channel remodeling, Circulation 128 (2013). [21] J.I. van Waning, K. Caliskan, M. Michels, A.F.L. Schinkel, A. Hirsch, M. Dalinghaus, et al., Cardiac phenotypes, genetics, and risks in familial noncompaction cardiomyopathy, J. Am. Coll. Cardiol. 73 (2019) 1601–1611. [22] D.L. Halaney, A. Sanyal, N.A. Nafissi, D. Escobedo, M. Goros, J. Michalek, et al., The effect of trabeculae carneae on left ventricular diastolic compliance: improvement in compliance with trabecular cutting, J. Biomech. Eng. 139 (2017). [23] F. Zemrak, M.A. Ahlman, G. Captur, S.A. Mohiddin, N. Kawel-Boehm, M.R. Prince, et al., The relationship of left ventricular trabeculation to ventricular function and structure over a 9.5-year follow-up the MESA study, J. Am. Coll. Cardiol. 64 (2014) 1971–1980. [24] G. Nucifora, G.D. Aquaro, A. Pingitore, P.G. Masci, M. Lombardi, Myocardial fibrosis in isolated left ventricular non-compaction and its relation to disease severity, Eur. J. Heart Fail. 13 (2011) 170–176. [25] J.A. Towbin, A. Lorts, J.L. Jefferies, Left ventricular non-compaction cardiomyopathy, Lancet. 386 (2015) 813–825.
Please cite this article as: S. Liu, Y. Xie, H. Zhang, et al., Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2019.12.001