Whole-exome sequencing reveals two de novo variants in the RBM20 gene in two Chinese patients with left ventricular non-compaction cardiomyopathy.

IMPORTANCE: Pathogenic variants in the RBM20 gene are associated with aggressive dilated cardiomyopathy (DCM). Recently, RBM20 was found to be associated with left ventricular non-compaction cardiomyopathy (LVNC). Thus far, only five families with LVNC have been reported to carry variants in RBM20. It remains unknown whether the variants in RBM20 associated with DCM can also cause LVNC.
OBJECTIVE: To elucidate the causative RBM20 variant in two unrelated patients with both LVNC and DCM, and to identify the clinical characteristics associated with variants in RBM20.
METHODS: Trio whole-exome sequencing (WES) was performed. Variants were filtered and classified in accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG).
RESULTS: We identified two distinct de novo variants in RBM20 (one per patient) in these two patients with LVNC. Both variants have been reported in patients with DCM, without the LVNC phenotype. Patient 1 was an 11-year-old girl who had DCM, LVNC, and heart failure; the ratio of noncompacted-to-compacted myocardium was 2.7:1. A de novo heterozygous variant c.1907G>A (p.Arg636His) in exon 9 was identified in this patient. Patient 2 was a 13-year-old boy who had clinical phenotypes identical to those of Patient 1; the ratio of noncompacted-to-compacted myocardium was 3.2:1 in this patient. WES revealed a de novo heterozygous variant c.1909A>G (p.Ser637Gly) in exon 9. Both variants were previously characterized as pathogenic, and our study classified them as pathogenic variants based on the ACMG guidelines.
INTERPRETATION: We found that two patients with LVNC had variants in RBM20. Our results extended the clinical spectrum of the two RBM20 variants and illustrated that the same variant in RBM20 can cause DCM, with or without the LVNC phenotype.

INTRODUCTION

Left ventricular non‐compaction cardiomyopathy (LVNC) is characterized by the presence of excessive left ventricular trabeculae, deep intratrabecular recesses, and a thin compacted myocardial layer. The LVNC phenotype may occur either in isolated form or in combination with additional cardiomyopathy phenotypes including dilated, hypertrophic, and restrictive cardiomyopathies, and/or other congenital heart diseases. , LVNC may be asymptomatic or manifest as arrhythmia, thromboembolism, and/or heart failure; compared with other phenotypes, LVNC with a dilated phenotype is associated with higher mortality and more severe cardiac events.

Most LVNC‐associated genes have also been reported in patients with hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Variants of sarcomere genes have been identified in a majority of patients with genetic LVNC; MYH7, MYBPC3, and TTN were the most common mutated genes in these patients. , Variants in RBM20, which encodes RNA‐binding protein 20, have been found in patients with clinically aggressive DCM. Recently, RBM20 was also identified as a novel causative gene in patients with LVNC ; to the best of our knowledge, only five variants in RBM20 have been identified in patients with LVNC thus far. , ,

Here, we used trio whole‐exome sequencing (WES) to identify two de novo heterozygous RBM20 variants in two unrelated patients with LVNC and DCM; these variants had previously been found only in patients with DCM who did not have the LVNC phenotype.

METHODS

Ethical Approval

The study was approved by the Ethics Committee of Henan Children’s Hospital. Written informed consents for participation and publication were obtained from the guardians of each patient.

Patients

Patient 1 was an 11‐year‐old girl, who was admitted to Henan Children’s Hospital because of chest tightness and hypodynamia. Patient 2 was a 13‐year‐old boy without a family history of cardiomyopathy; he was admitted to Henan Children’s Hospital because of chest tightness, cough, and tachypnea during sports. Clinical data, pedigree information, and blood samples were collected from the two probands, as well as the probands’ parents and other available family members. All participants underwent detailed physical evaluations, including blood examination, electrocardiography, chest radiography, and echocardiography. LVNC was diagnosed on the basis of a characteristic morphological appearance comprising: 1) multiple trabeculations, 2) deep intertrabecular recesses visible on color flow, and 3) a two‐layered myocardial structure with a > 2:1 ratio of non‐compacted to compacted myocardium in systole.

Sequencing

Trio WES of the two probands and their parents was performed as previously described. In brief, DNA was isolated from 200 μL blood samples from the two probands and their family members, using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Exon capture was performed using the Agilent SureSelect Human All Exome V6 kit (Agilent Technologies Inc., Santa Clara, CA, USA). WES was performed on an Illumina Hiseq X Analyzer (Illumina, San Diego, CA, USA) in 150‐bp paired‐end mode, in accordance with the manufacturer’s recommendations. Sequences obtained from WES were aligned to the GRCh37/hg19 human reference genome. Variants were filtered if they had a minor allele frequency > 0.5%, or if they had a minor allele frequency > 2% when the variant was homozygous or there was a second variant in the gene in three SNP databases (db‐SNP, gnomAD v2, and 1000 Genomes Project Database). SIFT (http://sift.jcvi.org/www/SIFT_enst_submit.html), Polyphen‐2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org), and CADD (http://cadd.gs.washington.edu/) were used to predict the potential impacts of the identified mutations on protein function. Variants were classified in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines. A virtual cardiomyopathy‐related gene panel was generated that included 409 genes based on the HPO term “cardiomyopathy” (HP:0001638; http://www.human-phenotype-ontology.org/); this panel was supplemented with 10 genes that were not included in this HPO term, but were associated with LVNC (Supplementary Table S1). Putative pathogenic variants in cardiomyopathy‐related genes detected by WES were confirmed by Sanger sequencing with a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) in the two probands, their parents, and available family members.

RESULTS

Clinical information

Chest radiography of Patient 1 revealed pneumonia and marked cardiomegaly. Echocardiography examination revealed dilation of both atria and both ventricles with prominent trabeculations, a depressed left ventricular ejection fraction of 22%, moderate to severe tricuspid regurgitation, and mild mitral regurgitation. The ratio of noncompacted‐to‐compacted myocardium was 2.7:1 (Figure 1A); trabeculations and deep intertrabecular recesses were observed (Figure 1B). The left and right ventricular diastolic dimensions were 66.6 mm and 16.7 mm, respectively; left and right atrial diameters were 52.8 mm and 52.4 mm, respectively. Electrocardiography revealed sinus rhythm with P‐wave abnormalities, ST‐T changes, transient atrial tachycardia, and atrial fibrillation (Figure 1C). Serum biochemistry tests showed high levels of N‐terminal fragment of pro‐B‐type natriuretic peptide (8482 pg/mL; normal range, < 100 pg/mL). The patient was treated with optimal doses of an angiotensin‐converting‐enzyme inhibitor (captopril), diuretics (hydrochlorothiazide and spironolactone), and potassium citrate, as well as oral anticoagulant (aspirin). She was diagnosed with LVNC, DCM and heart failure, and showed no response to treatment. Her parents were nonconsanguineous and reported no family history of cardiomyopathy. The patient’s parents and her younger brother had no symptoms of heart failure; their echocardiography findings were normal.

Figure 1

Echocardiography and electrocardiography findings in Patient 1. (A) Echocardiography examination revealed a 2.7:1 ratio of noncompacted‐to‐compacted myocardium. (B) Trabeculations and deep intertrabecular recesses were observed in echocardiography examination. (C) Electrocardiography revealed sinus rhythm with P‐wave abnormalities and ST‐T changes.

Echocardiography examination of Patient 2 revealed marked ventricular dilation (left and right ventricular diastolic dimensions of 71.3 mm and 23.7 mm, respectively) and atrial dilation (left and right atrial diameters of 58.4 mm and 42.4 mm, respectively). Diffuse hypokinesis was present with depressed systolic function (left ventricular ejection fraction of 25%). The ratio of noncompacted‐to‐compacted myocardium was 3.2:1 (Figure 2A); trabeculations and deep intertrabecular recesses were observed (Figure 2B). The patient was diagnosed with LVNC, DCM and heart failure. Electrocardiography revealed sinus rhythm with ST‐T changes and occasional premature ventricular contraction (Figure 2C). The patient was treated with an angiotensin‐converting‐enzyme inhibitor (captopril), diuretics (hydrochlorothiazide and spironolactone), and potassium citrate, as well as an oral anticoagulant (aspirin). The treatment was ineffective; subsequently, the patient died due to heart failure and respiratory infection. Echocardiography analyses of the patient’s other family members (his parents and his elder sister) did not demonstrate any clinical evidence of DCM or LVNC.

Figure 2

Echocardiography and electrocardiography findings in Patient 2. (A) Echocardiography examination revealed a 3.2:1 ratio of noncompact‐ed‐to‐compacted myocardium. (B) Trabeculations and deep intertrabecular recesses were observed in echocardiography examination. (C) Electrocardi‐ography revealed sinus rhythm with ST‐T changes.

Genetic findings

WES data were filtered by using db‐SNP, gnomAD v2, and the 1000 Genomes Project Database to exclude common variants. In total, 227 unique variants were detected in Patient 1, while 270 unique variants were detected in Patient 2. After cardiomyopathy‐related gene filtering, six variants in five genes were identified in Patient 1 (Supplementary Table S2); seven variants in five genes were identified in Patient 2 (Supplementary Table S3). All filtered data were predicted by four separate bioinformatics programs (SIFT, Polyphen‐2, MutationTaster, and CADD).

A de novo missense variant, c.1907G>A (p.Arg636His, rs267607004), was identified in RBM20 in Patient 1 (Figure 3). Sanger sequencing of this variant was performed in all four family members; the results showed that the proband’s unaffected parents and younger brother did not carry this variant. c.1907G>A was located in a highly conserved genomic region in exon 9; it was not present in gnomAD v2 or the 1000 Genomes Project Database. This variant was predicted to have a deleterious effect on the gene or gene product by SIFT, PolyPhen‐2, MutationTaster, and CADD. In accordance with the ACMG guidelines, c.1907G>A was classified as a pathogenic variant. Furthermore, no other pathogenic or likely pathogenic variants in cardiomyopathy disease genes were identified in Patient 1.

Figure 3

Family pedigree and Sanger sequencing analysis of RBM20 in Patient 1 and her family members. (A) Pedigree includes Patient 1 (II.1), her unaffected parents (I.1 and I.2), and her unaffected younger brother (II.2). (B) Sanger sequencing analysis of RBM20. Arrows indicate mutated nucleotides. The proband (II.1) carries a de novo missense variant, c.1907G>A (p.Arg636His), while her younger brother (II.2) does not.

Patient 2 carried a de novo missense variant, c.1909A>G (p.Ser637Gly, rs267607005), in RBM20 (Figure 4). Sanger sequencing was performed in the proband and his parents. A blood sample from the proband’s elder sister was not available. c.1909A>G was also located in exon 9 and not present in gnomAD v2 or the 1000 Genomes Project Database. This variant was considered to be “damaging” by multiple lines of computational evidence (SIFT, PolyPhen‐2, MutationTaster, and CADD). In accordance with the ACMG guidelines, c.1909A>G was also classified as a pathogenic variant. Also no pathogenic or likely pathogenic variants in any other cardiomyopathy disease genes were identified in Patient 2.

Figure 4

Family pedigree and Sanger sequencing analysis of RBM20 in Patient 2 and his family members. (A) Pedigree includes Patient 2 (II.2), his unaffected parents (I.1 and I.2), and his unaffected elder sister (II.1). (B) Sanger sequencing analysis of RBM20. Arrows indicate mutated nucleotides. The proband (II.2) carries a de novo missense variant, c.1909A>G (p.Ser637Gly). The proband’s elder sister did not undergo genetic testing

DISCUSSION

We identified two de novo heterozygous variants in RBM20, c.1907G>A and c.1909A>G, in two unrelated patients with LVNC and DCM. Although these variants (c.1907G>A , , , and c.1909A>G ) have been previously identified in patients with DCM, none of those patients have been reported to exhibit concurrent LVNC.

RBM20 is a key cardiac splice regulator that controls the processing of several important transcripts predominantly expressed in striated muscle, with the highest levels in heart tissue. , RBM20 mutations can cause mis‐splicing of several targets and often lead to progressive DCM with conduction diseases, as well as atrial or malignant ventricular arrhythmia. , Multiple human RBM20 pathogenic variants that cause DCM have been identified thus far, including a mutational hot spot in an arginine/serine‐rich domain. , , Recurrent pathogenic missense variants in the arginine/serine‐rich region disrupt binding with other splicing factors and alter transcript processing, causing highly penetrant DCM.

Recently, RBM20 was identified as a novel LVNC‐associated gene. To the best of our knowledge, only five LVNC‐associated variants in RBM20 have been reported thus far, including three missense variants: c.1901G>T (p.Arg634Leu), c.3416C>T (p.Pro1139Leu), and c.3584C>A (p.Ser1195Tyr) ; a splicing region variant: c.192‐3C>T ; and a frameshift variant: c.846_853 delTTACGGAC (p.Tyr283Glnfs*14). However, the ACMG guidelines indicate that only two of these variants, c.1901G>T (p.Arg634Leu) and c.846_853 delTTACGGAC (p.Tyr283Glnfs*14), can be classified as pathogenic or likely pathogenic. The two variants identified in this study, c.1907G>A and c.1909A>G, were both located in the arginine/serine‐rich region of RBM20. c.1907G>A has been reported to segregate with DCM in three families , , ; DCM was most noticeable during adolescence or young adulthood in each family. c.1909A>G was identified in four patients from a family with DCM, in which the age of diagnosis ranged from 20 to 44 years. However, none of the patients in these families were reported to exhibit LVNC. Our work extends the phenotypic spectra of these two variants by reporting their associations with the LVNC phenotype.

RBM20 is an important splice regulator of TTN; notably, RBM20 regulates alternative splicing of the TTN gene by exon skipping or exon shuffling. Functional studies have shown that the c.1901G>T variant in RBM20, identified in patients with LVNC, leads to an abnormal giant isoform of TTN. TTN is the most prevalent disease‐causing gene in patients with DCM and LVNC. It has been reported that variants in a single gene (e.g., SCN5A or PKP2) can lead to apparently unrelated phenotypes in inheritable cardiac disorders. Mutations in SCN5A have been previously identified in patients with DCM and long QT syndrome. Various phenotypes, ranging from purely arrhythmogenic (Brugada syndrome or catecholaminergic polymorphic ventricular tachycardia) to purely structural (DCM), as well as arrhythmogenic cardiomyopathy, could result from dysfunction in the PKP2 gene. In this study, the two probands exhibited clinical phenotypes distinct from those of previously described patients with identical variants in RBM20. There may be an underlying mutation in other genes that contributes to the LVNC phenotype (i.e., a causative mutation or genetic modifier). However, it remains unclear why these two patients exhibited LVNC with DCM, rather than DCM alone.

In conclusion, we have described two patients with distinct RBM20 variants that were not previously associated with LVNC; no pathogenic or likely pathogenic variants in any other cardiomyopathy disease genes were identified in either patient, which might have contributed to the deviant phenotype. This study extended the clinical spectrum of phenotypes associated with variants in RBM20 . Furthermore, although the mechanism remains unknown, we have shown that the same variant in RBM20 can cause distinct cardiac phenotypes.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

Supplementary Material

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