EXPERIMENTAL STUDY

A Novel Truncation Variant Linked to Familial Dilated Cardiomyopathy Found in a Japanese Family and Its Functional Analysis in Genome-Edited Model Cells Kayoko Hirayama-Yamada,1 MD, Natsuko Inagaki,2,3 MD, Takeharu Hayashi,1,4,5 MD and Akinori Kimura,1,4,6 MD

Summary Dilated cardiomyopathy (DCM) is a common cause of heart failure. TTN, which encodes titin , is a representative causative of DCM, and is presented mainly as a truncation variant. However, TTN truncation variants are also found in healthy individuals, and it is therefore important to evaluate the pathogenicity of each variant. In this study, we analyzed 67 cardiomyopathy-associated in a male Japanese patient who was hospitalized for recurrent severe heart failure and identified a novel truncation variant, TTN Ser17456Arg fs*14. This TTN truncation variant was located in the A-band region. Moreover, the patient’s mother with heart failure harbored the same variant, whereas the father and brother without heart failure did not harbor the variant. To examine the functional changes associated with the truncation variant, H9c2 cells were subjected to genome ed- iting to generate cells with a homologous truncation variant. The cells were differentiated using all-trans-retinoic acid, and the mRNA expression of skeletal and cardiac actin were found to be increased and decreased, respectively, consistent with known changes in patients with DCM or heart failure. In contrast, another cell with the titin truncation variant used as a control showed no changes in heart failure-related genes. In summary, we found a novel TTN truncation variant in familial DCM patients and confirmed its functional changes using a relatively simple cell model. The novel truncation variant was identified as a pathogenic and disease-causing mutation. (Int Heart J Advance Publication) Key words: Genome editing, Heart failure

eart failure is a life-threatening disease often cytoskeletal protein and plays important structural, devel- caused by cardiomyopathy. Primary cardio- opmental, and mechanical regulatory roles in striated mus- myopathy can be classified as dilated cardio- cle, i.e., heart and skeletal muscles, particularly with re- H 7,8) myopathy (DCM), hypertrophic cardiomyopathy, restric- gard to passive stiffness. Titin truncation variants are ex- tive cardiomyopathy, arrhythmogenic right ventricular car- pected to result in loss of functions, leading to passive diomyopathy, or left ventricular noncompaction tension and to the impairment of various titin-mediated (LVNC).1,2) DCM is characterized by ventricular enlarge- signaling pathways, and ultimately to DCM.5,9-11) However, ment and progressive heart failure with a prevalence of 1: 1-2% of the general population without DCM carry TTN 250-2500 adults.1-3) Additionally, 20-50% of DCMs have a truncation variants, and therefore the pathological signifi- family history of DCM (FDCM), predominantly showing cance of each TTN truncation variant should be investi- autosomal dominant traits.1,4) gated.5,12,13) More than 40 genes have been reported as causative In this study, we identified a novel TTN truncation genes for DCM. Among these genes, TTN, encoding the variant in Japanese patients with FDCM. In addition, cells titin protein, is frequently reported in patients with DCM; with the truncation variant in the same domain were con- in particular, heterozygous truncation variants of TTN are structed using a relatively simple method in order to ex- noted in 14-25% of all DCM cases.4-6) Titin is the largest amine whether changes in in the cells

From the 1Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 2Department of Cardiology, Tokyo Medical University, Tokyo, Japan, 3Department of Clinical Genetics Center, Tokyo Medical University, Tokyo, Japan, 4Laboratory for Integrated Research Projects on Intractable Diseases, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 5Department of Physiology, Tokai University School of Medicine, Kanagawa, Japan and 6Research Core, Tokyo Medical and Dental University (TMDU), Tokyo, Japan. This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant numbers 26460407 [TH], 17K08684 [TH], 15K15095 [AK], and 16H05296 [AK]), the Japan Society for the Promotion of Science, Nanken-Kyoten, Tokyo Medical and Dental University (TMDU), Tokai University School of Medicine, Project Research and the Japan Agency for Medical Research and Development (AMED) (grant number JA19ek0109292 [TH]). Address for correspondence: Takeharu Hayashi, MD, Department of Physiology, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kana- gawa 259-1193, Japan. E-mail: [email protected] or Akinori Kimura, MD, Research Core, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail: [email protected] Received for publication October 1, 2020. Revised and accepted November 20, 2020. Released in advance online on J-STAGE March 6, 2021. doi: 10.1536/ihj.20-664 All rights reserved by the International Heart Journal Association. 1 IntHeartJ 2 HIRAYAMA-YAMADA, ET AL Advance Publication were similar to those observed in general DCM. cardiomyocytes was used as a model system. H9c2 cells with TTN frameshift variants were generated using the Guide-it CRISPR/Cas9 system (Clontech, CA, USA). Tar- Methods get genomic sequences in single guide RNAs (sgRNAs) Cardiac imaging and histological examinations: To were customized by designing pairs of oligos (5’-TACCA evaluate the patient’s cardiac function, echocardiography CACAACTCGAATTTC-3’ and 5’-GAAATTCGAGTTGT (EPIQ 7G; Phillips Healthcare, MA, USA) was per- GTGGTA-3’ for generating variants in TTN I6334fs, and formed. In addition, cardiac catheterization and myocar- 5’-CACTATTCTGTCTCTGACGC-3’ and 5’-GCGTCAG dial biopsy at the right ventricular myocardium were per- AGACAGAATAGTG-3’ for TTN V17602fs). After hy- formed when the proband patient was 46 years old. The bridization of the target oligo, it was cloned into pGuide- specimen was fixed with 10% formaldehyde, embedded in it-ZsGreen1 vector with the cas9 sequence. H9c2 cells conventional-dehydrated paraffin after dehydration, sec- were transfected with pGuide-it-ZsGreen1 vector having tioned, stained with hematoxylin and eosin (HE) and Mas- the generated target gRNA sequence using Lipofectamine son trichrome, and observed under an optical microscope. TM 3000 Reagent (Thermo Fisher Scientific). After 48 Genetic analysis and evaluation of genetic variants: hours, H9c2 cells expressing ZsGreen were selected from Genomic DNA was extracted from the peripheral blood of cells transfected with the pGuide-it-ZsGreen1 vector using the patient using a Wizard DNA purification kit (Promega MoFlo XDP (Beckman Coulter, CA, USA) and cultured Corporation, WI, USA). Genetic analysis of the genomic as single cells. Each single cell clone was cultured and DNA was performed using an Ion Torrent PGM™ system subjected to Sanger sequencing for validation of gene ed- (Thermo Fisher Scientific, CA, USA) for 67 genes re- iting. The mutant and wild-type H9c2 cells were seeded ported as inherited primary and secondary in 24-well plates at a density of 3×104 cells/cm2 and cul- cardiomyopathy-associated genes: ABCC9, ACTC1, tured at 37°C and 5% CO2 in Dulbecco’s modified ACTN2, ANKRD1, BAG3, CALR3, CAV3, CRYAB, CSRP3, Eagle’s medium (Thermo Fisher Scientific) supplemented DES, DOLK, DSGH2, JUP, LAMA4, LAMP2, LDB3, with 10% fetal bovine serum (FBS) and 1% penicillin/ LMNA, MTO1, MURC, MYBPC3, MYH6, MYH7, MYL2, streptomycin. After reaching 70-80% confluence, the cells MYL3, MYL2, DSP, DTNA, EMD, EYA4, FHL1, FHL2, were cultured for 1 week in medium supplemented with FHOD3, FKTN, GAA, GATAD1, GLA, ILK, ISL1, JPK2, 1% FBS with 100 nM all-trans-retinoic acid (ATRA; MYOZ2, MYPN, NEBL, NEXN, OBSCN, PKP2, PLN, Sigma-Aldrich, MO, USA) for differentiation.17) Cells cul- PRDM16, PRKAG2, PSEN1, PSEN2, RBM20, SCN5A, tured in 10% FBS without ATRA were used as controls. SDHA, SGCD, TAZ, TCAP, TGFB3, TIEG1, TMEM43, Total RNA was extracted from cultured cells using a Nu- TMPO, TNNC1, TNNI3, TNNT2, TPM1, TTN, TTR, and cleoSpin RNA kit (Takara Bio). The extracted RNA was VCL.14) Sequenced lead data were mapped to the reference converted into cDNA using a PrimeScript II first strand genome, hg19, using Torrent Suite 4.4 (Thermo Fischer cDNA synthesis kit (Takara Bio) according to the manu- Scientific) to extract the variants. Next, variants with a facturer’s instructions. Quantitative real-time PCR (qPCR) minor allele frequency (MAF) of less than 0.2% were se- was performed on the obtained cDNA using an Applied lected from the following databases: the 1000 Genome Biosystems 7500 Real-Time PCR System (Thermo Fisher Project, the Genome Aggregation Database (gnomAD), Scientific). The relative expression of the target gene was and the Human Genetic Variations Database (HGVD), evaluated using glyceraldehyde-3-phosphate dehydroge- which is a general Japanese variant database. In addition, nase (GAPDH) as a reference gene. The target genes and nonsynonymous variants were selected using CLC primers are listed in the Table. For relative gene expres- Genomic Workbench (Qiagen, Hilden, Germany).15,16) To sion analysis, calibration curves for the target gene and confirm the identified TTN variant, Sanger sequencing was reference gene were prepared using a dilution series of performed using the following primers: TTN 17456F, 5’-G standard samples, and the relative expression levels of the ACCATGGATGAGCTAATGG-3’ (forward), TTN 17456R, target and reference genes were calculated based on the 5’-CATTGCTGGCATTGGAGAAC-3’ (reverse). The PCR calibration curves. Statistical analysis was performed us- products were purified using ExoSAP-IT (Affymetrix, ing the unpaired or Welch’s t-tests; statistical significance CA, USA) and sequenced using a BigDye terminator v3.1 was indicated by P values lower than 0.05. For the TTN Cycle Sequencing Kit (Thermo Fisher Scientific) on an gene sequence, the following accession numbers in Gene- ABI 3130 (Thermo Fisher Scientific).14) In addition, the Bank were used: human TTN, NM_001256850; rat TTN, PCR product of the TTN variant was cloned into the pMD XM_017592328. 20-T vector using a Mighty TA-cloning Kit (Takara Bio, Shiga, Japan), and each allele of the gene mutation was Results confirmed by sequencing. Written informed consent was obtained from patients who agreed to undergo genetics Clinical course of a proband patient with DCM: The analysis. Segregation analyses were performed for the pro- proband male patient (Figure 1, IV-1) was admitted to To- band patient’s family members. This study was approved kyo Medical University Hospital because of acute heart by the Ethics Review Committee of Medical Research In- failure associated with left ventricular systolic dysfunction stitute, Tokyo Medical and Dental University (TMDU) (NYHA class IV) at 46 years of age. Echocardiography and Tokyo Medical University School of Medicine. findings showed marked dilation of the left ventricle and Functional analysis using genome-edited cell line: H9c2 decreased ejection fraction; coronary angiography showed cell line (ATCC, VA, USA) derived from rat embryonic no stenosis of the coronary artery. Myocardial biopsy IntHeartJ Advance Publication TTN TRUNCATION MUTATION IN FAMILIAL DCM 3

Table. Information on Primers and Amplicons for qPCR

Gene Amplicon Covering Sequence (5´ to 3´) Ref Seq Symbol (bp) Exons TTN FW: AACCTGGAGAAGTCACAGAC 144 Exon 249-250 XM_017592328.1 RV: GTGGCTTCCCAGACACATAG TNNT2 FW: ATGTCTGACGCCGAGGAAGA 101 Exon 1-4 NM_012676.1 RV: TCCTCCACTGCCTCCTCTTG ACTC1 FW: GCTGTCTTCCCATCCATCGT 100 Exon 2-3 NM_019183.1 RV: GCTTGCTCTGAGCCTCGTCA ACTA1 FW: GCTGTGTTCCCATCCATCGT 113 Exon 2-3 NM_019212.2 RV: GTCAGGATACCTCGCTTGCT GAPDH FW: CACAGTCAAGGCTGAGAATG 123 Exon 3-4 NM_017008.4 RV: GTAGACTCCACGACATACTC FW indicates forward; RV, reverse; and bp, .

Figure 1. Pedigree showing the inheritance of dilated cardiomyopathy or heart failure. Squares denote males, and circles denote females. Black symbols indicate affected individuals, and open symbols indicate unaffected individuals. The arrow indicates the proband. P indicates proband; d, death; E, genetic evaluation; +, presence of the TTN truncation variant; and -, absence of the TTN truncation variant. showed abnormal arrangement and vacuolation of cardio- observed around the apex of the left ventricle, and LVNC myocytes and interstitial fibrosis, suggesting DCM (Figure was also suspected but did not match the diagnostic crite- 2A, B). Two years later, at the age of 48, despite appro- ria (Figure 3C).18) The patient received an infusion of a priate medical treatment and a heart failure management phosphodiesterase inhibitor, the dose of beta-blocker was program, his heart failure suddenly worsened, and the pa- gradually increased, and he continued to receive ACE in- tient was readmitted to hospital. An echocardiogram hibitors and aldosterone antagonists. Fortunately, he was showed left ventricular diffuse hypokinesis and 4-chamber able to leave the hospital without a heart transplantation. enlargement (Figure 3A, B). The left ventricular end- About 3 years have passed since the patient was dis- diastolic/end-systolic dimension was 69/65 mm, and the charged from the hospital; the echocardiographic findings left ventricular ejection fraction was 17%. The thicknesses did not show any improvement in the cardiac contractility of the interventricular septum and posterior wall were 7 or ventricular diameter, and no reverse remodeling was and 8 mm, respectively, and there was no cardiac hy- obtained. pertrophy. In addition, marked trabeculae formation was Genetic analysis of the proband and family members: IntHeartJ 4 HIRAYAMA-YAMADA, ET AL Advance Publication

Figure 2. A: Histopathological sections of the right ventricular myocardium from a proband patient, stained with hematoxylin and eosin, showing moderate to severe hypertrophy, disarrangement, and vacuola- tion of cardiomyocytes. The magnified view of the square part in the figure is shown on the right. Scale bar: 50 μm. B: Myocardium stained with Masson trichrome, showing mild endocardial thickening, and moderate perivascular fibrosis. Scale bar: 100 μm.

Mutation analysis of 67 reported cardiomyopathy- low titin expression; however, when differentiated using a associated genes in the patient’s genome identified a low concentration of bovine serum plus ATRA, the ex- frameshift variant, TTN c. 52368_52370 delinsGCAAA, p. pression levels of titin (TTN) and cardiac actin (ACTC1) Ser17456Arg fs* 14. This variant consists of a 3-base de- increased, whereas those of actin (ACTA1) letion, a 5-base insertion, and a frameshift, resulting in a remained low, resulting in an enhanced cardiac phenotype truncation (Figure 4), in the 7-domains super-repeat seg- (Figure 6A). Cardiac T in H9c2 cells were ex- ment (D-zone) of the A-band region of titin (Figure 5). pressed before differentiation (data not shown). This region is evolutionarily conserved, and the variant H9c2 cells with the rat TTN V17602fs truncation was novel because it was not found in multiple genome variant were generated by genome editing. Codon 17602 variation databases including gnomAD, which has accu- in rat titin is a homologous sequence site on the same mulated more than 140,000 individuals from multiple eth- exon as codon 17456 of human titin, and rat TTN V17602 nic groups, and HGVD, which contains data from the fs is considered a homologous truncation variant to human general Japanese population.15,16) The proband’s maternal TTN S17456Rfs found in the proband case (Figure 5). In family members had also experienced heart disease, and addition, as the external control, H9c2 cells with the rat his mother (III-7) developed heart failure in her 40s (Fig- TTN I6334fs (homolog of human TTN V6382fs) were ure 1). The truncation variant was observed in his mother, generated. This variation, located in the titin I-band re- but both the proband’s father (III-4) and brother (IV-2), gion, has been reported to be on an exon with a relatively who did not have heart disease, were confirmed not to low transcription level owing to and is carry the variant (Figure 1). Thus, this TTN truncation considered to be a benign genetic variant11) (Figure 5). variant was co-segregated with the heart failure in his H9c2 cells with the respective truncation variants, family. TTN V17602fs and TTN I6334fs, were selected as het- Functional analysis using a genome-edited cell line: To erozygotes, as was observed in humans, and differentiated investigate the functional alterations associated with the using ATRA. Notably, TTN mRNA expression was ele- TTN truncation variant, a cell model was constructed us- vated in both cell lines (Figure 6B). The mRNA expres- ing genome editing. H9c2 rat cardiomyoblasts exhibited sion of ACTA1, which is usually increased in human heart IntHeartJ Advance Publication TTN TRUNCATION MUTATION IN FAMILIAL DCM 5

Figure 3. Two-dimensional echocardiography from the proband patient at the age of 48 years old, showing the parasternal long-axis view (A) and apical 4-chamber view (B, C). All 4 chambers of the heart were enlarged. Marked trabeculae carneae were observed in the left ventricular apex (circled area).

Figure 4. Nucleotide sequence of TTN from the proband patient. The upper panel shows the results of Sanger se- quencing, and a frameshift was identified (arrow). The middle and lower panels show the sequences of the cloned wild-type and mutant alleles. A 3-base (TGT) deletion followed by a 5-base (GCAAA) insertion resulted in a hetero- zygous frameshift variant, yielding the truncation variant TTN Ser17456Arg fs*14, as shown in the box. IntHeartJ 6 HIRAYAMA-YAMADA, ET AL Advance Publication

Figure 5. Schematic representation of the titin protein with the location of variants form human and rat homolo- gous truncation variants used in this study. The TTN S17456R fs*14 truncation variant was identified in patients with DCM. TTN V6382fs was used as the control.11) TTN I6334fs and TTN V17602fs were selected as heterozygous homol- ogous variants in H9c2 cells. Human and rat amino acid sequences corresponding to the truncation sites are also shown. failure and DCM, was not changed in H9c2 cells carrying truncation variant should be investigated separately.5,12,13) TTN I6334fs compared with wild-type, but was increased Currently identified TTN truncation variants in DCM may in H9c2 cells with TTN V17602fs found in this FDCM explain the differences in terms of alternative splicing. In- (Figure 6B). In addition, the mRNA expression of ACTC1 deed, DCM tends to occur in patients with TTN truncation was not changed in the cells with the TTN I6334fs from variants in regions with a high percent spliced in (PSI) the wild-type, but was decreased in the cells with TTN V value, i.e., the fraction of mRNAs that contain an exon, 17602fs (Figure 6B). and truncation variants found in healthy individuals tend to occur in regions with low PSI values.13,23-25) However, TTN truncation variants in regions of high PSI have also Discussion been reported in healthy individuals, and therefore DCM In this study, we identified a TTN truncation variant, cannot be explained by PSI alone; thus, the pathogenicity TTN Ser17456Arg fs*14 in FDCM, by genetic analysis of of individual truncating mutants should be assessed.13) cardiomyopathy-associated genes. The male proband with In this study, genome editing for truncation variants DCM presented with severe heart failure, and echocar- was performed in rat H9c2 cells, which are inexpensive to diographic findings similar to LVNC were suggestive of a maintain and relatively easy to handle, in order to assess severe form of DCM. The same variant was found in his whether the TTN truncation variant identified in this case mother, who had heart failure, but not in his father or could indeed be associated with the phenotype of DCM or brother, who did not have heart failure, indicating a ge- heart failure. H9c2 cells with the rat TTN V17602fs, netically co-segregated finding. This truncation variant equivalent to the TTN S17456fs found in the FDCM fam- was not found in multiple gene variant databases of the ily, showed increased expression of skeletal actin and de- general population and was considered to be novel. creased expression of cardiac actin. The altered gene ex- Titin, also called connectin, is a large cytoskeletal pression pattern, which increases fetal gene expression, is protein that extends from the Z-disc to the M-line and common in patients with heart failure.26-29) In contrast, H9c contributes to the maintenance of and 2 cells harboring rat TTN I6334fs, homologous to human elasticity and to various signaling pathways.8,19,20) TTN mu- TTN V6382fs, were used as control. This variant has been tations in patients with DCM were found for the first time reported to be present in exons at a relatively low PSI in in the Z-disc and A-band regions; thereafter further ge- the adult myocardium.11,13) In these control cells, expres- netic analysis of TTN was carried out with the develop- sion of TTN was increased, but no significant changes in ment of next-generation sequencing (NGS) technology, in- skeletal or cardiac actin expression were observed. dicating that TTN truncation variants were the most fre- Considering clinical applications, there is a need for quent cause of DCM.5,9,13,21) Although the mechanism for a simple assessment of the pathogenicity for each muta- the development of DCM from titin truncation variants is tion; for example, in secondary cardiomyopathies with still unclear, the major mechanism is thought to involve therapeutics, such as cardiac Fabry disease, the individual nonsense-mediated mRNA decay.11,13,22) However, TTN gene mutations identified were introduced into cells, and truncation variants are also found in approximately 1-2% the potential for therapeutic effects was assessed at the of the healthy population, and the pathogenicity of each cellular level.30) In terms of cell types, it is ideal to induce IntHeartJ Advance Publication TTN TRUNCATION MUTATION IN FAMILIAL DCM 7

Figure 6. A: The mRNA expressions levels of titin (TTN), cardiac actin (ACTC1), and skeletal actin (ACTA1) were measured using qPCR. The expression levels were normalized to those of GAPDH expres- sion. Gene expression levels in undifferentiated H9c2 cells cultured in normal medium (10% FBS) were used as a control, and differentiated H9c2 cells cultured in medium with reduced FBS concentrations plus all-trans-retinoic acid (ATRA; 1% FBS + ATRA) are shown. Expression levels of each mRNA are presented as the relative quantification (RQ) values relative to the control levels (means ± SEMs (error bars); n = 3). Statistical differences were examined using the unpaired and Welch’s t-tests (*P < 0.05, **P < 0.01). B: mRNA expression of TTN, ACTC1, and ACTA1 in H9c2 cells with wild-type, or with the heterozygous TTN truncation variants, I6334fs and V17602fs, after differentiation. The expression levels were normalized to GAPDH expression and are presented relative to that in the wild-type cell (WT). Data are presented as the mean ± SEM (error range); n = 3, except for the expression of ACTA1 in TTN V17602fs (n = 2). Statistical differences were tested using the unpaired and Welch’s t-tests (*P < 0.05, **P < 0.01). the differentiation from human-induced pluripotent stem be used for evaluation of human TTN truncation vari- cells or other stem cells; however, the cost and time for ants.25) This approach could be used as a convenient differentiation in culture are problematic. In rats, TTN screening method if cells with mutations equivalent to truncation mutation models had the phenotype of heart those of humans would be generated to evaluate their failure and a cell line from rat origin, such as H9c2, may pathogenicity with a limited number of heart failure- IntHeartJ 8 HIRAYAMA-YAMADA, ET AL Advance Publication associated markers. functional spring. 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