Targeted panel sequencing in pediatric primary cardiomyopathy supports a critical role of TNNI3

The underlying genetic mechanisms and early pathological events of children with primary cardiomyopathy (CMP) are insufficiently characterized. In this study, we aimed to characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel‐based next‐generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation—not only in adult—but also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP.


Peer Review
The peer review history for this article is available at https://publons.com/publon/10. 1111/cge.13645. characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel-based next-generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation-not only in adultbut also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP.

| Clinical evaluation
Unrelated patients with CMP were recruited at the Charité -Universitätsmedizin Berlin and the German Heart Center Berlin, Berlin, Germany between November 2011 and February 2017 (study design: Figure S1). Written informed consent was obtained from each subject or their legal guardians according to the Declaration of Helsinki.
Probands ≤18 years and available family members were evaluated by medical history, physical examination, 12-lead electrocardiography, and transthoracic echocardiography. For patients recruited after heart transplantation (HTX), the clinical and echocardiographic data were collected by retrospective review of patients' records. Patients with a primary diagnosis of HCM, DCM, RCM, LVNC, or ARVC were included in the study. 4,5 Patients who had secondary CMP (syndromic, neuromuscular, metabolic, or myocarditis) or CMP in combination with a structural congenital heart defect were excluded. ARVC was not included in the analysis of subgroups.

| Targeted NGS
NGS was carried out in genomic DNA samples isolated from patient blood samples with the NucleoSpin Blood kit according to the manufacturer's protocol (Macherey-Nagel, Germany). DNA quantification occurred with a Qubit 3 fluorometer using appropriate Qubit dsDNA reagents (Thermo Fisher Scientific, Invitrogen).
NGS was performed with a panel-based approach using the Illumina TruSight Cardio Sequencing Kit (Illumina). 6 With this approach, exonic regions of 174 cardiovascular disease genes specifically implicated in structural heart disease and arrhythmias were sequenced (https://support.illumina.com/sequencing/sequencing_ kits/trusight-cardio-sequencing-kit.html). Library preparation and enrichment was performed according to the manufacturer's protocol. Quantification of DNA libraries was performed with a Bioanalyzer 2100 using the high sensitivity DNA kit or DNA 1000 kit (Agilent Technologies). Patient DNA libraries were sequenced on a NextSeq 500 platform with mid output cartridge v2 with paired end sequencing (150 cycles) and dual indexing. Sanger sequencing was applied to validate variants detected by NGS in index patients and in family members for segregation analysis. Sanger sequencing was performed according to standard laboratory protocols. 7 The primer for genomic amplification of cardiac troponin I (TNNI3) polymerase chain reaction (PCR) products and Sanger sequencing is available in Table S1.

| Bioinformatic evaluation and variant classification
The called variants were evaluated with Variant Studio (Illumina) for their minor allele frequency (<0.001) and mutation specification. We used Genome Aggregation Database (gnomAD) as genetic reference database for unaffected individuals (http://gnomad. broadinstitute.org/). 12 We evaluated 89 CMP disease genes that have been ascertained in a series of patients, preferably including family segregation data; CMP genes suggested from single case reports were excluded (Table S2) database, ClinVar. 16 The most recent variant classification was used in the final analysis (https://www.ncbi.nlm.nih.gov/clinvar/). 17 For functional classification, the 89 genes evaluated in this study were arranged into functional groups (Table S2). 18

| Protein and mRNA expression analysis
Age-matched human ventricular biopsies obtained at time of cardiac surgery (age range 5 months to 2.5 years) were subjected to protein and total RNA isolation after liquid nitrogen treatment and mechanical crashing. Powdered heart tissue was subjected to RNA isolation with Trizol reagent (Invitrogen, Carlsbad, CA) and protein lysates were obtained after incubation with RIPA buffer. Protein samples were analyzed by Western blot after sodium dodecylsulfate polyacrylamide gel electrophoresis and transfer onto polyvinylidene difluoride membrane For mRNA analysis, total RNA was isolated according to standard protocols and transcribed into cDNA with SuperscriptII (Invitrogen).
Quantitative PCR (qPCR) was performed on a Taqman 7500 (Applied Biosystems) using GAPDH as endogenous control. The relative mRNA level was calculated according to the ΔΔCT method. The primers used for qPCR are listed in Table S1.

| Patient characteristics
During the study period, 80 individuals were recruited at our tertiary center in the pediatric cardiology clinics (German Heart Center and Charité -Universitätsmedizin Berlin, both in Berlin) with a diagnosis of primary CMP at age ≤ 18 years. The cohort of unrelated Caucasian individuals (42 males and 38 females; mean age at diag-  Table S3). The largest group was composed of DCM patients (43%) of which 50% underwent HTX. In addition, the DCM group had the highest rate of implanted ventricular assist device systems (47%), extracorporeal membrane oxygenation systems (15%), and death (6%). Within the whole cohort, one-third of patients (n = 26; 33%) already had a HTX at the time of enrollment or underwent HTX during the study period ( Figure 1D).

| Spectrum of detected rare variants in pediatric primary CMP
To identify a potential genetic defect, we screened all index patients with a panel-based NGS approach detecting genetic variants in 89 CMP-associated genes. In 46 of these 89 genes, we identified at least one rare variant. In total, 126 detected variants were classified as pathogenic (n = 17, 14%), probably pathogenic (n = 15, 12%), or VUS (n = 94, 74%) ( Table 2). Forty-two percentage (53/126) of all variants were novel and not annotated in disease reference databases; novel variants were mostly classified as VUS (42/53). One hundred three Missense variants were found in >80% of cases (103/126) and 37 of them were novel (37/103). In addition, 9 indel/frameshift, 3 stop gain, and 11 splice site rare variants were detected. From 75/126 rare variants, for which inheritance was tested in the parents, 7 (10%) arose de novo and 5/7 were classified as pathogenic and 2/7 were probably pathogenic. A detailed summary of all VOI is supplied in

| TTN variants
VOI inducing truncations of the TTN protein (n = 4, 3% of all VOI) were rarely found and occurred in three RCM as well as in one DCM patient, two probably pathogenic, and two VUS (Table S5). In RCM patients, three TTN splice-site variants were identified in three individuals, respectively, two VUS and one probably pathogenic ( Figure 2E).

| Homozygous loss of TNNI3 protein is compensated by upregulation of fetal TNNI1
Within the pediatric CMP cohort, VOI were most frequently  Table S6).
The TNNI3 protein is composed of a cardiac specific region, the IT arm region, and the flexible C-terminal tail ( Figure 4A). 20 All heterozygous TNNI3 missense variants detected in this study cluster in exon 8 ( Figure 4B). To further explore the molecular consequences of the truncating TNNI3 variants (c.240delG, p.Arg69Alafs*8; c.24+2T>A; c.624dupT, p.Glu209*), we determined mRNA and protein levels in heart biopsies. The TNNI3 protein is absent in patients 1-II:1 and 2-II:3 ( Figure 4C,D). Of note, the protein level of TNNI1, the fetal TNNI variant, is markedly higher in patients 1-II:1 and 2-II:3. The TNNI3 mRNA level is markedly reduced in patient 1-II:1, suggesting degradation by non-sense mediated decay, or not different from controls in patient 2-II:3 ( Figure 4E). Levels of the sarcomeric proteins TNNT2 and MYBPC3 were not significantly affected. The TNNI3 protein level in a heart biopsy of patient 3-II:1 (p.E209*) was normal ( Figure S6A-C). In contrast, the TNNI3 and TNNI1 transcript levels were higher in heart tissue of patient 3-II:1.

| DISCUSSION
In this study, we report the genetic basis of a cohort of 80 pediatric patients with pediatric primary CMP referred to a tertiary center. VOI were most frequently detected in the MYH7, MYBPC3, TNNI3, and DSP genes. TNNI3 is the third most important disease gene in this cohort and complete loss of myocardial TNNI3 was associated with severe CMP and a compensatory increase in TNNI1.
Our study serves to assess the impact of genetic testing for counseling and management of pediatric primary CMP patients, and it shows wide genetic heterogeneity, depending on the stringency of phenotypes included in the cohort. 2 The strength of this pediatric study is that it included only patients with primary CMP.

| Spectrum of genetic variants in pediatric primary CMP
In the entire cohort, we mainly identified patients with DCM and HCM, which is consistent with the established incidence of pediatric CMP. 5 Less frequently, we detected patients with LVNC and RCM.
F I G U R E 3 Families with CMP and mutation of TNNI3. A-F, Pedigrees of all families with rare genetic TNNI3 variants. Open symbols depict unaffected individuals and filled symbols affected family members. The phenotype is indicated for each pedigree. The TNNI3 genotype is presented for each pedigree, with the mutated allele marked as mut and the reference allele as wt. Individuals without genotype were not available for genetic testing. CMP, cardiomyopathy Over all CMP subtypes, genetic variants were most frequently detected in sarcomeric genes (MYH7, MYBPC3, TNNI3) followed by desmosomal (DSP) and z-disc genes (LDB3, MYPN).

| Pediatric CMP: Implications for genetic testing?
It is a matter of ongoing debate whether whole genome sequencing (WGS) or whole exome sequencing (WES) in CMP would be necessary to provide more insight into the genetic disease mechanisms. 42 The incremental yield of clinically actionable variants by WGS is limited by a paucity of genetic and functional evidence. 43  In summary, with an NGS panel-based approach, we identified the genetic cause in 38% of pediatric patients with primary CMP. Most frequently, we detected pathogenic or probably pathogenic missense variants in sarcomere genes such as MYH7, MYBPC3, and TNNI3.
Homozygous deactivation of TNNI3 leads to severe CMP in pediatric patients and compensatory expression of TNNI1. We propose that this study advances the general genetic understanding of pediatric primary CMP and highlights certain genetic defects with severe clinical courses.