Skip to main content
PLOS ONE logoLink to PLOS ONE
. 2021 Feb 3;16(2):e0245681. doi: 10.1371/journal.pone.0245681

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy

Juha W Koskenvuo 1,*, Inka Saarinen 1, Saija Ahonen 1, Johanna Tommiska 1, Sini Weckström 2, Eija H Seppälä 1, Sari Tuupanen 1, Tiia Kangas-Kontio 1, Jennifer Schleit 1, Krista Heliö 1, Julie Hathaway 3, Anders Gummesson 4, Pia Dahlberg 5, Tiina H Ojala 6, Ville Vepsäläinen 7, Ville Kytölä 1, Mikko Muona 1, Johanna Sistonen 1, Pertteli Salmenperä 1, Massimiliano Gentile 1, Jussi Paananen 1, Samuel Myllykangas 1, Tero-Pekka Alastalo 3, Tiina Heliö 2
Editor: Amanda Ewart Toland8
PMCID: PMC7857588  PMID: 33534821

Abstract

Background

Familial dilated cardiomyopathy (DCM) is typically a monogenic disorder with dominant inheritance. Although over 40 genes have been linked to DCM, more than half of the patients undergoing comprehensive genetic testing are left without molecular diagnosis. Recently, biallelic protein-truncating variants (PTVs) in the nebulin-related anchoring protein gene (NRAP) were identified in a few patients with sporadic DCM.

Methods and results

We determined the frequency of rare NRAP variants in a cohort of DCM patients and control patients to further evaluate role of this gene in cardiomyopathies. A retrospective analysis of our internal variant database consisting of 31,639 individuals who underwent genetic testing (either panel or direct exome sequencing) was performed. The DCM group included 577 patients with either a confirmed or suspected DCM diagnosis. A control cohort of 31,062 individuals, including 25,912 individuals with non-cardiac (control group) and 5,150 with non-DCM cardiac indications (Non-DCM cardiac group). Biallelic (n = 6) or two (n = 5) NRAP variants (two PTVs or PTV+missense) were identified in 11 unrelated probands with DCM (1.9%) but none of the controls. None of the 11 probands had an alternative molecular diagnosis. Family member testing supports co-segregation. Biallelic or potentially biallelic NRAP variants were enriched in DCM vs. controls (OR 1052, p<0.0001). Based on the frequency of NRAP PTVs in the gnomAD reference population, and predicting full penetrance, biallelic NRAP variants could explain 0.25%-2.46% of all DCM cases.

Conclusion

Loss-of-function in NRAP is a cause for autosomal recessive dilated cardiomyopathy, supporting its inclusion in comprehensive genetic testing.

Introduction

Dilated cardiomyopathy (DCM) is characterized by left ventricular enlargement and systolic dysfunction in the absence of other etiological causes [1]. It is typically an adult-onset disease but disease onset may take place as early as in infancy. Genetic DCM has incomplete, age-dependent penetrance and presentation may vary even within the same family ranging from asymptomatic to end-stage heart failure and sudden cardiac death (SCD). The prevalence of DCM in the general population is estimated int the range of 1:500 to 1:3,000 [24].

Familial DCM is typically considered to be a monogenic disorder following most commonly an autosomal dominant pattern of inheritance [1,2,5]. However, X-linked, recessive and mitochondrial inheritance patterns have been observed [6]. As much as 30–50% of DCM is thought to be genetic or familial [6,7]. Over 40 genes encoding proteins of cytoskeleton, sarcomere, nuclear envelope, ion channels, and intercellular junction such as TTN, LMNA, MYH7, FLNC, DSP, TNNT2, RBM20, DES, TPM1 and DMD contribute to the monogenic forms of DCM [711].

Recently, biallelic protein-truncating variants (PTVs) in the nebulin-related anchoring protein gene (NRAP) have been identified in a few patients with severe sporadic DCM [1215], and have been proposed to cause low-penetrant recessive DCM (Table 1). However, two healthy individuals (age 33 and 35) in these families had the same homozygous PTV, which was considered to partially question the variants’ pathogenicity. NRAP is not yet officially a morbid OMIM gene and has not yet been curated by ClinGen (NIH) or Genomics England PanelApp [16]. Thus, it is absent from most commercially available gene panels at the moment.

Table 1. Previously reported patients with biallelic truncations in NRAP.

Chromosomal position Transcript; exon Variant GnomAD allele frequency Pheno-type Age at onset (years)gender LVEDD (mm) EF (%) Other clinical features References
1 10:115355414 NM_198060.3 Exon 38/42 c.4504 C>T, p.(Arg1502*)# 92/282804 DCM 26 M 71 15 Biventricular heart failure after prolonged viral-like illness, ventricular tachycardia, CK 68 U/l (normal: 39–308), NT-proBNP 5154 pg/ml, and TnT 15.04 ng/ml. 36- year old brother# is healthy. Truszkowska 2017 (12)
2 10:115413838–115413845 NM_001261463 Exon 5/42 c.400_407del,p.(Cys134Serfs*12)# 0/251404 DCM NA NA NA NA Monies 2017 (13)
3 10:115400070 NM_001261463 Exon 14/42 c.1344T>A, p.(Tyr448)*# 34/263614 DCM 3.5 F >3SD 15 DCM after mild respiratory viral infection, suspected myocarditis, LVEDV 245ml/m2. Died before planned Htx. In autopsy no signs of myocarditis. Vasilescu 2018 (14)
4 10:115413838–115413845 NM_001261463 Exon 5/42 c.400_407del,p.(Cys134Serfs*12)# 0/251404 DCM 1 F 15 Cardiac arrest. CK 163 U/l (normal 26–168), Brother died at 17 months for cardiomyopathy, genotype unknown. Father# is healthy Ahmed 2019 (15)

Abbreviations: DCM, dilated cardiomyopathy; F, female; GnomAD; Allele frequency in gnomAD, Htx, heart transplantation; LVEDD; Left ventricular end-diastolic diameter; M, male; NA, not available; Pheno, phenotype

#, homozygous. No homozygotes for these variants are present in the gnomAD reference population cohort.

Since both enrichment and co-segregation of NRAP variants in DCM are unknown, our aims were to 1) evaluate whether patients who underwent genetic testing due to DCM have a higher frequency of NRAP variants compared to controls, 2) to study co-segregation of the NRAP variants, and 3) to define genotype-to-phenotype associations in NRAP-associated cardiomyopathy.

Materials and methods

Patients

The cohort represents 31,639 consecutive patients referred to genetic testing relying either on whole exome sequencing platform (WES; n = 24,630) or 4,600 gene high-quality next generation sequencing assay (HQSA; n = 7009) after January 2017. The inclusion criteria for DCM group (see later) was referral to genetic testing due to diagnosis or clinical suspicion of DCM.

This registry study complies with the Declaration of Helsinki. Patients who consented for Blueprint Genetics to contact them in relation to future research findings after initial testing, were contacted through their referring healthcare professional when possibly diagnostic biallelic variants in NRAP gene were found in sequence data. Patients living in the Helsinki University Hospital (HUS) region in Southern Finland were recruited to the Inherited Cardiomyopathies Study or KidCMP Study, and segregation studies were carried out when possible. Participants of the Inherited Cardiomyopathies study gave written informed consent, and the study was approved by the Ethical Review Committee of The Department of Medicine, University of Helsinki (Dnro 307/13/03/01/2011, HUS/3225/2018, TMK11§274,16.12.2015). This study has permission from Statistics Finland and Ministry of Social Affairs and Health to obtain clinical data from deceased patients for research purposes.

Sequencing

Sample preparation including DNA isolation, fragmentation, library preparation techniques, bioinformatics, and quality control were similar for both WES and HQSA.

When required, the total genomic DNA was extracted from the biological sample using bead-based method. DNA quality and quantity were assessed using electrophoretic methods. After assessment of DNA quality, qualified genomic DNA sample was randomly fragmented using non-contact, isothermal sonochemistry processing. Sequencing library was prepared by ligating sequencing adapters to both ends of DNA fragments. Sequencing libraries were size-selected with bead-based method to ensure optimal template size and amplified by polymerase chain reaction. Regions of interest (exons and intronic targets) were targeted using hybridization-based target capture method. The quality of the completed sequencing library was controlled by ensuring the correct template size and quantity and to eliminate the presence of leftover primers and adapter-adapter dimers. Ready sequencing libraries that passed the quality control were sequenced using the Illumina's sequencing-by-synthesis method using paired-end sequencing (150 by 150 bases). Primary data analysis converting images into base calls and associated quality scores was carried out by the sequencing instrument using Illumina's proprietary software, generating CBCL files as the final output.

Base called raw sequencing data was transformed into FASTQ format using Illumina's software (bcl2fastq). Sequence reads of each sample were mapped to the human reference genome (GRCh37/hg19). Burrows-Wheeler Aligner (BWA-MEM) software was used for read alignment. Duplicate read marking, local realignment around indels, base quality score recalibration and variant calling were performed using GATK algorithms (Sentieon) for nuclear DNA. Variant data was annotated using a collection of tools (VcfAnno and VEP) with a variety of public and private variant databases including but not limited to gnomAD, ClinVar and HGMD. The median sequencing depth and coverage across the target regions for the tested sample were calculated based on MQ0 aligned reads. The sequencing run included in-process reference sample(s) for quality control, which passed our thresholds for sensitivity and specificity. The patient's sample was subjected to thorough quality control measures including assessments for contamination and sample mix-up.

Analysis in this study was limited to single-nucleotide variants, and small insertions/deletions and their combinations (INDELs) up to 220 bps within protein coding exons and exon-intron boundaries (± 20 bps). Copy number variations were excluded from the analysis. Performance metrics were as follows: WES: Median coverage 174x, >20x depth at target region 99.4%, >20x depth at NRAP gene 100%, sensitivity for SNVs 99.65%, indels 1–50 bps 99.1%, and specificity >99.9% and HQSA: median coverage 143x, >20x depth at target region 99.86%, >20x depth at NRAP gene 100%, sensitivity for SNVs 99.89%, indels 1–50 bps 99.2% and specificity >99.9%). Both assays have been validated in a CAP and ISO accredited laboratory (Blueprint Genetics, Finland).

NRAP variants

Since our aim to evaluate the role of potentially disease causing NRAP variants, the analysis was limited only to the variants with the highest potential to cause disease, specifically PTVs (nonsense, frameshift, canonical splice site, start lost) and missense variants as most of the synonymous and intronic variants are less likely to be disease causing. In addition, variants were included into further analysis only if no homozygous carriers were present in the Genome Aggregation Database control cohort (gnomAD) [17] and missense variants with 100 or less heterozygous individuals in gnomAD. Frequency of such high-quality variants were compared between patients with clinical or suspected dilated cardiomyopathy (DCM group), other cardiology indication (Non-DCM cardiac group consisting patients tested due diagnosis or suspicion inherited aortopathy, channelopathy or cardiomyopathy other than DCM) or any other clinical indication for panel or exome testing (Control group).

Statistics

Comparisons between groups were performed with either Fisher’s exact or Chi-Square test for categorical variables and unpaired T-test for normally distributed continuous variables. Odds ratios (ORs) for DCM and non-DCM cardiac group vs. control group were calculated, and 95% confidence intervals (CIs) were determined using the conditional maximum likelihood/Fishers’ method. Normally distributed parameters are presented as mean ± standard deviation.

Results

Whole exome sequencing (WES) data set and NRAP variants

All variant calls from the NRAP gene were queried from the internal variant database in 31,639 individuals who underwent genetic testing using NGS-panels or direct WES approach. Of these patients, 577 were tested due to DCM or suspected DCM (DCM group), 5,150 due to suspicion of other monogenic cardiac disease (Non-DCM cardiac group) and 25,912 served as controls (control group).

Enrichment of NRAP variants in DCM

We identified cases with two rare NRAP variants, of which at least one was a PTV in 11 out 577 (1.91%) patients in the DCM group but none were in either the non-DCM cardiac group or control group (Table 2). Frequency of such variant combination was significantly greater in the DCM group vs. controls (OR 1052, 95%CI 62–17876, p<0.0001; Table 3). Three of the patients had familial cardiomyopathy and eight had a sporadic disease. In these 11 individuals, four had a homozygous PTV, one had two heterozygous PTVs (phase unknown) and two were compound heterozygous for a PTV and a missense variant (in trans). In five patients, the phase of the NRAP variants was unknown. Two presumably unrelated patients had the same frameshift/missense variant combination (c.4371del, p.Thr1458Glnfs*36 and c.72G>C, p.Gln24His) and two had the same nonsense/missense variant combination (c.4504C>T, p.Arg1502* and c.72G>C, p.Gln24His). Thus, the p.(Gln24His) missense variant was observed altogether in four presumably unrelated patients. This variant may in fact lead to splicing defect as it affects the last nucleotide of the exon 1. Alamut Visual Splicing software v2.11 (Interactive Biosoftware, France) predicts that this variant either leads to loss of the native splice donor (NNSPLICE) or significant weakening of this site (SSF, MaxEnt). One patient had a start lost variant, NRAP p.(Met1?), which is expected to cause loss-of-function as there is an alternative out-of-frame start codon 5-bp down-stream from the wild type initiation codon.

Table 2. NRAP variants observed in the patients with dilated cardiomyopathy.

Patient Variants HGVS nomenclature Variant type Exon gnomAD AC SIFT Cons. ACMG Class
1 10:115374685 c.3099G>A, p.(Trp1033*) Nonsense 28/42 18 LP
2 10:115356904 c.4371del, p.(Thr1458Glnfs*36) Frameshift 37/42 100 P
10:115423570 c.72G>C, p.(Gln24His) Missense 1/42 34 Delet. (0.01) Full P
3 10:115356904 c.4371del, p.(Thr1458Glnfs*36) Frameshift 37/42 100 P
10:115423570 c.72G>C, p.(Gln24His) Missense 1/42 34 Delet. (0.01) Full P
4 10:115400070 c.1344T>A, p.(Tyr448*) Nonsense 14/42 35 Delet. (0.01) Full P
10:115423593 c.49G>A, p.(Glu17Lys) Missense 1/42 5 VUS
5 10:115356904 c.4371del, p.(Thr1458Glnfs*36) Frameshift 37/42 100 P
10:115355414 c.4504C>T, p.(Arg1502*) Nonsense 38/42 95 P
6 10:115423570 c.72G>C, p.(Gln24His) Missense 1/42 34 Delet. (0.01) Full P
10:115355414 c.4504C>T, p.(Arg1502*) Nonsense 38/42 95 P
7 10:115423570 c.72G>C, p.(Gln24His) Missense 1/42 34 Delet. (0.01) Full P
10:115355414 c.4504C>T, p.(Arg1502*) Nonsense 38/42 95 P
8 10:115356904 c.4371del, p.(Thr1458Glnfs*36) Frameshift 37/42 100 P
9 10:115374675 c.3109C>T, p.(Arg1037*) Nonsense 28/42 2 LP
10 10:115364570 c.4025G>A, p.(Ser1342Asn) Missense 35/42 3 Delet. (0.01) Full VUS
10:115423640 c.2T>C, p.(Met1?) Start lost 1/42 21 LP
11 10:115400070 c.1344T>A, p.(Tyr448*) Nonsense 14/42 35 P

Genomic coordinates refer to human reference genome (GRCh37/hg19) and mutation nomenclature is based on GenBank accession NM_001261463.1 (NRAP). Homozygotes and compound heterozygous patients are marked with bold font. Cons, Conservation in mammals; Delet., Deleterious; GnomAD AC, Allele count in gnomAD reference population cohort; LP, Likely pathogenic; P, Pathogenic; VUS, Variant of Uncertain Significance. No homozygotes for these variants are present in the gnomAD reference population cohort.

Table 3. Enrichment of rare NRAP variants in patients with dilated cardiomyopathy (DCM).

Control group Non-DCM cardiac group OR (95% CI), P-value DCM group OR (95% CI), p-value
Individuals (n) 25912 5150 577
Dominant hypothesis
Only one PTV variant 75 (0.29%) 24 (0.47%) 1.61 (1.02–2.56), p = 0.04 11 (1.91%) 6.71 (3.5–12.7), p<0.0001
Only one missense variant 698 (2.45%) 132 (2.56%) 1.05 (0.86–1.26), p = 0.65 10 (1.74%) 0.70 (0.4–1.3), p = 0.27
Recessive hypothesis
Two missense variants 27 (0.10%) 1 (0.02%) 0.18 (0.02–1.35), p = 0.10 0 (0.0%) 0.81 (0.05–13.4), p = 0.89
One PTV + one missense 0 0 NA 6 (1.04%) 590 (33–10494), p<0.0001
Two PTV variants 0 0 NA 5 (0.87%) 407 (22–7575), p<0.0001
PTV + missense or 2 PTVs 0 0 NA 11 (1.91%) 1052(62–17876), p<0.0001

Control group patients underwent genetic testing due to non-cardiac reasons and non-DCM group patients due to cardiological reasons excluding patients with DCM or suspected DCM. DCM group includes patients tested with a DCM Panel or other panels because of confirmed or suspected DCM. Abbreviations: pts, patients; OR, odds ratio; 95% CI, 95% confidence interval, DCM, dilated cardiomyopathy, PTV, Protein-truncating variant (means here nonsense, frameshift variant, consensus splice site, start lost). Only variants with 100 or fewer heterozygous individuals in a gnomAD reference population cohort were included in calculations.

None of these 11 patients had an alternative molecular diagnosis identified in either large NGS panel (n = 9) or exome sequencing (n = 2). Six (55%) of the patients have had major endpoints, defined as history of cardiac transplantation (n = 2), death on waiting list for heart transplantation (n = 1) or during left ventricular assist device (LVAD) treatment (n = 2), and previous cardiac arrest (n = 1). The mean age at the time of the major endpoint was 22.8±19.4 years (Table 4). Four of these six patients had homozygous PTV in NRAP and one patient had two PTVs (phase unknown). Patients with two PTVs (n = 5) were younger at disease onset than patients with PTV + missense variant (n = 6) combination (19.6±20.4 vs. 48.3±12.3 years, p = 0.018). None of the patients had known skeletal muscle involvement.

Table 4. Clinical characteristics of patients with biallelic or potentially biallelic NRAP variants.

Patient Age Gender Phenotype LVEDD(mm)/EF% Age at onset Htx Died Other
1 19 F DCM NA <19 Yes, at age of 19
2 36 F DCM 71/10-15% NA 36
3 31 M DCM 70/13% 28 Mild LGE
4 57 M DCM NA 56 Maximum treatment for heart failure
5 4 F DCM 58/20% 4 LVAD
6 59 F DCM 63/34% 46
7 59 M DCM NA/30% NA LBBB
8 43 M DCM NA 22 Yes, at age 43
9 53 F DCM NA NA History of cardiac arrest
10 48 M DCM/HF NA NA
11 2 M DCM NA NA 2

Age means age at last follow-up. Abbreviations: DCM, Dilated cardiomyopathy; EF, Ejection fraction; F, Female; HF, Heart failure; Htx, Heart transplantation; LBBB, Left bundle branch block; LGE, Late gadolinium enhancement at cardiac MRI; LVEDD, Left ventricular end-diastolic diameter in mm; M, Male; NA, Not available or not known.

A single heterozygous PTV without another rare NRAP variant was observed in 11 patients (1.91%) and they were also enriched in the DCM group (OR 6.71, 95% CI 3.5–12.7, p<0.0001; Table 3). The single heterozygous PTV group excludes all patients with two rare NRAP variants as defined earlier. However, one of these patients also had another moderately rare (500 heterozygotes in gnomAD) missense variant in NRAP (c.2963A>C, p.(Gln988Pro); phase unknown) in addition to a start-lost variant. The patient had no alternative molecular diagnosis in established cardiomyopathy genes. Of the 11 patients with only one heterozygous PTV in NRAP, three had another molecular diagnosis including three PTVs affecting A-band of TTN and one had an additional frameshift variant in DSP.

Familial segregation

We were able to recruit two out of the three probands with familial disease and one with sporadic disease for additional screening. Co-segregation was assessed from 18 family members who underwent screening of familial variants and clinical history, as well as and clinical evaluation including ECG and echocardiography. Cardiac MRI was performed as needed.

In family 2, the proband died at the age of 38 years from severe biventricular heart failure. She was compound heterozygous for c.4371del, p.(Thr1458Glnfs*36) and c.72G>C, p.(Gln24His) in NRAP (Table 2, Fig 1). At the time of last imaging study, her LVEDD was 71 mm, LVEF was 13% and RVEF was 17%, and she had elevated levels of TnI and proBNP and a widened QRS (132 ms). One of the proband’s brothers was diagnosed with DCM at the age of 24 years and he died at age of 34 years of severe biventricular heart failure. No DNA sample was available from this individual for genetic testing. Two of the family members were compound heterozygous for the same variants. One had a diagnosis of mild DCM at age 20 and no progression since initiating ACE inhibitor treatment, and the other had upper normal LV size despite of treatment initiation at the age of 21 years. All five heterozygous siblings and one with wild type allele were healthy. The parents of the proband were both heterozygous for one the variants and had normal echocardiography.

Fig 1.

Fig 1

Pedigree of the family-2 where the index patient and her affected brother were compound heterozygous for c.4371del, p.(Thr1458Glnfs*36) and c.72G>C, p.(Gln24His) in NRAP similarly as her 21-year brother who were on medication initiated before the results of genetic testing were available due to borderline imaging findings suggesting cardiomyopathy. He did not fulfill diagnostic criteria of DCM at the time of the study. DNA was not available from one affected individual who died for DCM at age of 34. All family members who were heterozygous only for the other variant or were homozygous for the wild type allele were unaffected.

In family 6, the proband was diagnosed with DCM at the age of 47 years due to dilated LV and reduced LV function (LVEDD 63mm, EF 34%). She was compound heterozygous for c.4504C>T, p.(Arg1502*) and c.72G>C, p.(Gln24His) in NRAP (Table 2, Fig 2). Mild improvement in LV size and function were observed with medical treatment. The proband’s sister died at the age 40 years due to DCM. She was an obligate compound heterozygote for the same variants as the proband, which was discovered after the testing of her children. Three of the proband’s siblings have died during childhood, but no samples were available from any of them for genetic testing. In the extended family, two heterozygous individuals and two with wild type alleles were healthy. The proband’s parents, who were both obligatory heterozygotes for one variant, had no known cardiomyopathy and had a normal life span.

Fig 2. Pedigree of the family-6 where the index patient and her affected brother were compound heterozygous for c.4504C>T, p.(Arg1502*) and c.72G>C, p.(Gln24His) in NRAP.

Fig 2

All family members who were heterozygous only for the other variant were unaffected.

In family 11, the proband was diagnosed with DCM at age 2 due to dilated LV and reduced LV function. The patient was homozygous for c.1344T>A, p.(Tyr448*) in NRAP (Table 2, Fig 3). He received LVAD soon after hospitalization due to severe heart failure but he died before planned transplantation. The proband’s parents were heterozygotes for the variant and his older sister was homozygous for a wild type allele. All family members were healthy.

Fig 3. Pedigree of the family-11 where the index patient was homozygous for c.1344T>A, p.(Tyr448*) in NRAP whereas all other family members who were heterozygous for the variant or had homozygous wild type allele were unaffected.

Fig 3

Estimating frequency of biallelic protein truncating NRAP variants in general population

As we discovered significant new evidence supporting the role of biallelic NRAP variants in DCM, we decided to further estimate the potential contribution of this gene on DCM at a global scale. We queried the count of NRAP-PTV in gnomAD reference population v2.1.1. In total 733 high-quality PTVs were present in the database. The average number of alleles reported at these positions was 233,756 indicating a cumulative allele frequency of 0.31%. Thus, the probability of homozygosity or compound heterozygosity is approximately 0.000983% at the individual level. This is equal to 1 case per 101,700 individuals if we assume that only PTV variants would be disease causing.

Discussion

Our data suggest that the variants in the NRAP gene are associated with DCM and may explain up to 1.91% of DCM cases in an unselected clinical cohort consisting of patients with either clinically diagnosed DCM or suspected DCM. Because small disease cohorts may provide inaccurate estimates due to non-random inclusion and pure coincidence, we decided to estimate the prevalence of potentially biallelic NRAP-PTV in a large population data set (gnomAD). This analysis yielded a frequency of 0.000983%, equal to 1 case per 101,700 individuals. If all of these variants were fully penetrant, NRAP might explain up to 0.34%-2.03% of all DCM cases when relying on variable (1:3,000 to 1:500) estimates of DCM prevalence in the general population. Thus, our DCM patient cohort and population data cohort provide essentially similar estimates of the contribution of NRAP in DCM.

In general, non-syndromic familial cardiomyopathies follow dominant inheritance [1,18]. In 2016, the ALPK3 gene was discovered to be a rare cause of a recessive pediatric cardiomyopathy, which typically presents with DCM and non-compaction that progress to hypertrophic cardiomyopathy and possibly some syndromic features [19]. Some other genes such as GATAD1 [20], PLEKHM2 [21], and PPCS [22] have been shown to associate with recessive non-syndromic DCM. However, not much evidence has been gathered after initial reports, possibly reflecting the rarity of such gene-to-phenotype associations. Classic genes encoding cardiac desmosome proteins initially connected to ARVC/arrhythmogenic cardiomyopathy are now considered established causes of the DCM phenotype [10,23]. Notably, some variants in DSG2 gene cause recessive ARVC that may be difficult to distinguish from DCM [24]. However, based on the numbers of reported patients and mutation database submissions (e.g. ClinVar) of patients carrying variants in previously described recessive cardiomyopathy genes, it seems likely that NRAP has a more prominent contribution to the etiology.

Previous reports involving NRAP gene did not include segregation analysis [13] had insufficient data obtained from the family studies to fully support segregation or were inconsistent with co-segregation [12,14,15]. In the first study suggesting association of NRAP with cardiomyopathy, the proband’s 35-year-old brother who was homozygous for PTV in NRAP was considered unaffected while being asymptomatic and having normal echocardiography and ECG [12]. Thus, the authors concluded that NRAP may be a low penetrance genetic risk factor for DCM even though the previous observation can also be explained by age-dependent penetrance of cardiomyopathies. Later, Ahmed et al. published a consanguineous pedigree in which the index patient was a baby girl who presented at the age of 13 months with heart failure, easy fatigability, weakness, irritability, and shortness of breath and was diagnosed with DCM [15]. Whole exome sequencing revealed that her healthy 33-year-old father was homozygous for the same frameshift variant identified in the proband whereas the mother was heterozygous. The proband’s family history included one stillbirth and another brother who was diagnosed with cardiomyopathy at the age of 12 months and died at 17 months without a molecular diagnosis (samples were not available for genetic testing). Otherwise the extended pedigree did not reveal any known cardiomyopathy cases, which also suggests recessive inheritance. Our previous study reported a family in which the index patient who was diagnosed with DCM at the age of 3 years [14]. The proband was homozygous for NRAP p.(Tyr448*). Three family members were heterozygous for the variant and one had a homozygous wildtype allele, and all of them were considered healthy. Individuals who are heterozygous for a single LoF variant in NRAP are cardiologically healthy in all previously published reports as well as in our study suggesting that NRAP does not cause dominantly inherited monogenic DCM. However, we cannot exclude the possibility that it would increase susceptibility to cardiomyopathy even when heterozygous due to observed enrichment of single LoF variants in our DCM cohort.

NRAP seems to associate with severe DCM as the proportion of patients with major cardiac endpoint (death, cardiac arrest, transplant and LVAD) is similar or higher compared to LMNA related cardiac laminopathy (54.5% vs. 58.3%) [25]. NRAP patients also seem to have an earlier onset of major cardiac end-points when compared to cardiac laminopathy or DCM in general (22.8±19.4 vs. 51.0±8.7 and was 59.0±14.2 years) [25]. In addition, the rate of cardiac transplantation and LVAD utilization was higher in our NRAP group compared to a Norwegian LMNA cohort (45% vs. 33%) [26].

Our data also suggest that two PTVs in NRAP cause more severe disease than PTV + missense combination in NRAP. There are no previous observations on the PTV + missense variant combination in DCM, thus further studies are needed to confirm whether the previous assumption is correct. Given that our data did not show enrichment of potentially biallelic missense variants in the DCM group, these variants may not contribute to the DCM pathogenesis alone. However, at this time we cannot exclude the possibility that a small proportion of biallelic missense variants are disease causing alone.

NRAP appear to play important role in myocardial architecture and sarcomere function, supporting the biological plausibility of our findings. The NRAP gene on chromosome 10q25.3 encodes the nebulin related anchoring protein. This protein is involved in anchoring terminal actin filaments to the membrane, tension transmission from myofibrils to extracellular matrix, as well as having a significant role in myofibrillogenesis during cardiomyocyte development, and it is involved in the sarcomeric contraction cycle in adult heart [27,28]. The N-terminal LIM domain of NRAP interacts with α-actinin and talin [29,30], while the domain with single repeats interacts also with actin, the Kelch-like family member 41 (KLHL41) [31], and cysteine and glycine-rich protein 3 (CSRP3) [27], and the C-terminal super repeats interact with filamin C (FLNC) [31] and vinculin (VCL) [29]. Experimentally, upregulation of NRAP expression was observed in DCM mice models and human DCM patients [27,32]. This has been suggested to be an adaptive response to correct for disorganized actin thin filament architecture at intercalated disc junctions. NRAP is expressed in the myocardium and in striated muscle. Truszkowska et al. previously reported an absence of NRAP protein in the myocardium of a DCM proband with biallelic PTV in NRAP whileNRAP protein was clearly present in a control heart [12].

Study limitations

In one of the three probands with familial DCM, we were unable to obtain samples from the parents and other family members to further prove segregation of the phenotype with the genotype. Similarly, DNA samples were available only in one of the eight probands with sporadic DCM. Even though the data supported recessive inheritance since heterozygous individuals were unaffected, more thorough segregation studies would have brought depth to the scientific message especially by clarifying penetrance of NRAP related DCM. Moreover, no functional studies were carried out, nor animal models were generated for any of the identified variants. Four of the patients carried the same splice region missense variant, NRAP p.(Gln24His), but we did not perform transcriptional analysis to determine this variant's effect on splicing that would have increase our understanding on disease mechanisms. None of the NRAP variants detected via NGS were confirmed with Sanger sequencing since all of them had high variant call quality score, fulfilled several other quality control criteria for true positive call, and the reporting followed CLIA/CAP/ISO-15189 approved policy. This study provides the first statistical association between the NRAP gene and DCM without mechanistic insights or evidence that have been partially provided in the initial case reports.

The results of this study demonstrate significant enrichment of NRAP variants in DCM patients with severe clinical events and their co-segregation in multiple families support an inclusion of NRAP in genetic testing of cardiomyopathies.

Supporting information

S1 File

(XLSX)

Acknowledgments

We thank all the healthcare professional sending their samples to Blueprint Genetics for genetic testing and providing clinical information on patients’ phenotype that enabled this registry study. We also thank index patients and their family members who were recruited to either the Inherited Cardiomyopathies Study or KidCMP Study for the participation.

Data Availability

There are both ethical and legal restrictions on sharing the full data set. Data is owned by Blueprint Genetics, which has no consent to share the data so that individuals can be recognized. WES data is each individual's genetic fingerprint and provide information allowing to distinguish each person in the world by any other individual, thus the data can't be legally shared as full. Also the ethical permit acquired for this study does not allow evaluation of non-cardiac genes from any of the individuals, thus the WES data can't be shared. However, all relevant data necessary to replicate the study's results are within the paper and its Supporting Information files.

Funding Statement

This work was supported by grants from the Finnish Foundation for Cardiovascular Research (TH), Aarne Koskelo Foundation (TH), Special Governmental Subsidy (EVO) grants (TH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi F, Charron P, et al. Classification of the cardiomyopathies: A position statement from the european society of cardiology working group on myocardial and pericardial diseases. Eur Heart J. 2008;29: 270–276. 10.1093/eurheartj/ehm342 [DOI] [PubMed] [Google Scholar]
  • 2.Codd MB, Sugrue DD, Gersh BJ, Melton LJ. Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy. A population-based study in Olmsted County, Minnesota, 1975–1984. Circulation. 1989;80: 564–72. 10.1161/01.cir.80.3.564 [DOI] [PubMed] [Google Scholar]
  • 3.Mestroni L, Maisch B, McKenna WJ, Schwartz K, Charron P, Rocco C, et al. Guidelines for the study of familial dilated cardiomyopathies. Eur Heart J. 1999;20: 93–102. 10.1053/euhj.1998.1145 [DOI] [PubMed] [Google Scholar]
  • 4.McNally EM, Golbus JR, Puckelwartz MJ. Genetic mutations and mechanisms in dilated cardiomyopathy. J Clin Invest. 2013;123: 19–26. 10.1172/JCI62862 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Cahill TJ, Ashrafian H, Watkins H. Genetic Cardiomyopathies Causing Heart Failure. Circ Res. 2013;113: 660–675. 10.1161/CIRCRESAHA.113.300282 [DOI] [PubMed] [Google Scholar]
  • 6.Hershberger RE, Hedges DJ, Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol. 2013;10: 531–547. 10.1038/nrcardio.2013.105 [DOI] [PubMed] [Google Scholar]
  • 7.Mestroni L, Taylor MRG. Genetics and genetic testing of dilated cardiomyopathy: a new perspective. Discov Med. 2013;15: 43–9. [PMC free article] [PubMed] [Google Scholar]
  • 8.Pugh TJ, Kelly MA, Gowrisankar S, Hynes E, Seidman MA, Baxter SM, et al. The landscape of genetic variation in dilated cardiomyopathy as surveyed by clinical DNA sequencing. Genet Med. 2014;16: 601–608. 10.1038/gim.2013.204 [DOI] [PubMed] [Google Scholar]
  • 9.Herman DS, Lam L, Taylor MRG, Wang L, Teekakirikul P, Christodoulou D, et al. Truncations of titin causing dilated cardiomyopathy. N Engl J Med. 2012;366: 619–28. 10.1056/NEJMoa1110186 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Akinrinade O, Ollila L, Vattulainen S, Tallila J, Gentile M, Salmenperä P, et al. Genetics and genotype-phenotype correlations in Finnish patients with dilated cardiomyopathy. Eur Heart J. 2015;36: 2327–37. 10.1093/eurheartj/ehv253 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Walsh R, Thomson KL, Ware JS, Funke BH, Woodley J, McGuire KJ, et al. Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples. Genet Med. 2017;19: 192–203. 10.1038/gim.2016.90 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Truszkowska GT, Bilińska ZT, Muchowicz A, Pollak A, Biernacka A, Kozar-Kamińska K, et al. Homozygous truncating mutation in NRAP gene identified by whole exome sequencing in a patient with dilated cardiomyopathy. Sci Rep. 2017;7: 1–5. 10.1038/s41598-016-0028-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Monies D, Abouelhoda M, AlSayed M, Alhassnan Z, Alotaibi M, Kayyali H, et al. The landscape of genetic diseases in Saudi Arabia based on the first 1000 diagnostic panels and exomes. Hum Genet. 2017;136: 921–939. 10.1007/s00439-017-1821-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Vasilescu C, Ojala TH, Brilhante V, Ojanen S, Hinterding HM, Palin E, et al. Genetic Basis of Severe Childhood-Onset Cardiomyopathies. J Am Coll Cardiol. 2018;72: 2324–2338. 10.1016/j.jacc.2018.08.2171 [DOI] [PubMed] [Google Scholar]
  • 15.Ahmed HA, Al-ghamdi S, Mutairi F Al. Dilated cardiomyopathy in a child with truncating mutation in NRAP gene. JBCGenetics. 2018;1: 77 LP– 80. Available: https://www.jbcgenetics.com//?mno = 17290. [Google Scholar]
  • 16.Martin AR, Williams E, Foulger RE, Leigh S, Daugherty LC, Niblock O, et al. PanelApp crowdsources expert knowledge to establish consensus diagnostic gene panels. Nat Genet. 2019;51: 1560–1565. 10.1038/s41588-019-0528-2 [DOI] [PubMed] [Google Scholar]
  • 17.Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv. 2019. 10.1101/531210 [DOI] [Google Scholar]
  • 18.Haas J, Frese KS, Peil B, Kloos W, Keller A, Nietsch R, et al. Atlas of the clinical genetics of human dilated cardiomyopathy. Eur Heart J. 2015;36: 1123–1135. 10.1093/eurheartj/ehu301 [DOI] [PubMed] [Google Scholar]
  • 19.Almomani R, Verhagen JMA, Herkert JC, Brosens E, van Spaendonck-Zwarts KY, Asimaki A, et al. Biallelic Truncating Mutations in ALPK3 Cause Severe Pediatric Cardiomyopathy. J Am Coll Cardiol. 2016;67: 515–525. 10.1016/j.jacc.2015.10.093 [DOI] [PubMed] [Google Scholar]
  • 20.Theis JL, Sharpe KM, Matsumoto ME, Chai HS, Nair AA, Theis JD, et al. Homozygosity mapping and exome sequencing reveal GATAD1 mutation in autosomal recessive dilated cardiomyopathy. Circ Cardiovasc Genet. 2011;4: 585–9. 10.1161/CIRCGENETICS.111.961052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Muhammad E, Levitas A, Singh SR, Braiman A, Ofir R, Etzion S, et al. PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction. Hum Mol Genet. 2015;24: 7227–40. 10.1093/hmg/ddv423 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Iuso A, Wiersma M, Schüller HJ, Pode-Shakked B, Marek-Yagel D, Grigat M, et al. Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy. Am J Hum Genet. 2018;102: 1018–1030. 10.1016/j.ajhg.2018.03.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Garcia-Pavia P, Syrris P, Salas C, Evans A, Mirelis JG, Cobo-Marcos M, et al. Desmosomal protein gene mutations in patients with idiopathic dilated cardiomyopathy undergoing cardiac transplantation: a clinicopathological study. Heart. 2011;97: 1744–1752. 10.1136/hrt.2011.227967 [DOI] [PubMed] [Google Scholar]
  • 24.Qadri S, Anttonen O, Viikilä J, Seppälä EH, Myllykangas S, Alastalo T-P, et al. Case reports of two pedigrees with recessive arrhythmogenic right ventricular cardiomyopathy associated with homozygous Thr335Ala variant in DSG2. BMC Med Genet. 2017;18: 86 10.1186/s12881-017-0442-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ollila L, Nikus K, Holmström M, Jalanko M, Jurkko R, Kaartinen M, et al. Clinical disease presentation and ECG characteristics of LMNA mutation carriers. Open Hear. 2017;4: e000474 10.1136/openhrt-2016-000474 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hasselberg NE, Haland TF, Saberniak J, Brekke PH, Berge KE, Leren TP, et al. Lamin A/C cardiomyopathy: young onset, high penetrance, and frequent need for heart transplantation. Eur Heart J. 2018;39: 853–860. 10.1093/eurheartj/ehx596 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ehler E, Horowits R, Zuppinger C, Price RL, Perriard E, Leu M, et al. Alterations at the intercalated disk associated with the absence of muscle LIM protein. J Cell Biol. 2001;153: 763–72. 10.1083/jcb.153.4.763 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Henderson CA, Gomez CG, Novak SM, Mi-Mi L, Gregorio CC. Overview of the muscle cytoskeleton. Compr Physiol. 2017;7: 891–944. 10.1002/cphy.c160033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Luo G, Herrera AH, Horowits R. Molecular Interactions of N-RAP, a Nebulin-Related Protein of Striated Muscle Myotendon Junctions and Intercalated Disks. Biochemistry. 1999;38: 6135–6143. 10.1021/bi982395t [DOI] [PubMed] [Google Scholar]
  • 30.Zhang JQ, Elzey B, Williams G, Lu S, Law DJ, Horowits R. Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart. Biochemistry. 2001;40: 14898–906. 10.1021/bi0107445 [DOI] [PubMed] [Google Scholar]
  • 31.Lu S, Carroll SL, Herrera AH, Ozanne B, Horowits R. New N-RAP-binding partners α-actinin, filamin and Krp1 detected by yeast two-hybrid screening: Implications for myofibril assembly. J Cell Sci. 2003;116: 2169–78. 10.1242/jcs.00425 [DOI] [PubMed] [Google Scholar]
  • 32.Perriard JC, Hirschy A, Ehler E. Dilated cardiomyopathy: A disease of the intercalated disc? Trends Cardiovasc Med. 2003;13: 30–38. 10.1016/s1050-1738(02)00209-8 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Amanda Ewart Toland

31 Jul 2020

PONE-D-20-18740

Biallelic loss-of-function in NRAP is a common cause of recessive dilated cardiomyopathy

PLOS ONE

Dear Dr. Koskenvuo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1. Address reviewer 1's concern on the lack of detail on the clinical phenotyping of patients and include more details on these.

2. Provider more details for the pipeline used in the bioinformatics analysis.

3  Address reviewer's comments on improvements for Tables 1 and 2.

4. Fix discrepancies in numbers identified by reviewer 2.

5. Respond to reviewer 4's comments related to exome sequencing and lack of detection of other DCM genes.

6. Consider adding in the reference suggested by Reviewer 4.

7.  Provide greater detail on the inclusion and exclusion criteria for variants.  See comments from reviewers.  Also address how these variants meet ACMG criteria.

8.  Address the other points raised by the reviewers.

Please submit your revised manuscript by Sep 14 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Amanda Ewart Toland, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests section:

"Drs. Koskenvuo, Saarinen, Ahonen, Tommiska, Seppälä, Tuupanen, Kangas-Kontio, Schleit, Hathaway, Kytölä, Muona, Sistonen, Salmenperä, Gentile, Paananen, Myllykangas, Alastalo are full-time employees of Blueprint Genetics, which offers genetic diagnostics for cardiomyopathies. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose."

We note that one or more of the authors are employed by a commercial company: Blueprint Genetics, a Quest Diagnostics Company.

2.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. 

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript by Koskenvuo and colleagues utilize a large genetic testing database to identify a number of rare variants in NRAP associated with DCM. Overall, this is an appropriately concise report that is well written. It attempts to tackle a common, yet challenging problem – what is the cause of genotype negative DCM?

The size of the cohort analyzed, and the limited co-segregation present are strengths of this paper. However, there are a number of points which should be expanded or clarified in my opinion.

In the introduction, a brief explanation of the physiologic role of NRAP would be helpful. Is it expressed in the heart? Solely in the heart? Would its expression pattern lend itself to a cardiac myocardium only disease?

A major limitation of this manuscript is the lack of clinical phenotyping of the patients with DCM. This should be addressed. What is the evidence that the cohort that was screened had non-ischemic, congenital DCM? This is even more of any issue for the NRAP-positive individuals.

The bioinformatic pipeline used to identify these variants is not well delineated. How were other variants that localized to genes which are known to cause DCM excluded as possibilities?

Were copy number variants identified? This is important to address as there are CNVs associated with DCM (1p36 deletion syndrome) which may confound this data.

The variant inclusion and exclusion data is unclear. For example, having an exclusionary criteria which appears to be <100 individuals is not appropriate as coverage in gnomAD is variable. Thus, a poorly covered area with fewer individuals sequenced may thus have fewer minor allele positive individuals that are nonetheless a high minor allele frequency. Minor allele frequency should be used.

Inclusion criteria for missense variants are particularly unclear. What criteria is used to determine whether a missense variant is included?

Table 1 seems to be extraneous and seems odd. I think it should be removed and potentially referenced in the Discussion

What was the pathogenicity assessment of the NRAP variants by 2015 ACMG guidelines?

Reviewer #2: Koskenvuo et al. presented interesting novel data on NRAP gene’s implication in the pathogenesis of autosomal recessive dilated cardiomyopathy (DCM).

Authors reviewed genetic results of 577 patients either with a confirmed or suspected diagnosis of DCM and compared them with data of control cohort of 31062 individuals, including both non-cardiac controls (n>25000) and non DCM cardiac controls (n>5000).

They identified two rare variants in NRAP gene [(four homozygous truncating (PTV), or compound heterozygous (one patient had two different PTV and 6 had PTV/missense)] in 11 unrelated probands with DCM (1.9%) but none in the controls, with OR of 1052 in comparison to controls. They concluded that NRAP variants could explain 0.25%-2.46% of all DCM cases.

Also, Authors stress the fact that NRAP patients seem to have an earlier onset of major cardiac end-points compared to cardiac laminopathy or DCM, and higher requirement for cardiac transplantation/LVAD.

This is a very important, well designed and rigorously performed study, rather nicely presented.

However, page 10:

Authors state:

“In these 11 individuals four had a homozygous PTV, one had two heterozygous PTVs and two were compound heterozygous for a PTV/missense variant.” This adds up to 7 , and not to 11.

Although later we can learn that the same variants were present in other patients, and the Table 2 lists them, it is unclear and should be corrected.

Page 19

Authors state:

"In the first study suggesting association of NRAP with cardiomyopathy, the proband’s 35-year-old brother who was compound heterozygous for two PTVs in NRAP”. This is untrue. As Authors correctly state in the Table 1, the first identified variant was homozygous nonsense NRAP variant (p.Arg1502*).

Reviewer #3: Koskenvuo et al report a study where they identified 4 cases with biallelic NRAP protein truncating variants (PTV), 2 cases with NRAP compound heterozygous PTV and a missense variant, 1 case with two PTVs (phase unknown) and 4 cases with a PTV and a missense variant (phase unknown), in a large dilated cardiomyopathy cohort (n=577). The aim was to assess the association of bi-allelic loss of function (LoF) variants in NRAP with autosomal recessive cardiomyopathy, since previously, only 4 such cases have been reported. Large disease cohorts are a useful resource to assess for the presence of any gene’s variants and to study their association with the disease. This study provides supports the role of NRAP biallelic loss of function variants and their association with autosomal recessive cardiomyopathy, though with reduced penetrance. The data presented in the study does not support for a causal role of NRAP LoF variants in autosomal dominant dilated cardiomyopathy. However, this manuscript in its current form needs substantial revision before it is suitable for publication in PLos One. This report can benefit from addition of phenotype details of the affected individuals and further discussion on incomplete penetrance or age related penetrance etc.

Major comments:

In this study the authors found 6 cases with biallelic NRAP variants (4 homozygous & 2 with PTV and a missense variant in trans/compound heterozygous). This needs to be clearly stated in the text as the other 5 sets of NRAP variants may or may not be in trans.

Were the variants Sanger confirmed in the 11 cases ? If so, please mention that in the methods.

Table 1

• It is best to list the variants in the “chr-genomic coordinate- Ref allele- Alt allele” format. This format is most useful along with the HGVS nomenclature for the cDNA , protein and the coding exon to which the LoF variants maps to (for example 3/20).

• Please add the units and reference ranges for the CK & ProBNP levels listed

• Addition of the gender of the affected individual will also be helpful

• In the footnotes, it needs to be mentioned that homozygotes for these variants in Gnomad were not seen.

• Please mention the NRAP NM_ID used by the published reports listed in this table.

Table 2

• Table 2 contains information on the cases reported in the current study; however, the organization of this table is not reader friendly.

• Please reword the legend for clarity and make it concise

Suggested Legend “NRAP variants identified in individual with confirmed or suspected dilated cardiomyopathy”

• It is best to have one row each for one affected individual

• Please add available phenotype information for each individual including gender, age of onset, CK levels, available cardiac biopsy

• It is best to list the variants in the “chr-genomic coordinate- Ref allele- Alt allele” format

• List the variants in Hgvs nomenclature (cDNA and protein)

• Add a column to clarify whether it is compound heterozygous or phase unknown

• The cases where unambiguous biallelic NRAP variants were seen (6/11) should be listed first

• the number of coding exon where the variant is located will be a useful addition especially, for the C terminal LoF variants, to assess whether they are predicted to undergo NMD or will result in truncation

• The “type of variant” column is redundant and may be removed

• SIFT and conservation data can be listed in the footnotes

• In the footnotes, it needs to be mentioned that homozygotes for these variants in Gnomad were not seen

For calculation of the population based prevalence using the frequency of NRAP LoF variants in Gnomad v.2.1, were the NRAP LoF variants seen in the last coding exon also included? The LOF variants in the last coding exon of NRAP may not undergo NMD and may result in a truncated product. Hence if these were included, the prevalence estimate derived for NRAP associated recessive cardiomyopathy might be a slight overestimate. Further, there were two high quality homozygous LoFs seen for NRAP in gnomad v.2.1.1 – were they included/removed for the prevalence calculation?

Discussion

If the age of onset data is available for the 6 individuals, along with the 4 published reports the heterogeneity seen should be discussed. If a large variability is seen, it might be supportive of age related penetrance. In addition, the age of onset or its variability, if seen for other AR non syndromic dilated cardiomyopathy genes, is also worth a mention.

Since NRAP is predominantly expressed in the heart & skeletal muscle, in the 6 cases with bialleleic variants were there any muscle issues seen in the affected individuals? Were any muscle issues noted in the 4 previously published cases? If so, this needs to be mentioned in the discussion

The authors list a handful of genes, which cause AR non-syndromic cardiomyopathy. Is age related penetrance or incomplete penetrance a feature seen for these genes as well ? in individuals who carry biallelic variants in these genes. Or this is uniquely seen for NRAP?

This will be insightful; Further, Gnomad V.2.1.1 data has two high quality homozygous LoF variants in NRAP. The authors should attempt to provide a potential explanation for this observation.

It is interesting that a single missense variant (p.Gln24His) which could potentially affect splicing is seen in 4 affected individuals.

Minor comments

Several sentences lack clarity and I urge the authors to read through the manuscript carefully and edit the manuscript for clarity and typos.

For example,

Title

“ Biallelic loss-of-function in NRAP is a common cause of recessive dilated cardiomyopathy”

Instead the following title is clearer

“ Biallelic loss-of-function variants in NRAP are a common cause of recessive dilated cardiomyopathy”

Abstract

“Confirmed or potentially biallelic NRAP variants were enriched in DCM”

the usage of the word confirmed is confusing ; instead it is suggested to use “Biallelic or potential bi-allelic”

“Biallelic (n=6) or two (n=5) NRAP variants (two PTVs or PTV+missense) were”

(two PTVs or PTV+missense)

Instead the following addition will make it more clear.

“Biallelic (n=6) or two (n=5) NRAP variants (two PTVs or PTV+missense) were”

(two PTVs or PTV+missense, phase unknown )

Methods: Patients

Please clearly mention whether the HQSA also included NRAP.

Page 5

estimated int the range of 1:500 to 1:3,000 [2–4].

Page 10:

“Visual Splicing software v2.11 (Interactive Biosoftware, France) predicts that this variant either leads to either loss of the native splice donor”

Instead

“Visual Splicing software v2.11 (Interactive Biosoftware, France) predicts that this variant either leads to loss of the native splice donor”

“Enrichment of NRAP variants in DCM

We identified two rare NRAP variants, of which at least one was a PTV in 11 out”

Instead

We identified cases, which carried two rare NRAP variants, of which atleast one was a PTV

Gene names should be italicized in the manuscript text.

Reviewer #4: The authors identified biallelic loss-of-function in NARP as a common cause of recessive dilated cardiomyopathy with next generation sequencing. However, there are some issues to address.

Major issues:

1. The study didn’t detect the variants of well-established causal genes of DCM. Is the DCM phenotype caused by biallelic loss-of-function in NARP or variants of the known causal genes, such as TTN, RBM20, et, al? It would better to perform whole exome sequencing or targeted next generation sequencing with causal genes of DCM on the cohort.

2. It would be better to confirm the variants in NARP by Sanger sequencing, especially in family segregation analysis due to the false positive rate of next generation sequencing.

Minor issues:

1. Vasilescu et, al reported NRAP underlying childhood dilated cardiomyopathy. It would be better to mention their work in discussion section. (J Am Coll Cardiol. 2018 Nov 6;72(19):2324-2338.) in NARP.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Andrew Landstrom

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Decision Letter 1

Amanda Ewart Toland

16 Dec 2020

PONE-D-20-18740R1

Biallelic loss-of-function in NRAP is a common cause of recessive dilated cardiomyopathy

PLOS ONE

Dear Dr. Koskenvuo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1.  Soften language suggesting that biallelic NRAP loss is a common cause of AR DCM throughout manuscript and in title.

2.  Expand description of bioinformatics analysis of NGS data in the methods.

3. Include details of inclusion/exclusion criteria for study population.  See reviewer's comments.

4. Consider doing Sanger to confirm the 11 variants found.  If Sanger is not done, list lack of confirmation of these variants as a study limitation in the Discussion.

5.  Check for spelling errors.  See reviewer's comments.

Please submit your revised manuscript by Jan 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Amanda Ewart Toland, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The revisions to this manuscript appear reasonable and the manuscript is greatly improved. I have one lingering concern.

Overall, I think the language associating biallelic loss of NRAP is too strong. For example, the title stating that it is the most common cause of AR DCM, the conclusion in the abstract and discussed in the paper is outside the scope of the data. Given the heterogeneity of the genetic substrate of DCM, additional, independent validation studies are needed to corroborate this finding. This should be highlighted in the manuscript, including in the title, and the language appropriately softened.

Reviewer #2: The manuscript is sound, concise and interesting. Although clinical data are not robust, the report nicely underlies severe clinical endpoints related to the biallelic loss-of-function NRAP variants causing autosomal recessive DCM.

Spelling mistakes:

fatigability

wildtype

Reviewer #4: The authors found that rare NRAP variants were associated with the pathogenesis of autosome recessive dilated cardiomyopathy through genetic testing performed on a large cohort. Moreover, co-segregation analysis provided a strong supporting evidence for this conclusion.

However, there are some issues to address.

1. Have the authors performed Sanger sequencing to validate the 11 variants as NGS possess relatively high error rate?

2. Please described the bioinformatics analysis in Materials and methods section in details.

3. It is better to list out the inclusion and exclusion criteria of the recruited subjects. How to define a DCM patient and what kind of individuals can be classified as suspected DCM instead of non-DCM cardiac group?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Andrew Landstrom

Reviewer #2: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 3;16(2):e0245681. doi: 10.1371/journal.pone.0245681.r004

Author response to Decision Letter 1


4 Jan 2021

PONE-D-20-18740R1

Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

(Ed1.1). Soften language suggesting that biallelic NRAP loss is a common cause of AR DCM throughout manuscript and in title.

RESPONSE: <<New title: Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy>>.

(Ed1.2). Expand description of bioinformatics analysis of NGS data in the methods.

RESPONSE: <<We have added information on bioinformatics pipeline into methods section as much as possible. Most of the detailed settings are proprietary, and are thus intentionally left out.>>

(Ed1.3). Include details of inclusion/exclusion criteria for study population. See reviewer's comments.

RESPONSE: << This information is now available in two places at methods section:

1) The patients paragraph: The inclusion criteria for DCM group (see later) was referral to genetic testing due to diagnosis or clinical suspicion of DCM.

2) NRAP variants paragraph: Frequency of such high-quality variants were compared between patients with clinical or suspected dilated cardiomyopathy (DCM group), other cardiology indication (Non-DCM cardiac group consisting patients tested due diagnosis or suspicion inherited aortopathy, channelopathy or cardiomyopathy other than DCM) or any other clinical indication for panel or exome testing (Control group).>>.

(Ed1.4). Consider doing Sanger to confirm the 11 variants found. If Sanger is not done, list lack of confirmation of these variants as a study limitation in the Discussion.

RESPONSE: <<We decided to continue without Sanger confirmation and added the required comment into the limitations section. To our opinion, this request would have been appropriate 7-10 years ago and in other circumstranmces. We have performed all tests in accredited laboratory by qualified personnel, fully documented workflows, validated assays, CLIA lab sign-off process etc. This is very different world compared to research laboratory.>>

(Ed1.5). Check for spelling errors. See reviewer's comments.

RESPONSE: <<Done>>.

Reviewer #1: The revisions to this manuscript appear reasonable and the manuscript is greatly improved. I have one lingering concern.

(Rev1.1). Overall, I think the language associating biallelic loss of NRAP is too strong. For example, the title stating that it is the most common cause of AR DCM, the conclusion in the abstract and discussed in the paper is outside the scope of the data. Given the heterogeneity of the genetic substrate of DCM, additional, independent validation studies are needed to corroborate this finding. This should be highlighted in the manuscript, including in the title, and the language appropriately softened.

RESPONSE: <<We have modified the title and conclusions throughout the manuscript as requested>>.

Reviewer #2:

(Rev2.1).The manuscript is sound, concise and interesting. Although clinical data are not robust, the report nicely underlies severe clinical endpoints related to the biallelic loss-of-function NRAP variants causing autosomal recessive DCM.

Spelling mistakes:

fatigability

wildtype

RESPONSE: <<Done>>.

Reviewer #4:

(Rev4.1).The authors found that rare NRAP variants were associated with the pathogenesis of autosome recessive dilated cardiomyopathy through genetic testing performed on a large cohort. Moreover, co-segregation analysis provided a strong supporting evidence for this conclusion.

However, there are some issues to address.

1. Have the authors performed Sanger sequencing to validate the 11 variants as NGS possess relatively high error rate?

RESPONSE: <<We decided to continue without Sanger confirmation and added the required comment into the limitations section. >>

(Rev4.2). Please described the bioinformatics analysis in Materials and methods section in details.

RESPONSE: <<We have added information on bioinformatics pipeline into methods section as much as possible. Most of the detailed settings are proprietary, and are thus intentionally left out.>>

(Rev4.3). It is better to list out the inclusion and exclusion criteria of the recruited subjects. How to define a DCM patient and what kind of individuals can be classified as suspected DCM instead of non-DCM cardiac group?

RESPONSE: << This information is now available in two places at methods section:

1) The patients paragraph: The inclusion criteria for DCM group (see later) was referral to genetic testing due to diagnosis or clinical suspicion of DCM.

2) NRAP variants paragraph: Frequency of such high-quality variants were compared between patients with clinical or suspected dilated cardiomyopathy (DCM group), other cardiology indication (Non-DCM cardiac group consisting patients tested due diagnosis or suspicion inherited aortopathy, channelopathy or cardiomyopathy other than DCM) or any other clinical indication for panel or exome testing (Control group).>>.

Decision Letter 2

Amanda Ewart Toland

6 Jan 2021

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy

PONE-D-20-18740R2

Dear Dr. Koskenvuo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Amanda Ewart Toland, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Amanda Ewart Toland

12 Jan 2021

PONE-D-20-18740R2

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy

Dear Dr. Koskenvuo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Amanda Ewart Toland

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File

    (XLSX)

    Attachment

    Submitted filename: response to reviewers PONE-D-20-18740.docx

    Data Availability Statement

    There are both ethical and legal restrictions on sharing the full data set. Data is owned by Blueprint Genetics, which has no consent to share the data so that individuals can be recognized. WES data is each individual's genetic fingerprint and provide information allowing to distinguish each person in the world by any other individual, thus the data can't be legally shared as full. Also the ethical permit acquired for this study does not allow evaluation of non-cardiac genes from any of the individuals, thus the WES data can't be shared. However, all relevant data necessary to replicate the study's results are within the paper and its Supporting Information files.


    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES