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Autosomal recessive cardiomyopathy and sudden cardiac death associated with variants in MYL3

Purpose Variants in genes encoding sarcomeric proteins are the most common cause of inherited cardiomyopathies. However, the underlying genetic cause remains unknown in many cases. We used exome sequencing to reveal the genetic etiology in patients with recessive familial cardiomyopathy. Methods Exome sequencing was carried out in three consanguineous families. Functional assessment of the variants was performed. Results Affected individuals presented with hypertrophic or dilated cardiomyopathy of variable severity from infantile- to early adulthood–onset and sudden cardiac death. We identified a homozygous missense substitution (c.170C>A, p.[Ala57Asp]), a homozygous translation stop codon variant (c.106G>T, p.[Glu36Ter]), and a presumable homozygous essential splice acceptor variant (c.482-1G>A, predicted to result in skipping of exon 5). Morpholino knockdown of the MYL3 orthologue in zebrafish, cmlc1, resulted in compromised cardiac function, which could not be rescued by reintroduction of MYL3 carrying either the nonsense c.106G>T or the missense c.170C>A variants. Minigene assay of the c.482-1G>A variant indicated a splicing defect likely resulting in disruption of the EF-hand Ca2+ binding domains. Conclusions Our data demonstrate that homozygous MYL3 loss-of-function variants can cause of recessive cardiomyopathy and occurrence of sudden cardiac death, most likely due to impaired or loss of myosin essential light chain function.


Clinical evaluation
Medical history, physical examination and imaging were performed as part of routine clinical workup. For MYL3 mutation-positive individuals, extensive clinical follow-up was performed (Table S1). The clinical evaluation was performed by experienced cardiologists. The conventional, two-dimensional and tissue doppler echocardiographic measurements of the heart of the individuals were evaluated by an echocardiologic examination. Neurophysiological studies were carried out in the proband (III.7) (Fig. 1A) from Family A by an experienced neurologist.

DNA isolation
Blood samples were obtained from the patients, their parents, siblings and additional family members in some families. Extraction of genomic DNA was performed from whole blood, using DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.

Next generation sequencing
For Family A, exome sequencing was performed on DNA from the proband (III.7) and two clinically unaffected individuals in the family (III.1 and IV.3) (Fig. 1A), and data analysed through the use of the Ingenuity Variant Analysis (IVA) software (Qiagen, Hilden Germany), as previously described [1]. The filtering strategy focused on coding variants in known cardiac disease genes, selected based on variant databases Human Genome Mutation Database (HGMD) and ClinVar, and the most recent literature.
For Family C, exome sequencing was performed on DNA from the individual V.2, following the death of her daughter (VI.1), who was diagnosed with unclassified cardiomyopathy and died at the age of 8 of SCD (Fig. 1C). Variants were prioritized based on the following criteria: 1) The variant was predicted to perturb protein function. All synonymous and intronic variants were excluded unless the variant was within a predicted splice site (+ or -5 bp from splice junction). Any variation that was predicted to alter gene expression or protein function was included. These included nonsynonymous variations in coding regions (i.e. missense) or alterations resulting in frameshifts, premature stop codons, loss of stop codons, coding INDELS, and splice sites (i.e. ±2 nucleotides from an exon junction); 2) Homozygous variants; 3) The variant was rare as defined by allele frequency of less than 1% in either gnomAD or GME variomes; 4) The variant was present in a region of homozygosity as defined by HomozygosityMapper; 5) The variant was conserved evolutionarily as determined by a number of conservation scores including GERP (Score > 3), and PolyPhen2, SIFT, or predicted as disease causing by Mutation Taster for nonsense mutations.
Confirmatory bi-directional Sanger sequencing was performed in the probands in Families A and B and available family members from Families A, B and C ( Fig. 1 and Fig. S2). PCR primers are available on request.

Functional analysis of MYL3 in zebrafish
All zebrafish studies were conducted in compliance with the UK Animals (Scientific Antisense MO oligonucleotides (Genetools, LLC) were generated as previously described against the translational start site of cmlc1 (5'-TGGGTTCCACTTTTTTTGGTGCCAT-3') [2].
100pg of Morpholino was injected into the yolk of 1-2 cell staged embryos and incubated at 28.5 °C until the desired stage. A control morpholino was used for comparison, targeting an intronic sequence in the human beta-globin gene. For MYL3 rescue experiments, full human MYL3 was commercially synthesized with BamHI/XhoI linkers, 5' and 3' respectively, and directionally cloned into pCS2+ at the BamHI/XhoI sites (GenScript). Patient variants were commercially generated using site directed mutagenesis of the pCS2+_MYL3 construct (GenScript). DNA constructs were linearised using NotI restriction enzyme and RNA transcribed using the SP6 ambion MAXIscript kit, following the manufacturer's instructions.
Approximately 100 pg of RNA was injected into the cytoplasm of wildtype one-cell staged embryos. were performed using standard techniques on 4% paraformaldehyde (PFA) fixed embryos and primary antibodies were diluted 1 in 5 for optimal results. Appropriate secondary antibodies were used (ThermoFisher, cat # A21144 and A21121) at a 1 in 1000 dilution.
Embryos were mounted on a 27mm glass bottom dish (ThermoFisher, cat # 150682) and imaged using a Nikon A1R confocal microscope.
The ventricular percentage shortening fraction was calculated from ventrally orientated videos of the heart (Supplementary Movies S9-13). Videos were converted to TIFF files using Adobe Photoshop CS6 and then imported as an image sequence into ImageJ for analysis. Average area measurements of diastolic and systolic ventricular state were taken over five cycles per embryos. The percentage shortening fraction was calculated as the percentage area change between diastolic and systolic state using the equation 100− [ 100 diastolic area × systolic area].
Statistical significance was evaluated using an unpaired t test in Graphpad Prism version 8.4.3.

Minigene assay methods
Minigene assays were performed as previously described [3,4] with slight modifications. This assay relies on the use of a minigene vector which contains a fragment of the SERPING1 gene, with two exonic regions separated by an intron, cloned into the mammalian expression vector    (Fig. S1). The Holter monitor test showed no arrhythmias. Her younger brother, III.8, was affected at an earlier age. Her mother (II.4) and her younger brother (III.8) died at the age of 55 and 26, respectively, of SCD. In addition, her uncle (II.7) and her cousin (III.11) (Fig. 1A) died of SCD at the age of 57 and 24, respectively. There was, however, no record of cardiac evaluation available for these individuals. The echocardiography evaluation of individual III.6, who is from the same geographic region but not known to be related to the pedigree, indicated no signs of HCM (Table S1). The other apparently unaffected siblings and other family members showed no evidence of cardiac disease by history (Fig. 1A). The three asymptomatic children of the proband (IV.3, IV.4 and IV.5) 18, 13 and 6 years of age, respectively, were examined by a cardiologist and had normal echocardiograms at their current age (Table S1) Given MYL3 is also expressed in slow-twitch skeletal muscle fibers [5], a deleterious impact of MYL3 variants on skeletal muscle function is possible. Therefore neurological evaluation was performed in the proband (Case III.7) from Family A. Affected individuals showed, however, no evidence of muscle weakness by neurological examination or by history. The neurological examination of the probands indicated no apparent muscle weakness. Muscle tone was normal and no distal or proximal muscle atrophy or fasciculation was detected in the limbs. The strength of muscle in the lower and upper limbs was 5/5. Levels of creatine phosphokinase were normal.

Cardiac and neurological evaluations of the patients
As in Family A, affected individuals from Family B showed no evidence of muscle weakness by history, suggesting that the recessive myosin ELC variant has little impact on skeletal muscle function.

Genetic findings
In Family  Following the filtering strategy in Family C, the mother (V.2) of deceased individual (VI.1) ( Fig. 1C), was found to harbor a heterozygous splice acceptor variant in MYL3 (c.482-1G>A (rs727503300)). The variant was validated by PCR and bi-directional Sanger sequencing analysis, which confirmed the mother as a carrier and identified the father to also be a carrier.
DNA from two of the mothers' siblings (V.3 and V.10) was available for testing, one of whom was wildtype (V.10) and the other (V.3) was found to be a carrier of the variant (Fig. 1C and   S2C). Individual V.1 was also a carrier of the variant. Genetic testing of the two children VI.2 and VI.3 identified them as wild type and a carrier, respectively ( Fig. 1C and S2C).

Minigene assay of the MYL3 c.482-1G>A variant
The MYL3 c.482-1G>A variant predominantly leads to skipping of the 78 bp exon 5 and a subsequent protein change p.(Gly161_Glu186del) (Fig. 2E-F). This would disrupt the last 3 AA of the EF-hand 2 domain, and the majority of the EF-hand 3 domain (Fig. 2I). The second (less frequent) consequence is a single nucleotide deletion of the first nucleotide of exon 5, as result of the G>A change generating a new splice acceptor site adjacent to the previous one (offset by 1nt). The first nucleotide of exon 5 (G) is instead considered intronic and is spliced out (Fig.   2G). As this is a single nucleotide deletion, it causes a frameshift leading to the p.(Gly161Valfs*4 protein change (as per HGVS guidelines) (Fig. 2G). This would completely abolish the EF-hand 3 domain and disrupt the last 3 (of 36) AA of the EF-hand 2 domain (Fig.   2I).
Based on quantitation by densitometry, the exon skipping event is more frequent (around 75%) compared to the single nucleotide deletion (~25%) (Fig. 2F), presumably as the latter uses 'less preferred' nucleotides either side of the consensus splice nucleotides (AG). However, it should be noted that the RT-PCRs are only semi-quantitative, and the minigene assays may not completely recapitulate in vivo splice event frequencies.