Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. Here, we report a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, we discovered that one of the affected children had a homozygous frameshift variant in Myosin light chain 2 (MYL2:NM_000432.3:c.431_432delCT: p.Pro144Argfs*57;MYL2-fs), which alters the last 20 amino acids of the protein and is predicted to impact the most C-terminal of the three EF-hand domains in MYL2. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and the infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed a marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant (MYL2:p.Gly162Arg) and three other MYL2 stop-gain variants (p.E22*, p.K62*, p.E97*) that result in loss of the EF domains are stably expressed but show impaired localization. The degradation of the MYL2-fs can be rescued by inhibiting the cell's proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither the MYL2-fs nor the MYL2:p.Gly162Arg variant supports normal cardiac function. The tools that we have generated provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. Our study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants in infantile HCM and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathy.

Abnormal Longitudinal Growth of the Aorta in Children with Familial Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV) is the most common type of congenital heart defect (CHD) and is associated with clinically significant cardiovascular complications including valve calcification and ascending aortopathy (AscAo), predominantly occurring in adulthood. While a limited number of genetic etiologies for BAV have been defined, family members of affected individuals display BAV along with other left-sided CHD. This has led to guidelines from the American Heart Association and American College of Cardiology that recommend echocardiographic screening of first-degree relatives of affected adults. While potentially beneficial in adults, the yield of such screening in children is unknown. The purpose of this study was to investigate a cohort of children with familial BAV to determine the frequency of development of AscAo, and to identify risk factors that contribute to abnormal aortic growth. Echocardiograms over a 10-year follow-up period were reviewed on 26 patients with familial BAV [22 male, 4 female; 22 with isolated BAV, 6 with BAV and aortic coarctation (CoA)]. All had a family history of CHD and were recruited from 2005 to 2010 as part of a genetics research study. Four aortic segments (annulus, root, sinotubular junction, ascending aorta) on parasternal long-axis echocardiographic images were measured by a single observer. The mean age at first echocardiogram was 7.1 ± 5.5 and that was 13.8 ± 6.2 years at the last echocardiogram. Only patients with > 2 echocardiograms in the 10-year period were included. Z score measurements of the aorta were plotted over time and based on these the cohort was divided into two groups: Group 1 (abnormal)-Z score for any segment > 2 or a change in Z score > 2 over follow-up; Group 2 (normal)-Z score < 2 throughout follow-up and change in Z score < 2. Nineteen out of 26 children displayed abnormal aortic growth or dilation of the aorta. BAV with right/left cusp fusion was more frequent in Group 1 (15/18) versus Group 2 (3/7) (p < 0.05). There were no significant differences in gender, aortic valve dysfunction, presence of CoA, family history, cardiac function, presence of left ventricular hypertrophy, or medication use between the 2 groups. In our longitudinal study of children with familial BAV, the majority display evidence of abnormal growth of the ascending aorta during the follow-up period consistent with AscAo and support the extension of current adult guidelines to the pediatric population. While we find that right/left cusp fusion is a risk factor for abnormal aortic growth, additional studies are needed to identify other factors to better select children who require serial screening.

A cohort study of multiple families with FBN1 p.R650C variant, ectopia lentis, and low but not absent risk for aortopathy.

Marfan syndrome is a multisystem disease with cardiovascular, ophthalmologic, and skeletal features. Diagnosis is made clinically with emphasis on presence of aortic root dilation and ectopia lentis (EL). Most individuals meeting these criteria have a pathogenic variant in FBN1, usually unique or observed rarely. Individuals with EL alone may also have FBN1 pathogenic variants, and the risk for aortic disease is not well known. We identified a unique cohort of 31 individuals (mean age 29, range 2-78) from nine families ascertained by a proband with EL alone, who had the same FBN1 p.R650C variant. Comparison was made to individuals with Marfan syndrome (n = 103 from 97 families) at our institution. Those with the p.R650C variant had few skeletal features of Marfan syndrome. Age of onset of EL was later compared to others with cysteine variant changes. Aortic root dilation occurred in 4/16 (25%) of the p.R650C group versus 71/83 (86%) in the comparator group (p < 0.001) and dissection or replacement in 1/31 (3%) versus 20/103 (19%; p < 0.04). Aortic root Z scores were much lower in the p.R650C (0.34 ± 1.70) versus the comparator (2.99 ± 2.54; p < 0.0002). Kaplan-Meier failure curves for aortic root dilation demonstrated later age of onset and differed significantly for incidence rate ratio (comparator vs. p.R650C = 5.35, CI 1.84-21.17; p = 0.0001). Individuals with p.R650C predominantly have EL, but do have risk for aortic dilation at ages later than typical for Marfan syndrome in general and for cysteine changes specifically. Surveillance for aortic dilation is required but may occur less frequently.

Utilization of Whole Exome Sequencing to Identify Causative Mutations in Familial Congenital Heart Disease.

BACKGROUND: Congenital heart disease (CHD) is the most common type of birth defect with family- and population-based studies supporting a strong genetic cause for CHD. The goal of this study was to determine whether a whole exome sequencing (WES) approach could identify pathogenic-segregating variants in multiplex CHD families. METHODS AND RESULTS: WES was performed on 9 kindreds with familial CHD, 4 with atrial septal defects, 2 with patent ductus arteriosus, 2 with tetralogy of Fallot, and 1 with pulmonary valve dysplasia. Rare variants (<1% minor allele frequency) that segregated with disease were identified by WES, and variants in 69 CHD candidate genes were further analyzed. These selected variants were subjected to in silico analysis to predict pathogenicity and resulted in the discovery of likely pathogenic mutations in 3 of 9 (33%) families. A GATA4 mutation in the transactivation domain, p.G115W, was identified in familial atrial septal defects and demonstrated decreased transactivation ability in vitro. A p.I263V mutation in TLL1 was identified in an atrial septal defects kindred and is predicted to affect the enzymatic functionality of TLL1. A disease-segregating splice donor site mutation in MYH11 (c.4599+1delG) was identified in familial patent ductus arteriosus and found to disrupt normal splicing of MYH11 mRNA in the affected individual. CONCLUSIONS: Our findings demonstrate the clinical utility of WES to identify causative mutations in familial CHD and demonstrate the successful use of a CHD candidate gene list to allow for a more streamlined approach enabling rapid prioritization and identification of likely pathogenic variants from large WES data sets. CLINICAL TRIAL REGISTRATION: URL: https://clinicaltrials.gov; Unique Identifier: NCT0112048.

Novel familial dilated cardiomyopathy mutation in MYL2 affects the structure and function of myosin regulatory light chain.

Dilated cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. Here we describe a novel DCM mutation in the myosin regulatory light chain (RLC), in which aspartic acid at position 94 is replaced by alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To obtain insight into the functional significance of this pathogenic MYL2 variant, we cloned and purified the human ventricular RLC wild-type (WT) and D94A mutant proteins, and performed in vitro experiments using RLC-mutant or WT-reconstituted porcine cardiac preparations. The mutation induced a reduction in the α-helical content of the RLC, and imposed intra-molecular rearrangements. The phosphorylation of RLC by Ca²âº/calmodulin-activated myosin light chain kinase was not affected by D94A. The mutation was seen to impair binding of RLC to the myosin heavy chain, and its incorporation into RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared with ATPase of wild-type. No changes in the myofibrillar ATPase-pCa relationship were observed in wild-type- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle, with no change in the calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with wild-type. Our data indicate that subtle structural rearrangements in the RLC molecule, followed by its impaired interaction with the myosin heavy chain, may trigger functional abnormalities contributing to the DCM phenotype.

Association of common variants in ERBB4 with congenital left ventricular outflow tract obstruction defects.

BACKGROUND: The left ventricular outflow tract (LVOT) defects aortic valve stenosis (AVS), coarctation of the aorta (COA), and hypoplastic left heart syndrome (HLHS) represent an embryologically related group of congenital cardiovascular malformations. They are common and cause substantial morbidity and mortality. Prior evidence suggests a strong genetic component in their causation. METHODS: We selected NRG1, ERBB3, and ERBB4 of the epidermal growth factor receptor (EGFR) signaling pathway as candidate genes for investigation of association with LVOT defects based on the importance of this pathway in cardiac development and the phenotypes in knockout mouse models. Single nucleotide polymorphism (SNP) genotyping was performed on 343 affected case-parent trios of European ancestry. RESULTS: We identified a specific haplotype in intron 3 of ERBB4 that was positively associated with the combined LVOT defects phenotype (p=0.0005) and in each anatomic defect AVS, COA, and HLHS separately. Mutation screening of individuals with an LVOT defect failed to identify a coding sequence or splice site change in ERBB4. RT-PCR on lymphoblastoid cells from LVOT subjects did not show altered splice variant ratios among those homozygous for the associated haplotype. CONCLUSION: These results suggest ERBB4 is associated with LVOT defects. Further replication will be required in separate cohorts to confirm the consistency of the observed association.