Olson, Timothy M.

Hypoplastic left heart syndrome (HLHS) is a genetically complex disorder. Whole genome sequencing enables comprehensive scrutiny of single nucleotide variants and small insertions/deletions, within both coding and regulatory regions of the genome, revolutionizing susceptibility-gene discovery research. Because millions of rare variants comprise an individual genome, identification of alleles linked to HLHS necessitates filtering algorithms based on various parameters, such as inheritance, enrichment, omics data, known genotype–phenotype associations, and predictive or experimental modeling. In this brief review, we highlight family and cohort-based strategies used to analyze whole genome sequencing datasets and identify HLHS candidate genes. Key findings include compound and digenic heterozygosity among several prioritized genes and genetic associations between HLHS and bicuspid aortic valve or cardiomyopathy. Together with findings of independent genomic investigations, MYH6 has emerged as a compelling disease gene for HLHS and other left-sided congenital heart diseases.

Leucine-rich repeat containing (LRR) proteins facilitate protein-protein interactions critical in a number of cellular functions including ion channel regulation. Leucine-rich repeat containing protein 10 (LRRC10), a cardiac-specific protein expressed in zebrafish, mice and humans, is essential for normal cardiac physiology with loss of Lrrc10 in Lrrc10 -/- mice resulting in dilated cardiomyopathy (DCM). However, the mechanism by which LRRC10 contributes to DCM is unknown. Further, the functional role of LRRC10 in the regulation of ion channels in the heart has not been explored. Here we recorded L-type Ca 2+ channel currents (I Ca,L ) from isolated ventricular myocytes of WT and Lrrc10 -/- mice, demonstrating a significant reduction in I Ca,L in Lrrc10 -/- myocytes (-4.9 ± 0.19 pA/pF, n=10) compared to WT (-6.5 ± 0.24 pA/pF, n=8). Co-immunoprecipitation (co-IP) experiments showed an association between LRRC10 and Ca v 1.2 in WT, but not Lrrc10 -/- mouse lysates, with immunocytochemistry studies further demonstrating colocalization of LRRC10 and Ca v 1.2 in isolated ventricular myocytes from wild-type (WT) mice. Additionally, whole exome sequencing revealed a novel homozygous recessive missense variant in LRRC10 , I195T, found in a pediatric DCM patient. To determine whether the I195T variant impacts Ca v 1.2 L-type Ca 2+ channels, whole-cell patch clamp experiments were performed using HEK293 cells transiently expressing the L-type Ca 2+ channel complex (LTCC) alone or with WT or I195T LRRC10. These electrophysiology experiments demonstrated a significant increase in I Ca,L with WT LRRC10 coexpression (-81 ± 5.3 pA/pF, n=12), but a decrease in I Ca,L when I195T (-18.2 ± 3.3 pA/pF, n=9) was coexpressed compared to the LTCC alone (-34.1 ± 2.2 pA/pF, n=17). Parallel surface biotinylation experiments demonstrated that WT and I195T LRRC10 did not alter L-type Ca 2+ channel expression on the plasma membrane, while co-IP experiments using lysates prepared from the transiently transfected HEK293 cells showed the association between Ca v 1.2 and I195T LRRC10 variant. Overall, these findings newly identify LRRC10 as a cardiac-specific component of the Ca v 1.2 macromolecular complex and demonstrate dysregulation of I Ca,L by the DCM associated I195T LRRC10 variant.

Background: Whole-genome sequencing in families enables deciphering of congenital heart disease causes. A shared genetic basis for familial bicuspid aortic valve (BAV) and hypoplastic left heart syndrome (HLHS) was postulated. Methods: Whole-genome sequencing was performed in affected members of 6 multiplex BAV families, an HLHS cohort of 197 probands and 546 relatives, and 813 controls. Data were filtered for rare, predicted-damaging variants that cosegregated with familial BAV and disrupted genes associated with congenital heart disease in humans and mice. Candidate genes were further prioritized by rare variant burden testing in HLHS cases versus controls. Modifier variants in HLHS proband-parent trios were sought to account for the severe developmental phenotype. Results: In 5 BAV families, missense variants in 6 ontologically diverse genes for structural ( SPTBN1 , PAXIP1 , and FBLN1 ) and signaling ( CELSR1 , PLXND1 , and NOS3 ) proteins fulfilled filtering metrics. CELSR1 , encoding cadherin epidermal growth factor laminin G seven-pass G-type receptor, was identified as a candidate gene in 2 families and was the only gene demonstrating rare variant enrichment in HLHS probands ( P =0.003575). HLHS-associated CELSR1 variants included 16 missense, one splice site, and 3 noncoding variants predicted to disrupt canonical transcription factor binding sites, most of which were inherited from a parent without congenital heart disease. Filtering whole-genome sequencing data for rare, predicted-damaging variants inherited from the other parent revealed 2 cases of CELSR1 compound heterozygosity, one case of CELSR1 - CELSR3 synergistic heterozygosity, and 4 cases of CELSR1 - MYO15A digenic heterozygosity. Conclusions: CELSR1 is a susceptibility gene for familial BAV and HLHS, further implicating planar cell polarity pathway perturbation in congenital heart disease.

Hypoplastic Left Heart Syndrome (HLHS) is a complex multifactorial disease for which no definitive genetic causes have been found. Current genetic filtering strategies render lists of genes with unknown relevance in terms of pathogenesis. A complementary filter based on biological evidence would create a new approach to prioritize relevant candidate genes and mutations. In our study, 5 members of a nuclear family including a child with HLHS were evaluated using echocardiography and their genetic information was obtained through whole genome sequencing (WGS). Data filtering including rarity, functional impact and mode of inheritance was implemented, resulting in identification of 34 genes with recessive or de novo variants potentially involved in the pathogenesis of HLHS. Additionally, iPSC were derived from proband and parents and subjected to RNA-sequencing at the undifferentiated state and following spontaneous differentiation. Comparative transcriptional analyses identified genes differentially expressed in proband samples at each stage. These gene sets were used as an additional filter for the previously generated WGS data. This strategy revealed that out of 34 mutated genes originally identified, 10 displayed transcriptional differences in undifferentiated iPSC from the HLHS-affected individual while 16 out of 34 mutated genes showed significantly different expression levels in differentiated cells from proband. Furthermore, expression dynamics were studied during guided cardiac differentiation for the 9 genes fulfilling all applied criteria. Two genes not previously linked to HLHS, ELF4 and HSPG2 were found to behave significantly different in HLHS-iPSC when compared to control counterparts. In summary, filtering WGS data according to a new layer of transcriptional information that leverages iPSC plasticity allows prioritization of genes associated with HLHS in an in vitro model of disease.

Background: Spontaneous coronary artery dissection (SCAD) typically presents as acute coronary syndrome in young women with no cardiovascular risk factors. It is associated with high morbidity and mortality, and is underdiagnosed. Despite this, risk factors and pathophysiology associated with SCAD are not completely understood. The objective of the present case series was to assess family history as a risk factor in patients with SCAD. Methods: We reviewed the medical records and questionnaires of 335 participants in the Mayo Clinic SCAD Registry for history of MI, SCAD or CAD in another family member. Coronary angiograms of all affected family members were reviewed by a senior interventional cardiologist. Results: We identified 4 familial cases of SCAD comprised of affected mother-daughter, identical twin sister, sister and aunt-niece pairs, implicating both recessive and dominant modes of inheritance. The mother-daughter pair also reported history of fatal MI in 3 maternal relatives including grandmother, great grandmother and great aunt. One of the affected twin sisters died of SCAD, which was confirmed on autopsy. None of the subjects had other potential risk factors for SCAD including extreme exercise, polycystic kidney disease, connective tissue disorder, peripartum status or diagnosed non-coronary fibromuscular dysplasia. The details of their demographics, risk factors, presentation, location of SCAD and management are presented in the table. Conclusions: This series is the first to identify a familial association in SCAD suggesting a genetic predisposition. Recognition of SCAD as a heritable disorder has implications for at-risk family members and furthers our understanding of the pathophysiology of this complex disease. Whole exome sequencing in these families provides a unique opportunity to identify the molecular underpinnings of SCAD susceptibility.

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.

Background: Hypoplastic left heart syndrome (HLHS) with risk of poor outcome has been linked to MYH6 variants, implicating overlap in genetic etiologies of structural and myopathic heart disease. Methods: Whole genome sequencing was performed in 197 probands with HLHS, 43 family members, and 813 controls. Data were filtered for rare, segregating variants in 3 index families comprised of an HLHS proband and relative(s) with cardiomyopathy. Whole genome sequencing data from cases and controls were compared for rare variant burden across 56 cardiomyopathy genes utilizing a weighted burden test approach, accounting for multiple testing using a Bonferroni correction. Results: A pathogenic MYBPC3 nonsense variant was identified in the first proband who underwent cardiac transplantation for diastolic heart failure, her father with left ventricular noncompaction, and 2 fourth-degree relatives with hypertrophic cardiomyopathy. A likely pathogenic RYR2 missense variant was identified in the second proband, a second-degree relative with aortic dilation, and a fourth-degree relative with dilated cardiomyopathy. A pathogenic RYR2 exon 3 in-frame deletion was identified in the third proband diagnosed with catecholaminergic polymorphic ventricular tachycardia and his father with left ventricular noncompaction and catecholaminergic polymorphic ventricular tachycardia. To further investigate HLHS-cardiomyopathy gene associations in cases versus controls, rare variant burden testing of 56 genes revealed enrichment in MYH6 ( P =0.000068). Rare, predicted-damaging MYH6 variants were identified in 10% of probands in our cohort—4 with familial congenital heart disease, 4 with compound heterozygosity (3 with systolic ventricular dysfunction), and 4 with MYH6 – FLNC synergistic heterozygosity. Conclusions: Whole genome sequencing in multiplex families, proband-parent trios, and case-control cohorts revealed defects in cardiomyopathy-associated genes in patients with HLHS, which may portend impaired functional reserve of the single-ventricle circulation.