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Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex’s role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6 . Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 ( goliath , E3 ubiquitin ligase), or SPTBN1 ( β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Introduction: Spontaneous coronary artery dissection (SCAD) is an understudied cause of acute myocardial infarction due to hematoma formation in coronary arteries primarily affecting women. It is not known to what extent SCAD is genetically distinct from other cardiovascular diseases, including atherosclerotic coronary artery disease (CAD). Methods and Results: Through a meta-analysis of 8 GWAS (1917 cases, 9292 controls of European ancestry), we identified 17 risk loci, including 12 new. Functional annotations pointed at enrichment in enhancer marks of arteries, specifically smooth muscle cells, and strong candidate genes, such as tissue factor gene ( F3 ) on Chr1 near rs1146473 (OR=1.32, P=5.8 х10 -9 ). F3 is novel for SCAD or any cardiovascular disease. The risk allele correlated with F3 lower expression in arteries, supporting a mechanism consistent with hematoma formation. Bayesian gene regulatory networks constructed from expression and genetics data indicated the extracellular matrix organization in arteries as the biological function where most prioritized genes clustered (e.g. COL4A1/A2, HTRA1, and TIMP3 ). Overall, we report substantial polygenicity for SCAD (LDSC: h 2 SNP = 0.71 ± 0.11) and shared genetics with several neurovascular diseases (e.g intracranial aneurysm). Intriguingly, for 6 loci, colocalization analyses showed that SCAD and CAD are likely to share the same causal variants but involve opposite risk alleles (e.g COL4A1/A2 ). In addition, a negative genetic correlation was found between SCAD and CAD (rg=-0.12; P=3.7х10 -3 ), including after conditioning on BP (mt-COJO: rg CAD/SBP =-0.19, P=4.6х10 -6 ). Mendelian randomization analyses indicated higher BP to associate with increased risk for SCAD (beta IVW-SBP =0.05, P=7.6х10 -6 , beta IVW-DBP =0.10, P=1.9х10 -6 ) and CAD (beta IVW-SBP =0.04, P=8.6х10 -49 ; DBP: beta IVW-DBP =0.06, P=1.6х10 -44 ) but not BMI, lipids, or type 2 diabetes, which we confirmed as genetic risk factors for CAD Conclusions: Our results set the stage for future investigation of novel biological pathways relevant to both SCAD and CAD and potential therapeutic and preventive strategies specifically targeting this ischemic disease predominantly affecting women.

Background: Deciphering hypoplastic left heart syndrome (HLHS) pathogenesis is confounded by its genetic heterogeneity and oligogenic underpinnings. Methods: Whole genome sequences were analyzed by 3 independent strategies to identify HLHS gene candidates, ranked by variant, gene, and disease-level metrics. Results: First, a genome-wide association study of 174 cases and 853 controls revealed suggestive association with a MYO18B intron 33 variant (rs2269628-G; frequency=0.55 versus 0.39; OR, 1.97 [95% CI, 1.54–2.52]; P =6.70×10 − 8 ). Second, transmission disequilibrium testing of 161 HLHS proband-parent trios revealed overrepresentation of a MYO18B intron 42 variant (rs73154186-A; frequency=0.05; OR, 24 [95% CI, 3.2–177.4]; P =4.23×10 − 6 ). Third, rare, predicted-damaging variants were filtered in 2 multiplex families. In 141H, 2 fifth-degree relatives with HLHS shared a paternally-inherited MYO5A missense variant (p.Arg801Trp; frequency=0.00003; combined annotation-dependent depletion score=29), each with a maternally-inherited or de novo candidate modifier variant in a MYO5A-interacting conventional myosin. In 442H, a HLHS proband was compound heterozygous for MYO15A variants—a maternally-inherited pathogenic stop-gain variant co-segregating with tetralogy of Fallot and bicuspid aortic valve in maternal relatives (p.Tyr2819Ter; frequency=0.00003) and a paternally-inherited intronic variant altering a canonical transcription factor binding site (rs1277068603; frequency=0.00001; position weight matrix score=0.98). Conclusions: Collectively, these findings suggest that common and rare alleles within unconventional myosin genes are associated with HLHS susceptibility. The identified candidate MYO18B regulates cardiac sarcomerogenesis, supporting the hypothesis of intrinsic myogenic perturbation in arrested left heart development.

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.