Olson, Timothy M

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD) whose genetic basis remains elusive, likely due to oligogenic complexity. To identify regulators of cardiomyocyte (CM) proliferation relevant to HLHS, we performed a genome-wide siRNA screen in human iPSC-derived CMs, revealing ribosomal protein (RP) genes as the most prominent effectors of CM proliferation. Whole-genome sequencing of 25 HLHS proband–parent trios similarly showed enrichment of rare RP gene variants, including a damaging RPS15A promoter variant shared in a familial CHD case. Cross-species functional analyses demonstrated that perturbation of RP genes impairs cardiac growth: knockdown of RPS15A, RPS17, RPL26L1, RPL39, or RPS15 reduced CM proliferation, caused cardiac malformations in Drosophila , and produced hypoplastic or dysfunctional hearts in zebrafish. Genetic interactions between RP genes and key cardiac transcription factors (TBX5 and NKX2–7) further support their developmental role. Importantly, p53 suppression or Hippo activation partially rescued RP deficiency phenotypes. Together, these findings implicate RP genes as critical regulators of cardiogenesis and candidate contributors to HLHS.

Rare familial spontaneous coronary artery dissection (SCAD) kindreds implicate genetic disease predisposition and provide a unique opportunity for candidate gene discovery. Whole-genome sequencing was performed in fifteen probands with non-syndromic SCAD who had a relative with SCAD, eight of whom had a second relative with extra-coronary arteriopathy. Co-segregating variants and associated genes were prioritized by quantitative variant, gene, and disease-level metrics. Curated public databases were queried for functional relationships among encoded proteins. Fifty-four heterozygous coding variants in thirteen families co-segregated with disease and fulfilled primary filters of rarity, gene variation constraint, and predicted-deleterious protein effect. Secondary filters yielded 11 prioritized candidate genes in 12 families, with high arterial tissue expression (n = 7), high-confidence protein-level interactions with genes associated with SCAD previously (n = 10), and/or previous associations with connective tissue disorders and aortopathies (n = 3) or other vascular phenotypes in mice or humans (n = 11). High-confidence associations were identified among 10 familial SCAD candidate-gene-encoded proteins. A collagen-encoding gene was identified in five families, two with distinct variants in COL4A2. Familial SCAD is genetically heterogeneous, yet perturbations of extracellular matrix, cytoskeletal, and cell–cell adhesion proteins implicate common disease-susceptibility pathways. Incomplete penetrance and variable expression suggest genetic or environmental modifiers.

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.

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.

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.

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.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to parents the proband’s iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

AbstractGenetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking ‘no heart’ phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.

Hypoplastic left heart syndrome is one of the most complex congenital heart diseases and requires several cardiac surgeries for survival. The diagnosis is usually established prenatally or shortly after birth. Each stage of surgery poses a unique hemodynamic situation that requires deeper understanding to manage common pediatric problems such as dehydration and respiratory infections. Careful multidisciplinary involvement in the care of these complex patients is improving their outcome; however, morbidity and mortality are still substantial. In this review, we focus on the hemodynamic aspects of various surgical stages that a primary care provider should know to manage these challenging patients.