Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

An ancient founder mutation located between ROBO1 and ROBO2 is responsible for increased microtia risk in Amerindigenous populations.

Microtia is a congenital malformation that encompasses mild hypoplasia to complete loss of the external ear, or pinna. Although the contribution of genetic variation and environmental factors to microtia remains elusive, Amerindigenous populations have the highest reported incidence. Here, using both transmission disequilibrium tests and association studies in microtia trios (parents and affected child) and microtia cohorts enrolled in Latin America, we map an ∼10-kb microtia locus (odds ratio = 4.7; P = 6.78e-18) to the intergenic region between Roundabout 1 (ROBO1) and Roundabout 2 (ROBO2) (chr3: 78546526 to 78555137). While alleles at the microtia locus significantly increase the risk of microtia, their penetrance is low (<1%). We demonstrate that the microtia locus contains a polymorphic complex repeat element that is expanded in affected individuals. The locus is located near a chromatin loop region that regulates ROBO1 and ROBO2 expression in induced pluripotent stem cell­derived neural crest cells. Furthermore, we use single nuclear RNA sequencing to demonstrate ROBO1 and ROBO2 expression in both fibroblasts and chondrocytes of the mature human pinna. Because the microtia allele is enriched in Amerindigenous populations and is shared by some East Asian subjects with craniofacial malformations, we propose that both populations share a mutation that arose in a common ancestor prior to the ancient migration of Eurasian populations into the Americas and that the high incidence of microtia among Amerindigenous populations reflects the population bottleneck that occurred during the migration out of Eurasia.

Discordant clinical features of identical hypertrophic cardiomyopathy twins.

Hypertrophic cardiomyopathy (HCM) is a disease of heart muscle, which affects ∼1 in 500 individuals and is characterized by increased left ventricular wall thickness. While HCM is caused by pathogenic variants in any one of eight sarcomere protein genes, clinical expression varies considerably, even among patients with the same pathogenic variant. To determine whether background genetic variation or environmental factors drive these differences, we studied disease progression in 11 pairs of monozygotic HCM twins. The twin pairs were followed for 5 to 14 y, and left ventricular wall thickness, left atrial diameter, and left ventricular ejection fraction were collected from echocardiograms at various time points. All nine twin pairs with sarcomere protein gene variants and two with unknown disease etiologies had discordant morphologic features of the heart, demonstrating the influence of nonhereditable factors on clinical expression of HCM. Whole genome sequencing analysis of the six monozygotic twins with discordant HCM phenotypes did not reveal notable somatic genetic variants that might explain their clinical differences. Discordant cardiac morphology of identical twins highlights a significant role for epigenetics and environment in HCM disease progression.

Damaging variants in FOXI3 cause microtia and craniofacial microsomia.

PURPOSE: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown. METHODS: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro. RESULTS: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3. CONCLUSION: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.

Left Ventricular Outflow Tract Gradient Is Associated With Coronary Artery Obstruction in Children With Williams-Beuren Syndrome.

OBJECTIVES: Patients with Williams-Beuren syndrome are associated with a high risk of hemodynamic collapse during sedation and/or anesthesia, presumably due to occult coronary obstruction. The objective of this study was to determine the association between transthoracic echocardiogram findings and the presence of coronary obstruction to examine if coronary obstruction can be predicted by transthoracic echocardiogram before anesthesia. DESIGN: Retrospective data analysis of patients with Williams-Beuren syndrome who underwent transthoracic echocardiogram, cardiac catheterization, and/or surgical interventions to determine the correlation between echocardiogram findings and the presence of coronary obstruction determined by cardiac catheterization and/or surgery. SETTING: Single-center university teaching hospital. PARTICIPANTS: The study included 49 patients with Williams-Beuren syndrome who underwent transthoracic echocardiogram, cardiac catheterization, and/or surgical interventions. MEASUREMENTS AND MAIN RESULTS: The only variable associated with coronary artery obstruction was the maximum instantaneous gradient (MIG) across the left ventricular outflow tract (LVOT) on a transthoracic echocardiogram. LVOT MIG ≥ 75 mmHg as the optimal cutoff value was associated with coronary artery obstruction (area under the curve 0.659, odds ratio 6.71, 95% CI 1.31-34.35, p = 0.022). CONCLUSION: LVOT gradient can serve as a good predictor of the presence of coronary obstruction in patients with Williams-Beuren syndrome.

High-Throughput Screening of Kawasaki Disease Sera for Antiviral Antibodies.

Clinical features of Kawasaki disease (KD) display overlap with common pediatric viral illnesses, leading some to hypothesize that a viral infection is the inciting event for KD. To investigate viral infection history in KD patients, we performed comprehensive serological profiling using a high-throughput phage immunoprecipitation sequencing assay covering the complete reference protein sequences of known viruses with human tropism. KD and matched febrile control sera did not demonstrate differences in antiviral antibody profiles. We conclude that in the acute and subacute phases of disease, KD patients do not exhibit serologic evidence of exposure to known viruses that differs from controls.

Concerted regulation of myofiber-specific gene expression and muscle performance by the transcriptional repressor Sox6.

In response to physiological stimuli, skeletal muscle alters its myofiber composition to significantly affect muscle performance and metabolism. This process requires concerted regulation of myofiber-specific isoforms of sarcomeric and calcium regulatory proteins that couple action potentials to the generation of contractile force. Here, we identify Sox6 as a fast myofiber-enriched repressor of slow muscle gene expression in vivo. Mice lacking Sox6 specifically in skeletal muscle have an increased number of slow myofibers, elevated mitochondrial activity, and exhibit down-regulation of the fast myofiber gene program, resulting in enhanced muscular endurance. In addition, microarray profiling of Sox6 knockout muscle revealed extensive muscle fiber-type remodeling, and identified numerous genes that display distinctive fiber-type enrichment. Sox6 directly represses the transcription of slow myofiber-enriched genes by binding to conserved cis-regulatory elements. These results identify Sox6 as a robust regulator of muscle contractile phenotype and metabolism, and elucidate a mechanism by which functionally related muscle fiber-type specific gene isoforms are collectively controlled.

Increased endothelial sclerostin caused by elevated DSCAM mediates multiple trisomy 21 phenotypes.

Trisomy 21 (T21), a recurrent aneuploidy occurring in 1:800 births, predisposes to congenital heart disease (CHD) and multiple extracardiac phenotypes. Despite a definitive genetic etiology, the mechanisms by which T21 perturbs development and homeostasis remain poorly understood. We compared the transcriptome of CHD tissues from 49 patients with T21 and 226 with euploid CHD (eCHD). We resolved cell lineages that misexpressed T21 transcripts by cardiac single-nucleus RNA sequencing and RNA in situ hybridization. Compared with eCHD samples, T21 samples had increased chr21 gene expression; 11-fold-greater levels (P = 1.2 × 10-8) of SOST (chr17), encoding the Wnt inhibitor sclerostin; and 1.4-fold-higher levels (P = 8.7 × 10-8) of the SOST transcriptional activator ZNF467 (chr7). Euploid and T21 cardiac endothelial cells coexpressed SOST and ZNF467; however, T21 endothelial cells expressed 6.9-fold more SOST than euploid endothelial cells (P = 2.7 × 10-27). Wnt pathway genes were downregulated in T21 endothelial cells. Expression of DSCAM, residing within the chr21 CHD critical region, correlated with SOST (P = 1.9 × 10-5) and ZNF467 (P = 2.9 × 10-4). Deletion of DSCAM from T21 endothelial cells derived from human induced pluripotent stem cells diminished sclerostin secretion. As Wnt signaling is critical for atrioventricular canal formation, bone health, and pulmonary vascular homeostasis, we concluded that T21-mediated increased sclerostin levels would inappropriately inhibit Wnt activities and promote Down syndrome phenotypes. These findings imply therapeutic potential for anti-sclerostin antibodies in T21.

Genetic Contribution to End-Stage Cardiomyopathy Requiring Heart Transplantation.

BACKGROUND: Many cardiovascular disorders propel the development of advanced heart failure that necessitates cardiac transplantation. When treatable causes are excluded, studies to define causes are often abandoned, resulting in a diagnosis of end-stage idiopathic cardiomyopathy. We studied whether DNA sequence analyses could identify unrecognized causes of end-stage nonischemic cardiomyopathy requiring heart transplantation and whether the prevalence of genetic causes differed from ambulatory cardiomyopathy cases. METHODS: We performed whole exome and genome sequencing of 122 explanted hearts from 101 adult and 21 pediatric patients with idiopathic cardiomyopathy from a single center. Data were analyzed for pathogenic/likely pathogenic variants in nuclear and mitochondrial genomes and assessed for nonhuman microbial sequences. The frequency of damaging genetic variants was compared among cardiomyopathy cohorts with different clinical severity. RESULTS: Fifty-four samples (44.3%) had pathogenic/likely pathogenic cardiomyopathy gene variants. The frequency of pathogenic variants was similar in pediatric (42.9%) and adult (43.6%) samples, but the distribution of mutated genes differed (P=8.30×10-4). The prevalence of causal genetic variants was significantly higher in end-stage than in previously reported ambulatory adult dilated cardiomyopathy cases (P<0.001). Among remaining samples with unexplained causes, no damaging mitochondrial variants were identified, but 28 samples contained parvovirus genome sequences, including 2 samples with 6- to 9-fold higher levels than the overall mean levels in other samples. CONCLUSIONS: Pathogenic variants and viral myocarditis were identified in 45.9% of patients with unexplained end-stage cardiomyopathy. Damaging gene variants are significantly more frequent among transplant compared with patients with ambulatory cardiomyopathy. Genetic analyses can help define cause of end-stage cardiomyopathy to guide management and risk stratification of patients and family members.

Genomic frontiers in congenital heart disease.

The application of next-generation sequencing to study congenital heart disease (CHD) is increasingly providing new insights into the causes and mechanisms of this prevalent birth anomaly. Whole-exome sequencing analysis identifies damaging gene variants altering single or contiguous nucleotides that are assigned pathogenicity based on statistical analyses of families and cohorts with CHD, high expression in the developing heart and depletion of damaging protein-coding variants in the general population. Gene classes fulfilling these criteria are enriched in patients with CHD and extracardiac abnormalities, evidencing shared pathways in organogenesis. Developmental single-cell transcriptomic data demonstrate the expression of CHD-associated genes in particular cell lineages, and emerging insights indicate that genetic variants perturb multicellular interactions that are crucial for cardiogenesis. Whole-genome sequencing analyses extend these observations, identifying non-coding variants that influence the expression of genes associated with CHD and contribute to the estimated ~55% of unexplained cases of CHD. These approaches combined with the assessment of common and mosaic genetic variants have provided a more complete knowledge of the causes and mechanisms of CHD. Such advances provide knowledge to inform the clinical care of patients with CHD or other birth defects and deepen our understanding of the complexity of human development. In this Review, we highlight known and candidate CHD-associated human genes and discuss how the integration of advances in developmental biology research can provide new insights into the genetic contributions to CHD.

Population Prevalence of Premature Truncating Variants in Plakophilin-2 and Association With Arrhythmogenic Right Ventricular Cardiomyopathy: A UK Biobank Analysis.

BACKGROUND: Truncating variants in the desmosomal gene PKP2 (PKP2tv) cause arrhythmogenic right ventricular cardiomyopathy (ARVC) yet display varied penetrance and expressivity. METHODS: We identified individuals with PKP2tv from the UK Biobank (UKB) and determined the prevalence of an ARVC phenotype and other cardiovascular traits based on clinical and procedural data. The PKP2tv minor allelic frequency in the UKB was compared with a second cohort of probands with a clinical diagnosis of ARVC (ARVC cohort), with a figure of 1:5000 assumed for disease prevalence. In silico predictors of variant pathogenicity (combined annotation-dependent depletion and Splice AI [Illumina, Inc.]) were assessed. RESULTS: PKP2tv were identified in 193/200 643 (0.10%) UKB participants, with 47 unique PKP2tv. Features consistent with ARVC were present in 3 (1.6%), leaving 190 with PKP2tv without manifest disease (UKB cohort; minor allelic frequency 4.73×10-4). The ARVC cohort included 487 ARVC probands with 144 distinct PKP2tv, with 25 PKP2tv common to both cohorts. The odds ratio for ARVC for the 25 common PKP2tv was 0.047 (95% CI, 0.001-0.268; P=2.43×10-6), and only favored ARVC (odds ratio >1) for a single variant, p.Arg79*. In silico variant analysis did not differentiate PKP2tv between the 2 cohorts. Atrial fibrillation was over-represented in the UKB cohort in those with PKP2tv (7.9% versus 4.3%; odds ratio, 2.11; P=0.005). CONCLUSIONS: PKP2tv are prevalent in the population and associated with ARVC in only a small minority, necessitating a more detailed understanding of how PKP2tv cause ARVC in combination with associated genetic and environmental risk factors.

Genome-Wide De Novo Variants in Congenital Heart Disease Are Not Associated With Maternal Diabetes or Obesity.

BACKGROUND: Congenital heart disease (CHD) is the most common anomaly at birth, with a prevalence of ≈1%. While infants born to mothers with diabetes or obesity have a 2- to 3-fold increased incidence of CHD, the cause of the increase is unknown. Damaging de novo variants (DNV) in coding regions are more common among patients with CHD, but genome-wide rates of coding and noncoding DNVs associated with these prenatal exposures have not been studied in patients with CHD. METHODS: DNV frequencies were determined for 1812 patients with CHD who had whole-genome sequencing and prenatal history data available from the Pediatric Cardiac Genomics Consortium's CHD GENES study (Genetic Network). The frequency of DNVs was compared between subgroups using t test or linear model. RESULTS: Among 1812 patients with CHD, the number of DNVs per patient was higher with maternal diabetes (76.5 versus 72.1, t test P=3.03×10-11), but the difference was no longer significant after including parental ages in a linear model (paternal and maternal correction P=0.42). No interaction was observed between diabetes risk and parental age (paternal and maternal interaction P=0.80 and 0.68, respectively). No difference was seen in DNV count per patient based on maternal obesity (72.0 versus 72.2 for maternal body mass index <25 versus maternal body mass index >30, t test P=0.86). CONCLUSIONS: After accounting for parental age, the offspring of diabetic or obese mothers have no increase in DNVs compared with other children with CHD. These results emphasize the role for other mechanisms in the cause of CHD associated with these prenatal exposures. REGISTRATION: URL: https://clinicaltrials.gov; NCT01196182.

Association of Damaging Variants in Genes With Increased Cancer Risk Among Patients With Congenital Heart Disease.

Importance: Patients with congenital heart disease (CHD), the most common birth defect, have increased risks for cancer. Identification of the variables that contribute to cancer risk is essential for recognizing patients with CHD who warrant longitudinal surveillance and early interventions. Objective: To compare the frequency of damaging variants in cancer risk genes among patients with CHD and control participants and identify associated clinical variables in patients with CHD who have cancer risk variants. Design, Setting, and Participants: This multicenter case-control study included participants with CHD who had previously been recruited to the Pediatric Cardiac Genomics Consortium based on presence of structural cardiac anomaly without genetic diagnosis at the time of enrollment. Permission to use published sequencing data from unaffected adult participants was obtained from 2 parent studies. Data were collected for this study from December 2010 to April 2019. Exposures: Presence of rare (allele frequency, <1 × 10-5) loss-of-function (LoF) variants in cancer risk genes. Main Outcomes and Measures: Frequency of LoF variants in cancer risk genes (defined in the Catalogue of Somatic Mutations in Cancer-Cancer Gene Consensus database), were statistically assessed by binomial tests in patients with CHD and control participants. Results: A total of 4443 individuals with CHD (mean [range] age, 13.0 [0-84] years; 2225 of 3771 with reported sex [59.0%] male) and 9808 control participants (mean [range] age, 52.1 [1-92] years; 4967 of 9808 [50.6%] male) were included. The frequency of LoF variants in regulatory cancer risk genes was significantly higher in patients with CHD than control participants (143 of 4443 [3.2%] vs 166 of 9808 [1.7%]; odds ratio [OR], 1.93 [95% CI, 1.54-2.42]; P = 1.38 × 10-12), and among CHD genes previously associated with cancer risk (58 of 4443 [1.3%] vs 18 of 9808 [0.18%]; OR, 7.2 [95% CI, 4.2-12.2]; P < 2.2 × 10-16). The LoF variants were also nominally increased in 14 constrained cancer risk genes with high expression in the developing heart. Seven of these genes (ARHGEF12, CTNNB1, LPP, MLLT4, PTEN, TCF12, and TFRC) harbored LoF variants in multiple patients with unexplained CHD. The highest rates for LoF variants in cancer risk genes occurred in patients with CHD and extracardiac anomalies (248 of 1482 individuals [16.7%]; control: 1099 of 9808 individuals [11.2%]; OR, 1.59 [95% CI, 1.37-1.85]; P = 1.3 × 10-10) and/or neurodevelopmental delay (209 of 1393 individuals [15.0%]; control: 1099 of 9808 individuals [11.2%]; OR, 1.40 [95% CI, 1.19-1.64]; P = 9.6 × 10-6). Conclusions and Relevance: Genotypes of CHD may account for increased cancer risks. In this cohort, damaging variants were prominent in the 216 genes that predominantly encode regulatory proteins. Consistent with their fundamental developmental functions, patients with CHD and damaging variants in these genes often had extracardiac manifestations. These data may also implicate cancer risk genes that are repeatedly varied in patients with unexplained CHD as CHD genes.

Retrospective Analysis of Clinical Genetic Testing in Pediatric Primary Dilated Cardiomyopathy: Testing Outcomes and the Effects of Variant Reclassification.

Background Genetic testing in pediatric primary dilated cardiomyopathy (DCM) patients has identified numerous disease-causing variants, but few studies have evaluated genetic testing outcomes in this population in the context of patient and familial clinical data or assessed the clinical implications of temporal changes in genetic testing results. Methods and Results We performed a retrospective analysis of all patients with primary DCM who presented to our institution between 2008 and 2018. Variants identified by genetic testing were reevaluated for pathogenicity on the basis of current guidelines for variant classification. A total of 73 patients with primary DCM presented to our institution and 63 (86%) were probands that underwent cardiomyopathy-specific gene testing. A disease-causing variant was identified in 19 of 63 (30%) of cases, with at least 9/19 (47%) variants occurring de novo. Positive family history was not associated with identification of a causal variant. Reclassification of variants resulted in the downgrading of a large proportion of variants of uncertain significance and did not identify any new disease-causing variants. Conclusions Clinical genetic testing identifies a causal variant in one third of pediatric patients with primary DCM. Variant reevaluation significantly decreased the number of variants of uncertain significance, but a large burden of variants of uncertain significance remain. These results highlight the need for periodic reanalysis of genetic testing results, additional investigation of genotype-phenotype correlations in DCM through large, multicenter genetic studies, and development of improved tools for functional characterization of variants of uncertain significance.

MicroRNAs in cardiovascular disease: from pathogenesis to prevention and treatment.

The management of cardiovascular risk through lifestyle modification and pharmacotherapy is paramount to the prevention of cardiovascular disease. Epidemiological studies have identified obesity, dyslipidemia, diabetes, and hypertension as interrelated factors that negatively affect cardiovascular health. Recently, genetic and pharmacological evidence in model systems has implicated microRNAs as dynamic modifiers of disease pathogenesis. An expanded understanding of the function of microRNAs in gene regulatory networks associated with cardiovascular risk will enable identification of novel genetic mechanisms of disease and inform the development of innovative therapeutic strategies.

A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance.

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.