Beyond gene-disease validity: capturing structured data on inheritance, allelic requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

BACKGROUND: As the availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including secondary findings. METHODS: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. RESULTS: For 36/65 gene-disease pairs, loss of function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using the CardiacG2P dataset as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. CONCLUSIONS: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is a pre-requisite for scalable genomic testing.

Beyond gene-disease validity: capturing structured data on inheritance, allelic-requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

Background: As availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including as secondary findings. Methods: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. Results: For 36/65 gene-disease pairs, loss-of-function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using CardiacG2P as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. Conclusions: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is pre-requisite for scalable genomic testing.

Synergistic effect on cardiac energetics by targeting the creatine kinase system: in vivo application of high-resolution 31P-CMRS in the mouse.

BACKGROUND: Phosphorus cardiovascular magnetic resonance spectroscopy (31P-CMRS) has emerged as an important tool for the preclinical assessment of myocardial energetics in vivo. However, the high rate and diminutive size of the mouse heart is a challenge, resulting in low resolution and poor signal-to-noise. Here we describe a refined high-resolution 31P-CMRS technique and apply it to a novel double transgenic mouse (dTg) with elevated myocardial creatine and creatine kinase (CK) activity. We hypothesised a synergistic effect to augment energetic status, evidenced by an increase in the ratio of phosphocreatine-to-adenosine-triphosphate (PCr/ATP). METHODS AND RESULTS: Single transgenic Creatine Transporter overexpressing (CrT-OE, n = 7) and dTg mice (CrT-OE and CK, n = 6) mice were anaesthetised with isoflurane to acquire 31P-CMRS measurements of the left ventricle (LV) utilising a two-dimensional (2D), threefold under-sampled density-weighted chemical shift imaging (2D-CSI) sequence, which provided high-resolution data with nominal voxel size of 8.5 µl within 70 min. (1H-) cine-CMR data for cardiac function assessment were obtained in the same imaging session. Under a separate examination, mice received invasive haemodynamic assessment, after which tissue was collected for biochemical analysis. Myocardial creatine levels were elevated in all mouse hearts, but only dTg exhibited significantly elevated CK activity, resulting in a 51% higher PCr/ATP ratio in heart (3.01 ± 0.96 vs. 2.04 ± 0.57-mean ± SD; dTg vs. CrT-OE), that was absent from adjacent skeletal muscle. No significant differences were observed for any parameters of LV structure and function, confirming that augmentation of CK activity does not have unforeseen consequences for the heart. CONCLUSIONS: We have developed an improved 31P-CMRS methodology for the in vivo assessment of energetics in the murine heart which enabled high-resolution imaging within acceptable scan times. Mice over-expressing both creatine and CK in the heart exhibited a synergistic elevation in PCr/ATP that can now be tested for therapeutic potential in models of chronic heart failure.

Subtle Role for Adenylate Kinase 1 in Maintaining Normal Basal Contractile Function and Metabolism in the Murine Heart.

AIMS: Adenylate kinase 1 (AK1) catalyses the reaction 2ADP ↔ ATP + AMP, extracting extra energy under metabolic stress and promoting energetic homeostasis. We hypothesised that increased AK1 activity would have negligible effects at rest, but protect against ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: Cardiac-specific AK1 overexpressing mice (AK1-OE) had 31% higher AK1 activity (P = 0.009), with unchanged total creatine kinase and citrate synthase activities. Male AK1-OE exhibited mild in vivo dysfunction at baseline with lower LV pressure, impaired relaxation, and contractile reserve. LV weight was 19% higher in AK1-OE males due to higher tissue water content in the absence of hypertrophy or fibrosis. AK1-OE hearts had significantly raised creatine, unaltered total adenine nucleotides, and 20% higher AMP levels (P = 0.05), but AMP-activated protein kinase was not activated (P = 0.85). 1H-NMR revealed significant differences in LV metabolite levels compared to wild-type, with aspartate, tyrosine, sphingomyelin, cholesterol all elevated, whereas taurine and triglycerides were significantly lower. Ex vivo global no-flow I/R, caused four-of-seven AK1-OE hearts to develop terminal arrhythmia (cf. zero WT), yet surviving AK1-OE hearts had improved functional recovery. However, AK1-OE did not influence infarct size in vivo and arrhythmias were only observed ex vivo, probably as an artefact of adenine nucleotide loss during cannulation. CONCLUSION: Modest elevation of AK1 may improve functional recovery following I/R, but has unexpected impact on LV weight, function and metabolite levels under basal resting conditions, suggesting a more nuanced role for AK1 underpinning myocardial energy homeostasis and not just as a response to stress.

The CHD8 overgrowth syndrome: A detailed evaluation of an emerging overgrowth phenotype in 27 patients.

CHD8 has been reported as an autism susceptibility/intellectual disability gene but emerging evidence suggests that it additionally causes an overgrowth phenotype. This study reports 27 unrelated patients with pathogenic or likely pathogenic CHD8 variants (25 null variants, two missense variants) and a male:female ratio of 21:6 (3.5:1, p < .01). All patients presented with intellectual disability, with 85% in the mild or moderate range, and 85% had a height and/or head circumference ≥2 standard deviations above the mean, meeting our clinical criteria for overgrowth. Behavioral problems were reported in the majority of patients (78%), with over half (56%) either formally diagnosed with an autistic spectrum disorder or described as having autistic traits. Additional clinical features included neonatal hypotonia (33%), and less frequently seizures, pes planus, scoliosis, fifth finger clinodactyly, umbilical hernia, and glabellar hemangioma (≤15% each). These results suggest that, in addition to its established link with autism and intellectual disability, CHD8 causes an overgrowth phenotype, and should be considered in the differential diagnosis of patients presenting with increased height and/or head circumference in association with intellectual disability.

Null variants and deletions in BRWD3 cause an X-linked syndrome of mild-moderate intellectual disability, macrocephaly, and obesity: A series of 17 patients.

BRWD3 has been described as a cause of X-linked intellectual disability, but relatively little is known about the specific phenotype. We report the largest BRWD3 patient series to date, comprising 17 males with 12 distinct null variants and 2 partial gene deletions. All patients presented with intellectual disability, which was classified as moderate (65%) or mild (35%). Behavioral issues were present in 75% of patients, including aggressive behavior, attention deficit/hyperactivity and/or autistic spectrum disorders. Mean head circumference was +2.8 SD (2.8 standard deviations above the mean), and mean BMI was +2.0 SD (in the context of a mean height of +1.3 SD), indicating a predominant macrocephaly/obesity phenotype. Shared facial features included a tall chin, prognathism, broad forehead, and prominent supraorbital ridge. Additional features, reported in a minority (<30%) of patients included cryptorchidism, neonatal hypotonia, and small joint hypermobility. This study delineates the clinical features associated with BRWD3 null variants and partial gene deletions, and suggests that BRWD3 should be included in the differential diagnosis of patients with an overgrowth-intellectual disability (OGID) phenotype, particularly in male patients with a mild or moderate intellectual disability associated with macrocephaly and/or obesity.

Fractal Analysis of Right Ventricular Trabeculae in Pulmonary Hypertension.

Purpose To measure right ventricular (RV) trabecular complexity by its fractal dimension (FD) in healthy subjects and patients with pulmonary hypertension (PH) and to assess its relationship with hemodynamic and functional parameters and future cardiovascular events. Materials and Methods This retrospective study used data acquired from May 2004 to October 2013 in 256 patients with newly diagnosed PH who underwent cardiac MRI, right-sided heart catheterization, and 6-minute walk distance testing, with median follow-up of 4.0 years. A total of 256 healthy control subjects underwent cardiac MRI only. Biventricular FD, volumes, and function were assessed on short-axis cine images. Reproducibility was assessed with the intraclass correlation coefficient, correlation between variables was assessed with the Pearson correlation test, and mortality prediction was compared by using uni- and multivariable Cox regression analyses. Results RV FD reproducibility had an intraclass correlation coefficient of 0.97 (95% confidence interval [CI]: 0.96, 0.98). RV FD was higher in patients with PH (median, 1.310; interquartile range [IQR], 1.281-1.341) than in healthy subjects (median, 1.264; IQR, 1.242-1.295; P < .001), with the greatest difference near the apex. RV FD was associated with pulmonary vascular resistance (r = 0.30, P < .001). At univariable Cox regression analysis, RV FD was a significant predictor of death (hazard ratio [HR], 1.256; 95% CI: 1.011, 1.560; P = .04); however, at multivariable analysis, RV FD did not enable prediction of survival independently of conventional parameters of RV remodeling (HR, 1.179; 95% CI: 0.871, 1.596; P = .29). Conclusion Fractal analysis of RV trabecular complexity is a highly reproducible measure of remodeling in patients with PH that is associated with afterload, although the gain in survival prediction over traditional markers is not significant. Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion.

Aims: Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury. Methods and results: Mice were created over-expressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localization of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1 H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20 min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture, and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55 ± 4% in WT vs. 29 ± 4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Conclusions: Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

Increasing creatine kinase activity protects against hypoxia / reoxygenation injury but not against anthracycline toxicity in vitro.

The creatine kinase (CK) phosphagen system is fundamental to cellular energy homeostasis. Cardiomyocytes express three CK isoforms, namely the mitochondrial sarcomeric CKMT2 and the cytoplasmic CKM and CKB. We hypothesized that augmenting CK in vitro would preserve cell viability and function and sought to determine efficacy of the various isoforms. The open reading frame of each isoform was cloned into pcDNA3.1, followed by transfection and stable selection in human embryonic kidney cells (HEK293). CKMT2- CKM- and CKB-HEK293 cells had increased protein and total CK activity compared to non-transfected cells. Overexpressing any of the three CK isoforms reduced cell death in response to 18h hypoxia at 1% O2 followed by 2h re-oxygenation as assayed using propidium iodide: by 33% in CKMT2, 47% in CKM and 58% in CKB compared to non-transfected cells (P<0.05). Loading cells with creatine did not modify cell survival. Transient expression of CK isoforms in HL-1 cardiac cells elevated isoenzyme activity, but only CKMT2 over-expression protected against hypoxia (0.1% for 24h) and reoxygenation demonstrating 25% less cell death compared to non-transfected control (P<0.01). The same cells were not protected from doxorubicin toxicity (250nM for 48h), in contrast to the positive control. These findings support increased CK activity as protection against ischaemia-reperfusion injury, in particular, protection via CKMT2 in a cardiac-relevant cell line, which merits further investigation in vivo.

A role for thioredoxin-interacting protein (Txnip) in cellular creatine homeostasis.

Creatine is important for energy metabolism, yet excitable cells such as cardiomyocytes do not synthesize creatine and rely on uptake via a specific membrane creatine transporter (CrT; SLC6A8). This process is tightly controlled with downregulation of CrT upon continued exposure to high creatine via mechanisms that are poorly understood. Our aim was to identify candidate endogenous CrT inhibitors. In 3T3 cells overexpressing the CrT, creatine uptake plateaued at 3 h in response to 5 mM creatine but peaked 33% higher (P < 0.01) in the presence of cycloheximide, suggesting CrT regulation depends on new protein synthesis. Global gene expression analysis identified thioredoxin-interacting protein (Txnip) as the only significantly upregulated gene (by 46%) under these conditions (P = 0.036), subsequently verified independently at mRNA and protein levels. There was no change in Txnip expression with exposure to 5 mM taurine, confirming a specific response to creatine rather than osmotic stress. Small-interfering RNA against Txnip prevented Txnip upregulation in response to high creatine, maintained normal levels of creatine uptake, and prevented downregulation of CrT mRNA. These findings were relevant to the in vivo heart since creatine-deficient mice showed 39.71% lower levels of Txnip mRNA, whereas mice overexpressing the CrT had 57.6% higher Txnip mRNA levels and 28.7% higher protein expression compared with wild types (mean myocardial creatine concentration 124 and 74 nmol/mg protein, respectively). In conclusion, we have identified Txnip as a novel negative regulator of creatine levels in vitro and in vivo, responsible for mediating substrate feedback inhibition and a potential target for modulating creatine homeostasis.

Moderate elevation of intracellular creatine by targeting the creatine transporter protects mice from acute myocardial infarction.

AIMS: Increasing energy storage capacity by elevating creatine and phosphocreatine (PCr) levels to increase ATP availability is an attractive concept for protecting against ischaemia and heart failure. However, testing this hypothesis has not been possible since oral creatine supplementation is ineffectual at elevating myocardial creatine levels. We therefore used mice overexpressing creatine transporter in the heart (CrT-OE) to test for the first time whether elevated creatine is beneficial in clinically relevant disease models of heart failure and ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: CrT-OE mice were selected for left ventricular (LV) creatine 20-100% above wild-type values and subjected to acute and chronic coronary artery ligation. Increasing myocardial creatine up to 100% was not detrimental even in ageing CrT-OE. In chronic heart failure, creatine elevation was neither beneficial nor detrimental, with no effect on survival, LV remodelling or dysfunction. However, CrT-OE hearts were protected against I/R injury in vivo in a dose-dependent manner (average 27% less myocardial necrosis) and exhibited greatly improved functional recovery following ex vivo I/R (59% of baseline vs. 29%). Mechanisms contributing to ischaemic protection in CrT-OE hearts include elevated PCr and glycogen levels and improved energy reserve. Furthermore, creatine loading in HL-1 cells did not alter antioxidant defences, but delayed mitochondrial permeability transition pore opening in response to oxidative stress, suggesting an additional mechanism to prevent reperfusion injury. CONCLUSION: Elevation of myocardial creatine by 20-100% reduced myocardial stunning and I/R injury via pleiotropic mechanisms, suggesting CrT activation as a novel, potentially translatable target for cardiac protection from ischaemia.

Chronic creatine kinase deficiency eventually leads to congestive heart failure, but severity is dependent on genetic background, gender and age.

The creatine kinase (CK) energy transport and buffering system supports cardiac function at times of high demand and is impaired in the failing heart. Mice deficient in muscle- and mitochondrial-CK (M/Mt-CK(-/-)) have previously been described, but exhibit an unexpectedly mild phenotype of compensated left ventricular (LV) hypertrophy. We hypothesised that heart failure would develop with age and performed echocardiography and LV haemodynamics at 1 year. Since all previous studies have utilised mice with a mixed genetic background, we backcrossed for >10 generations on to C57BL/6, and repeated the in vivo investigations. Male M/Mt-CK(-/-) mice on the mixed genetic background developed congestive heart failure as evidenced by significantly elevated end-diastolic pressure, impaired contractility, LV dilatation, hypertrophy and pulmonary congestion. Female mice were less severely affected, only showing trends for these parameters. After backcrossing, M/Mt-CK(-/-) mice had LV dysfunction consisting of impaired isovolumetric pressure changes and reduced contractile reserve, but did not develop congestive heart failure. Body weight was lower in knockout mice as a consequence of reduced total body fat. LV weight was not significantly elevated in relation to other internal organs and gene expression of LVH markers was normal, suggesting an absence of hypertrophy. In conclusion, the consequences of CK deficiency are highly dependent on genetic modifiers, gender and age. However, the observation that a primary defect in CK can, under the right conditions, result in heart failure suggests that impaired CK activity in the failing heart could contribute to disease progression.