Tatiana Yuzyuk

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.

Metabolic Remodeling in Moderate Synchronous versus Dyssynchronous Pacing-Induced Heart Failure: Integrated Metabolomics and Proteomics Study

Heart failure (HF) is accompanied by complex alterations in myocardial energy metabolism. Up to 40% of HF patients have dyssynchronous ventricular contraction, which is an independent indicator of mortality. We hypothesized that electromechanical dyssynchrony significantly affects metabolic remodeling in the course of HF. We used a canine model of tachypacing-induced HF. Animals were paced at 200 bpm for 6 weeks either in the right atrium (synchronous HF, SHF) or in the right ventricle (dyssynchronous HF, DHF). We collected biopsies from left ventricular apex and performed comprehensive metabolic pathway analysis using multi-platform metabolomics (GC/MS; MS/MS; HPLC) and LC-MS/MS label-free proteomics. We found important differences in metabolic remodeling between SHF and DHF. As compared to Control, ATP, phosphocreatine (PCr), creatine, and PCr/ATP (prognostic indicator of mortality in HF patients) were all significantly reduced in DHF, but not SHF. In addition, the myocardial levels of carnitine (mitochondrial fatty acid carrier) and fatty acids (12:0, 14:0) were significantly reduced in DHF, but not SHF. Carnitine parmitoyltransferase I, a key regulatory enzyme of fatty acid ß-oxidation, was significantly upregulated in SHF but was not different in DHF, as compared to Control. Both SHF and DHF exhibited a reduction, but to a different degree, in creatine and the intermediates of glycolysis and the TCA cycle. In contrast to this, the enzymes of creatine kinase shuttle were upregulated, and the enzymes of glycolysis and the TCA cycle were predominantly upregulated or unchanged in both SHF and DHF. These data suggest a systemic mismatch between substrate supply and demand in pacing-induced HF. The energy deficit observed in DHF, but not in SHF, may be associated with a critical decrease in fatty acid delivery to the ß-oxidation pipeline, primarily due to a reduction in myocardial carnitine content.

Effect of dietary lysine restriction and arginine supplementation in two patients with pyridoxine-dependent epilepsy

Pyridoxine-Dependent Epilepsy (PDE) is a recessive disorder caused by deficiency of α-aminoadipic semialdehyde dehydrogenase in the catabolic pathway of lysine. It is characterized by intractable seizures controlled by the administration of pharmacological doses of vitamin B6. Despite seizure control with pyridoxine, intellectual disability and developmental delays are still observed in some patients with PDE, likely due to the accumulation of toxic intermediates in the lysine catabolic pathway: alpha-aminoadipic semialdehyde (AASA), delta-1-piperideine-6-carboxylate (P6C), and pipecolic acid. Here we evaluate biochemical and clinical parameters in two PDE patients treated with a lysine-restricted diet and arginine supplementation (100-150mg/kg), aimed at reducing the levels of PDE biomarkers. Lysine restriction resulted in decreased accumulation of PDE biomarkers and improved development. Plasma lysine but not plasma arginine, directly correlated with plasma levels of AASA-P6C (p<0.001, r(2)=0.640) and pipecolic acid (p<0.01, r(2)=0.484). In addition, plasma threonine strongly correlated with the levels of AASA-P6C (p<0.0001, r(2)=0.732) and pipecolic acid (p<0.005, r(2)=0.527), suggesting extreme sensitivity of threonine catabolism to pyridoxine availability. Our results further support the use of dietary therapies in combination with pyridoxine for the treatment of PDE.

Mild orotic aciduria in UMPS heterozygotes: a metabolic finding without clinical consequences

BackgroundElevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue.Methods and resultsWe identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members.ConclusionsWe therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

Intra-individual variability of long-chain fatty acids (C12–C24) in plasma and red blood cells

Long-chain fatty acids (LCFA) play key roles in mammalian cells as sources of energy, structural components and signaling molecules. Given their importance in numerous physiological processes, the roles of LCFAs in health and disease have been extensively investigated. In the majority of studies, correlations are established using a single measurement in plasma or red blood cells (RBCs). Although a few studies have reported on reproducibility of individual fatty acid measurements, the comprehensive analysis of intra-individual LCFA variability has not been performed. Therefore, our goal was to determine intra-individual variability for the 22 most abundant LCFAs in both plasma and RBC samples collected from healthy individuals on a regular diet after overnight fasting. The measurements of LCFAs in RBCs were consistent throughout the course of study reflecting long-term nutritional status. In contrast, the results in plasma showed considerable LCFA intra-individual variability, even between fatty acids of the same type. Plasma intra-individual variability for omega-3 alpha-linolenic and eicosapentaenoic acids in some participants were >40% whereas the variability of docosahexaenoic acid was consistently <12.8%. Omega-6 linoleic and arachidonic acids also showed low variability in plasma. The results suggest that some LCFAs have less variability and would be more reliable as biomarkers. Reliability of biomarkers can have a profound impact on the research outcomes. Intra-individual variability of LCFAs should be taken into consideration in designing, conducting and interpreting results of clinical studies.

Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Significance Smyd1 is a muscle-specific histone methyltransferase, and its role in the regulation of growth and differentiation in skeletal and cardiac muscle is well established. However, despite the persistent expression of Smyd1 in postnatal cardiomyocytes, the role of Smyd1 in the adult heart is largely unknown. We show that Smyd1 regulates energy metabolism in the heart. Cardiac-specific ablation of Smyd1 in the mouse adult heart resulted in global downregulation of mitochondrial proteins involved in oxidative phosphorylation, concurrent with reduced mitochondrial respiration capacity. We further demonstrate that the regulation of Smyd1 in metabolism is through transcriptional control of PGC-1α, a key regulator of mitochondrial energetics. Thus, our data reveal a role for Smyd1 as a master regulator of cardiac energetics. Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1’s involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Exploring the perceived self-efficacy of genetic counselors as teachers

In addition to counseling and educating patients, genetic counselors are critical in teaching genetic concepts and counseling techniques to genetic counseling students, both as faculty within accredited programs or as supervisors of new graduates during the initial on-the-job training. The majority of counselors surveyed by the National Society of Genetic Counselors (72%) reported involvement in teaching and other education activities (NSGC 2016). Moreover, the Accreditation Council for Genetic Counseling (ACGC) included BEducation^ as one of the four core domains for the genetic counseling profession (ACGC 2013; Doyle et al. 2016). These four core domains help to categorize the 22 practice-based competencies (PBC) that genetic counselors should master to successfully practice, and to inform genetic counseling training (ACGC 2013). PBC#16 (III Domain) specifically addresses the genetic counselors’ educational role, which include goals assessment, curriculum development, and the use of feedback, and it is distinct from clinical supervision (PBC#21in IV Domain) (ACGC 2013; Doyle et al. 2016). Although their role supervising students in clinical settings (clinical supervision) has been explored (Atzinger et al. 2014; Finley et al. 2016; Atzinger et al. 2016), no research is available on the experience of genetic counselors in their role as teachers, the resources used to prepare for this role, or the tools used to give and receive feedback. Srinivasan et al. (2011) built the BTeaching as a Competency^ framework utilizing a variety of methods, which included review of teaching literature, a 2-day medical educator conference focusing on educational competencies, and presentations at regional and national meetings. This conceptual model for medical educators utilizes the six core physician competencies initially developed by the Accreditation Council for Graduate Medical Education to evaluate trainees in medical education programs (Srinavasan et al. 2011; Joyner 2004; Swing 2007; ACGME 2016). The ACGME competencies were modified to identify the skills critical for medical education: (1) medical (or content) knowledge, (2) learnercenteredness, (3) interpersonal and communication skills, (4) professionalism and role modeling, (5) practice-based reflection, and (6) systems-based practice. This framework aims to enhance teaching quality and promote dialog about medical educator training, development, and outcomes. Recently, this framework has been further evaluated by Brink et al. (2018) using a modified Delphi process. Data directly evaluating genetic counselors’ teaching is difficult to obtain from direct observation, when considering the limited size of each program and the lack of formalized tools for gathering information. In the past, self-efficacy, the personal belief in one’s capability of performing a task, has been investigated as predictor of performance within the context of medical education, both assessing students’ academic performance (de Saintonge and Dunn 2001; Valentine et al. 2004; Gore 2006; Burgoon et al. 2012) and physicians’ teaching (Dybowski et al. 2016). Self-efficacy has been postulated to influence behavior and have predictive value on outcomes (Bandura 1995, 1997, 2006). Perceived self-efficacy is an important measure because it affects behavior and influences goals, perceived barriers, and outcome expectations, which remain important in teaching (Bandura 1997). Although selfperceived efficacy does not guarantee actual ability, Bandura’s framework has shown an association to positive classroom Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12687-018-0380-6) contains supplementary material, which is available to authorized users.