Abstract Hereditary hemochromatosis (HH) is a common autosomal recessive disorder estimated to affect one out of every 250–400 Caucasian individuals. It is a disorder of iron metabolism, in which excessive iron accumulation in the body may induce serious clinical manifestations (e.g. liver cirrhosis, hepatocellular carcinoma, diabetes, and cardiomyopathy). HH is caused by mutations in the HFE gene, and HFE genotyping thus enables early diagnosis of the disease and detection of the individuals at risk for HH. HFE mutations have also been proposed to predispose to certain other diseases, such as various hematological malignancies and cardiomyopathy. The present evaluation of the clinical utility and outcome of HFE genotyping in search for HH was based on data obtained from 137 subjects referred for HFE mutation analysis during the years 1999–2001. The C282Y and H63D mutations were determined for each subject. HFE genotyping was also used to examine the association between HFE mutations with various hematological disorders and idiopathic dilated cardiomyopathy (IDCM). The C282Y and H63D mutations were determined from 232 patients with various hematological disorders and the C282Y, H63D, and S65C mutations from 91 patients with IDCM and 102 control subjects. High frequencies of C282Y homozygotes (16.8%) and C282Y/H63D compound heterozygotes (5.1%) were found among the subjects referred for HFE genotyping, and the rate of positive findings for HH increased steadily over the years 1999–2001. The frequencies of HFE mutations did not differ significantly in patients with various hematological disorders and IDCM compared to controls. At the end of the follow-up period, left ventricular end-diastolic diameter (LVEDD) was significantly higher in IDCM patients carrying the C282Y mutation than in those without this mutation (p = 0.037). The present study supports active testing for the HFE gene mutations C282Y and H63D in public health care. Serum transferrin saturation is considered the most useful test for selecting subjects for such analysis. Although increasing numbers of HH cases are recognized by physicians, it may still be an underdiagnosed disease. HFE mutations do not seem to significantly increase the risk for various hematological disorders or IDCM. The C282Y mutation may, nevertheless, mediate the progression of IDCM by modifying LV dilation and remodeling.

The E2F family of transcription factors regulate cellular growth, death and differentiation, but their role in cardiac biology remains to be fully explored. We hypothesized that the balance of the E2F pathway would determine cardiac development and function. We provide evidence for this via modulation of the E2F6 repressor, in a transgenic (Tg) mouse model. Targeted expression of E2F6 in the heart led to dilated cardiomyopathy (DCM) and death. Microarray analysis revealed that E2F responsive pathways were activated in Tg mice. Furthermore, we found that E2F6 and YY1 (E2F-co-factor) were translocated to the nucleus in Tg mice, providing a potential mechanism for the observed transcriptional activation. We also observed a marked decrease of Connexin43 protein in the myocardium, and reduced atrial conductivity in Tg mice which may lead to reduced cardiac function. The data demonstrates a novel role for E2F pathway outside of cell cycle control in the heart.

Cardiomyopathies are the most common form of genetic disorders featuring primary abnormalities in the structure and function of the heart. Over the past few decades, tremendous progress has been made in elucidating the genetic basis of cardiac disorders. However, the development of specific and effective therapies remains largely limited due to the lack of suitable therapeutic targets. Nucleoli are polyfunctional subnuclear domains that are heavily involved in ribosomal RNA production. Recent studies have identified nucleolar structure perturbations and functional defects associated with different types of cardiomyopathies. Additionally, several mutations have been identified in several ribosomal genes that are linked to cardiomyopathy in human patients. We previously identified a nucleolar DEAD-box RNA helicase, DDX27, as a critical regulator of myogenesis. This study aimed to investigate the role of ddx27 deficiency in cardiac muscle and expand the understanding of DDX27 mediated pathways that are involved in myopathies. In this study, we used zebrafish models to investigate ddx27 deficiency in cardiac muscle. Phenotype characterization, cardiac function testing, transmission electron microscopy and histological analysis of ddx27 mutants revealed corresponding dilated cardiomyopathy and skeletal muscle hypotrophy. Furthermore, knockdown of DDX27 ortholog, Rs1, in cardiac muscle was fatal for Drosophila larvae. However, other tissues (i.e., neural or gastrointestinal) were unaffected suggesting that abnormalities caused by Ddx27 deficiency are specific to cardiac and skeletal muscle. Immunofluorescence, northern blotting and polysomal profiling of ddx27 zebrafish myofibers revealed that DDX27 is necessary for preserving nucleolar architecture and ribosome biogenesis. Here we have shown that DDX27 is essential for normal function of cardiac and skeletal myogenic processes due to its critical role in ribosomal regulation. Additionally, we provide novel evidence for DdX27 deficiency contributing to dilated cardiomyopathy. Overall, the findings of this study provide further evidence for the role of RNA helicases, specifically DDX27, in cardiac and skeletal muscle pathogenesis as well as provide novel insight into the molecular pathways of therapeutic benefit for afflicted patients of these diseases. / 2022-06-04T00:00:00Z

Objectives: We sought to identify a novel gene for dilated cardiomyopathy (DCM). Background: DCM is a heritable, genetically heterogeneous disorder that remains idiopathic in the majority of patients. Familial cases provide an opportunity to discover unsuspected molecular bases of DCM, enabling pre-clinical risk detection. Methods: Two large families with autosomal-dominant DCM were studied. Genome-wide linkage analysis was used to identify a disease locus, followed by fine mapping and positional candidate gene sequencing. Mutation scanning was then performed in 278 unrelated subjects with idiopathic DCM, prospectively identified at the Mayo Clinic. Results: Overlapping loci for DCM were independently mapped to chromosome 10q25-q26. Deoxyribonucleic acid sequencing of affected individuals in each family revealed distinct heterozygous missense mutations in exon 9 of RBM20, encoding ribonucleic acid (RNA) binding motif protein 20. Comprehensive coding sequence analyses identified missense mutations clustered within this same exon in 6 additional DCM families. Mutations segregated with DCM (peak composite logarithm of the odds score >11.49), were absent in 480 control samples, and altered residues within a highly conserved arginine/serine (RS)-rich region. Expression of RBM20 messenger RNA was confirmed in human heart tissue. Conclusions: Our findings establish RBM20 as a DCM gene and reveal a mutation hotspot in the RS domain. RBM20 is preferentially expressed in the heart and encodes motifs prototypical of spliceosome proteins that regulate alternative pre-messenger RNA splicing, thus implicating a functionally distinct gene in human cardiomyopathy. RBM20 mutations are associated with young age at diagnosis, end-stage heart failure, and high mortality.

Heart disease has been the leading cause of death in the United States for more than 90 years. The leading cause of death in individuals aged 65 and older has remained diseases of the heart from 1950 to the current time. According to the CDC, once diagnosed with heart disease, individuals have an approximately 50% chance of dying within 5 years, regardless of race. Mortality related to heart disease increased dramatically from the start of the 1900s to 1921, but subsequently experienced a steady decline from the mid-1960’s to 2000. However, when the decrease in heart disease is examined at the level of race it is clear that the decrease is not equally shared. While the leading cause of death among both Caucasian American men and women and African American men and women remains heart disease, the decrease in incidence of coronary heart disease among African American men was only half of the decrease in incidence among Caucasian American men. Genetic variants in BAG3 (Bcl-2 associated athanogene 3), a highly evolutionarily conserved gene that has recently emerged as a major dilated cardiomyopathy locus, are prevalent in isolated populations. This led us to hypothesize that variants in BAG3 might contribute to the increased prevalence of IDC in individuals of African ancestry. Expressed predominantly in the heart, the skeletal muscle and in many cancers, BAG3 has pleotropic effects in the heart. It inhibits apoptosis by binding to Bcl-2, facilitates protein quality control by binding to both large and small heat shock proteins, mediates adrenergic responsiveness by coupling the β-adrenergic receptor and the L-type Ca2+ channel, and maintains the integrity of the sarcomere by anchoring actin filaments to the Z disc. However, a paucity of subjects of African ancestry have been included in cohorts of probands with familial dilated cardiomyopathy whose exomes or genomes have been sequenced. Based on our previous observations and reports from other groups we postulated: 1) that mice with haplo-insufficiency of BAG3 will re-capitulate disease seen in humans and serve as a model for studying the pathogenesis of BAG3. 2) The prevalence or identification of specific BAG3 variants will differ by race and/or ethnicity. 3) SNVs of BAG3 may contribute to disease progression and thereby be pathogenic. Our study points out that we cannot understand population-based differences without enhancing the diversity of populations included in genomic studies. Similarly, in the era of big data, efforts must be undertaken to assess the genetic profile of both probands and their family members as without the ability to measure segregation, penetrance and plasticity we can only ascribe associations to functional genetic variants. / Biomedical Sciences

Dilated cardiomyopathy (DCM) or round heart disease is a muscle disease of the heart characterized by left ventricular dilatation and abnormal systolic and diastolic ventricular function. In animals, including turkeys and humans, DCM is a major cause of morbidity and mortality that results in heart failure. In the turkey, DCM can be idiopathic or induced. Since idiopathic or spontaneous DCM occurs in about 2-4 % of normal turkeys, it is of significant concern to the poultry industry. This dissertation was designed to increase our understanding of the pathophysiology of DCM in commercial turkeys. Specific objectives included: evaluating the influence of dietary selenium (Se) and vitamin E on poults fed toxic levels of furazolidone (Fz). Evaluating differences among reciprocal crosses of turkey varieties in susceptibility to a toxic level of Fz that induces DCM were used to assess the role of genetics in DCM. Using glutathione (GSH), glutathione peroxidase (GPx), malondialdehyde (MDA), and plasma uric acid (PUA) as biomarkers, oxidative stress (OS) levels were evaluated. Oxidative stress was also evaluated in poults fed diets containing varying concentration and combinations of vitamin E (0, 50 and 100 IU/kg) and Se (0.0, 0.3 and 0.5 mg/kg). Results from echocardiography measurements at four weeks of age, for poults fed toxic levels of Fz, showed the Narragansett x Bourbon Red reciprocal cross had the lowest internal-diastolic (LVIDd) and systolic dimensions (LVISd), while the Bourbon Red x Narragansett reciprocal cross had the largest LVIDd and LVISd. Left ventricular internal-diastolic and systolic dimension were lower for cross bred than parental poults. In treatment poults, heterosis for ventricular dilation was most significant for Bourbon Red x Narragansett cross. The data suggest that reciprocal crosses respond differently to toxin that induces DCM and genetics may influence a turkey's response to toxic levels of Fz that causes DCM. Results from OS measurements in poults fed normal and those fed normal diets with Fz at two weeks of age, showed no significant differences in MDA and GPx levels. PUA and GSH levels were however significantly increased for poults fed Fz-containing diets. At four weeks of age, no differences were observed for MDA and GPx measurements between poults fed normal and Fz-containing diets. PUA levels increased for poults fed normal diets with Fz, while GSH levels increased only for those fed normal diets. Differences between poults fed normal and Fz-containing diets were significant for GPx measurements. Results of this study showed that, feeding diets with Fz does not increase OS. Measure of the influence of feeding diets supplemented with different concentrations and combinations of Se and vitamin E to poults fed either normal or normal diets with Fz at two and four wks of age, showed higher MDA levels for poults fed Fz-containing diets supplemented with 0.3 mg/kg Se and 100 IU/kg vitamin E. For antioxidant biomarkers, GPx activity were increased for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se and those fed 100 IU/kg vitamin E. Poults fed normal diets supplemented with 100 IU/kg vitamin E had the highest GPx. PUA levels were higher for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se at two wks of age. At four wks of age, PUA concentrations were higher for poults fed Fz-containing diets supplemented with 100 IU/kg vitamin E. Increased PUA were also observed for poults fed diets supplemented with 0.5 mg/kg Se and 50 IU/kg vitamin E and 0.5 mg/kg and 100 IU/kg vitamin E. Poults fed diets supplemented with 50 and 100 IU/kg vitamin E had the highest GSH at two wks of age. Measurements taken at 2 wks of age, for poults fed normal diets supplemented with different concentrations and combinations of Se and vitamin E had increased GSH levels when compared with those fed diets with Fz at four wks of age. In this study, we showed that supplementation of poults fed normal diets with Fz with different concentrations and combinations of Se and vitamin E did not reduce DCM at 2 wks of age. However, at 4 wks of age, though DCM was not decreased by feeding diets supplemented with different concentrations and combinations of Se and vitamin E, reduced oxidant and antioxidant biomarkers were observed. / Ph. D.

Dilated cardiomyopathy (DCM), a disease of the myocardium, causes morbidity and premature death in humans and other domestic animals including turkeys. Though DCM results from many different factors including those that are unknown or idiopathic, genetic factor is a major cause of idiopathic DCM. In this study, I assessed the molecular basis of toxin-induced DCM in turkeys by evaluating the association and effect of mutations in candidate genes in the nucleus and mitochondria on the incidence and severity of this disease. Echocardiographic measurements at 3 weeks of age showed that birds on furazolidone-containing diet exhibited a significant DCM phenotype (increased left ventricular end diastolic dimension and left ventricular end systolic dimension) with a marked decrease in the left ventricular shortening fraction. Pathological phenotype confirmed the dilated heart with extended cell necrosis. Two mutations, both in NADH dehydrogenase genes, were found to be associated with DCM. Real-time RT-PCR quantification indicated that mRNA expression of alpha cardiac actin gene (ACTC) were significantly different between control and treatment birds. While ACTC expression increased, though moderately, in control birds from week 1 to 3, it decreased significantly in treatment birds. These findings suggest that the mitochondrial DNA variation and ACTC expression may be associated with the turkey's response to toxin. Therefore, further research is needed to investigate the molecular mechanism of toxin-induced DCM in the turkey. / Master of Science

Dilated cardiomyopathy (DCM), a heart disease, affects many vertebrates including humans and poultry. The disease can be either idiopathic (IDCM) or toxin-induced. Idiopathic DCM often occurs without a consensus cause. Though genetic and other studies of IDCM are extensive, the specific etiology of toxin-induced is still unknown. Here, our objective was to compare the level of mRNA expression of two candidate genes including troponin T (cTnT) and phospholamban (PLN) using quantitative reverse transcription polymerase chain reaction (RT-PCR) in toxin-induced DCM affected and unaffected turkeys. Cardiac TnT and PLN were chosen because their spontaneous expression has been reported to be associated with IDCM. We also scanned these genes for single nucleotide polymorphisms (SNPs) that could be useful in evaluating their functions in the incidence and severity of toxin-induced DCM in turkeys. There were no significant differences between affected and unaffected birds in the expression of both cTnT and PLN. A total of 12 SNPs were detected in cTnT and PLN DNA sequences. One of the seven haplotypes detected in cTnT was the most frequent. Linkage analysis showed that cTnT gene was unlinked on the current turkey genetic map. Resources developed here, including SNPs, haplotypes, cDNA sequences, and the PCR-RFLP genotype procedure will be used for future investigations involving cTnT and PLN and toxin-induced DCM. / Master of Science

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Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.