Belén Bornstein

Wild-type transthyretin amyloidosis as a cause of heart failure with preserved ejection fraction

AIMS Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous clinical syndrome with multiple underlying causes. Wild-type transthyretin (TTR) amyloidosis (ATTRwt) is an underdiagnosed cause of HFpEF that might benefit from new specific treatments. ATTRwt can be diagnosed non-invasively by (99m)Tc-3,3-diphosphono-1,2-propanodicarboxylic acid ((99m)Tc-DPD) scintigraphy. We sought to determine the prevalence of ATTRwt among elderly patients admitted due to HFpEF. METHODS AND RESULTS We prospectively screened all consecutive patients ≥60 years old admitted due to HFpEF [left ventricular (LV) ejection fraction ≥50%] with LV hypertrophy (≥12 mm). All eligible patients were offered a (99m)Tc-DPD scintigraphy. The study included 120 HFpEF patients (59% women, 82 ± 8 years). A total of 16 patients (13.3%; 95% confidence interval: 7.2-19.5) showed a moderate-to-severe uptake on the (99m)Tc-DPD scintigraphy. All patients with a positive scan underwent genetic testing of the TTR gene, and no mutations were found. An endomyocardial biopsy was performed in four patients, confirming ATTRwt in all cases. There were no differences in age, gender, hypertension, diabetes, coronary artery disease, or atrial fibrillation between ATTRwt patients and patients with other HFpEF forms. Although patients with ATTRwt exhibited higher median N-terminal pro-brain natriuretic peptide (6467 vs. 3173 pg/L; P = 0.019), median troponin I (0.135 vs. 0.025 µg/L; P < 0.001), mean LV maximal wall thickness (17 ± 3.4 vs. 14 ± 2.5 mm; P = 0.001), rate of pericardial effusion (44 vs. 19%; P = 0.047), and rate of pacemakers (44 vs. 12%; P = 0.004), clinical overlap between ATTRwt and other HFpEF forms was high. CONCLUSION ATTRwt is an underdiagnosed disease that accounts for a significant number (13%) of HFpEF cases. The effect of emerging TTR-modifying drugs should be evaluated in these patients.

MITOCHONDRIAL DNA DEPLETION SYNDROME DUE TO MUTATIONS IN THE RRM2B GENE

Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy.

Genetic basis of end‐stage hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by a heterogeneous presentation and clinical course. A minority of HCM patients develop end‐stage HCM and require cardiac transplantation. The genetic basis of end‐stage HCM is unknown but small series, isolated case reports and animal models have related the most aggressive heart failure course with the presence of multiple mutations.

A double mutation (A8296G and G8363A) in the mitochondrial DNA tRNALys gene associated with myoclonus epilepsy with ragged-red fibers

Objective: To define potential pathogenic mitochondrial DNA (mtDNA) point mutations in a patient with myoclonus epilepsy with ragged-red fibers (MERRF) syndrome. Background: MERRF syndrome is typically associated with point mutations in the mtDNA tRNALys gene. Methods: We performed morphologic, biochemical, and genetic analysis of muscle samples from the patient and four relatives. Molecular genetic studies included sequencing, PCR, and restriction enzyme analysis on whole muscle, blood, and single muscle fibers. Results: Muscle biopsy showed cytochrome c oxidase (COX), negative ragged-red fibers (RRF), and a defect of complex I of the mitochondrial respiratory chain. We found an A8296G transition and a G8363A mutation in the mtDNA tRNALys gene. The A8296G was almost homoplasmic in muscle and blood from the propositus and his oligosymptomatic maternal relatives. The G8363A mutation was heteroplasmic and more abundant in muscle than in blood, and its proportion correlated with clinical severity. Single muscle fiber analysis showed significantly higher levels of G8363A genomes in COX-negative than in normal fibers, and almost homoplasmic levels of mutant A8296G mtDNA in both COX-negative and normal fibers. The two mutations affect highly conserved nucleotides and were not found in controls. Conclusions: The G8363A mutation is pathogenic; the co-ocurrence of the A8296G mutation is of unclear significance and is likely to be a rare polymorphism.

Mitochondrial dysfunction associated with a mutation in the Notch3 gene in a CADASIL family

Background: Cerebral autosomal arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by recurrent subcortical ischemic strokes and dementia caused by mutations in the Notch3 gene. In Drosophila melanogaster, Notch signaling has a pleiotropic effect, affecting most tissues of the organism during development. Objective: To characterize a potential mitochondrial dysfunction associated with mutations in the Notch3 gene. Methods: Biochemical, histochemical, molecular, and genetic analyses were performed on muscle biopsy specimens and fibroblasts obtained from patients of a Spanish family with CADASIL. Additional biochemical and molecular analyses of the N55e11 mutant of D. melanogaster were performed. Results: In muscle biopsy specimens, a significant decrease was found in the activity of complex I (NADH [reduced form of nicotinamide adenine dinucleotide] dehydrogenase), and in one patient, histochemical analysis showed the presence of ragged-red fibers with abnormal cytochrome c oxidase staining. Reduced fibroblast activity of complex V (ATP synthase) was found. Supporting data on patients with CADASIL, it was found that the mutation N55e11 in Drosophila decreases the activity of mitochondrial respiratory complexes I and V. Conclusions: Mitochondrial respiratory chain activity responds, directly or indirectly, to the Notch signaling pathway. Mitochondrial dysfunction in patients with CADASIL may be an epiphenomenon, but results of this study suggest that the pathophysiology of the disease could include a defect in oxidative phosphorylation.

Myocardial carnitine and carnitine palmitoyltransferase deficiencies in patients with severe heart failure.

We studied myocardial tissue from 25 cardiac transplant recipients, who had end-stage congestive heart failure (CHF), and from 21 control donor hearts. Concentrations of total carnitine (TC), free carnitine (FC), short-chain acylcarnitines, long-chain acylcarnitines (LCAC) as well as carnitine palmitoyltransferase (CPT) activities were measured in myocardial tissue homogenates and referred to the concentration of non-collagen protein. Compared to controls, the concentrations of TC and FC as well as total CPT activities were significantly lower in patients. LCAC levels and the LCAC to FC ratio values were significantly greater in patients than in controls. While the malonyl-CoA sensitive fraction of CPT, which represents CPT I activity, was similar in patients and controls, the residual CPT activity after inhibition by malonyl-CoA, representing CPT II activity, was significantly reduced in patients compared to controls. Moreover, the activity of CPT in the presence of Triton X-100, which also represents the activity of CPT II, was significantly lower in patients than in controls. Malonyl-CoA concentrations required for half-maximal inhibition of CPT activity were significantly greater in patients than in controls. There was a linear relationship between ejection fraction (EF) values and concentrations of TC, FC, or total CPT activities. Values for LCAC and the LCAC to FC ratio were inversely related to EF values. We conclude that failing heart shows decreased total CPT and CPT II activities and carnitine deficiency that may be related to ventricle function.

Early-onset multisystem mitochondrial disorder caused by a nonsense mutation in the mitochondrial DNA cytochrome C oxidase II gene.

We report the first nonsense mutation (G7896A) in the mtDNA gene for subunit II of cytochrome c oxidase (COX) in a patient with early‐onset multisystem disease and COX deficiency in muscle. The mutation was heteroplasmic in muscle, blood, and fibroblasts from the patient and abundantly present in COX‐deficient fibers, but less abundant in COX‐positive fibers; it was not found in blood samples from the patients asymptomatic maternal relatives. Immunoblot analysis showed a reduced concentration of both COX II and COX I polypeptides, suggesting impaired assembly of COX holoenzyme.

Genetics in dilated cardiomyopathy

Discoveries made during the last 20 years have revealed a genetic origin in many cases of dilated cardiomyopathy (DCM). Currently, over 40 genes have been associated with the disease. Mutations in DCM-causing genes induce the condition through a variety of different pathological pathways with complex and not completely understood mechanisms. Genes that encode for sarcomeric, cytoskeletal, nuclear membrane, dystrophin-associated glycoprotein complex and desmosomal proteins are the principal genes involved. In this review we discuss the most frequent DCM-causing genes. We propose a classification in which DCM genes are considered as being major or minor genes according to their mutation frequency and the available supporting evidence. The main phenotypic characteristics associated with each gene are discussed.

Structure and Regulated Expression of the δ-Aminolevulinate Synthase Gene from Drosophila melanogaster

The structure of the single copy gene encoding the putative housekeeping isoform of Drosophila melanogaster δ-aminolevulinate synthase (ALAS) has been determined. Southern and immunoblot analyses suggest that only the housekeeping isoform of the enzyme exists in Drosophila. We have localized a critical region for promoter activity to a sequence of 121 base pairs that contains a motif that is potentially recognized by factors of the nuclear respiratory factor-1 (NRF-1)/P3A2 family, flanked by two AP4 sites. Heme inhibits the expression of the gene by blocking the interaction of putative regulatory proteins to its 5′ proximal region, a mechanism different from those proposed for other hemin-regulated promoters. Northern and in situ RNA hybridization experiments show that maternal alas mRNA is stored in the egg; its steady-state level decreases rapidly during the first hours of development and increases again after gastrulation in a period where the synthesis of several mRNAs encoding metabolic enzymes is activated. In the syncytial blastoderm, the alasmRNA is ubiquitously distributed and decreases in abundance substantially through cellular blastoderm. Late in embryonic development alas shows a specific pattern of expression, with an elevated mRNA level in oenocytes, suggesting an important role of these cells in the biosynthesis of hemoproteins inDrosophila.

Reactive oxygen species mediate the down-regulation of mitochondrial transcripts and proteins by tumour necrosis factor-alpha in L929 cells.

In this study, we show that reactive oxygen species production induced by tumour necrosis factor alpha (TNF-alpha) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-alpha had no effect on the amount of mitochondrial DNA, the results suggested that TNF-alpha acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-alpha, indicating a direct link between these two phenomena.