Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay.

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes.

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop. The third base was a biotinylated uracil (U(B)) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [gamma-(33)P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45 degrees C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin-target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

Optimal myocardial preconditioning in humans.

We developed a model of ischemia and reperfusion (I and R) in human ventricular myocytes (CM). CM injury and metabolics were studied after various interventions: endogenous preconditioning (PC) with anoxia, hypoxia, and anoxic or hypoxic supernatants; endogenous PC with or without SPT or adenosine deaminase; and exogenous adenosine PC before, during, or after I or continuously, with or without SPT. To assess the clinical implications of PC and the possible mediating effects of adenosine, patients undergoing elective coronary bypass surgery (CABG) received either a high or low dose of adenosine. Patients not receiving adenosine served as controls. Adenosine levels, high-energy phosphate levels, the metabolic parameters were evaluated from blood samples and left ventricular biopsy samples. Our cellular model studies indicated that preconditioning conferred protection to human CM via an adenosine-mediated pathway. Adenosine simulated PC without a fall in ATP. Adenosine administered to patients during CABG stimulated myocardial metabolism while preventing the degradation of high energy phosphates. A prospective randomized trial of adenosine administered to high-risk patients for myocardial protection is required.

Smooth muscle cell transplantation into myocardial scar tissue improves heart function.

This study was designed to evaluate the effect of smooth muscle cell transplantation into myocardial ventricular scar formed by cryo-necrosis. The left ventricular free wall (LVFW) of adult rats was cryo-necrosed. At 4 weeks after cryo-injury cultured fetal rat stomach smooth muscle cells (transplanted group, n = 10) or culture medium (control, n = 10) were transplanted. Sham animals (n = 8) were similarly operated but without cryo-necrosis and transplantation. The animals were administered a daily maintenance dose of cyclosporin A (5 mg/kg). At 8 weeks after cryo-injury, heart function was evaluated using a Langendorff preparation. Myocardial scar and transplanted cells were assessed histologically. Transplanted smooth muscle cells survived and formed smooth muscle cell tissue, as assessed by immunostaining against smooth muscle cell actin, within the myocardial scar. In the control hearts no muscle tissue was found in the scar. Angiogenesis occurred (P < 0.001) in the transplanted scar compared to the control scar. The transplanted cells increased the scar thickness (P < 0.01) by hyperplasia and prevented (P < 0.001) the dilatation of the ventricular chamber size compared to the controlled hearts. For physiological left ventricular volumes of 0.04 to 0.28 ml, the systolic and developed pressures in the transplanted group were greater (P < 0.001) than the control group, but less (P < 0.001) than those of the sham group. Transplanted smooth muscle cells formed smooth muscle tissue in myocardial scar tissue and improved contractile function compared to control hearts.

Repopulation of rho0 cells with mitochondria from a patient with a mitochondrial DNA point mutation in tRNA(Gly) results in respiratory chain dysfunction.

Familial hypertrophic ventricular cardiomyopathy has been demonstrated to be associated with a number of mitochondrial DNA (mtDNA) mutations. A fibroblast cell line carrying a mutation in its mtDNA at position 9997 in the gene encoding tRNA glycine was obtained from a patient with hypertrophic cardiomyopathy. To demonstrate that the etiology of this disease was a result of the mtDNA mutation, cybrid clones were constructed by fusion of enucleated patient skin fibroblasts to rho0 osteosarcoma cells. Clones carrying high levels of mutant mtDNA showed predominantly cytochrome c oxidase and complex I deficiency, as well as an elevated lactate/pyruvate (L/P) ratio, a biochemical marker characteristic of respiratory chain deficiencies. Pulse-labeling experiments demonstrated a strong negative correlation between the levels of newly synthesized mtDNA-encoded polypeptides and glycine content. These data suggest that the T9997C mutation in mtDNA is causative of respiratory chain dysfunction when present at high levels of heteroplasmy.

Elevated insulin-like growth factor-I and transforming growth factor-beta 1 and their receptors in patients with idiopathic hypertrophic obstructive cardiomyopathy. A possible mechanism.

BACKGROUND: Idiopathic hypertrophic obstructive cardiomyopathy (HOCM) is characterized by regional myocardial hypertrophy. In our previous study, we demonstrated that mRNA levels for insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) were elevated in HOCM tissue. In this study, we investigated IGF-I and TGF-beta 1 protein levels and their respective receptor levels and localization. METHODS AND RESULTS: Myocardial growth factor protein levels were quantified with the use of chemiluminescent slot blot analysis with monoclonal antibodies against IGF-I and TGF-beta. The growth factor receptor binding sites were evaluated with 125I-labeled IGF-I and TGF-beta 1. The receptors were localized with immunohistochemistry. Data were expressed as mean +/- SEM. IGF-I and TGF-beta protein levels in HOCM myocardium (351.8 +/- 46.5 and 17.4 +/- 2.0 ng/g tissue, respectively; n = 6) were significantly higher (P < 0.01 for all groups) than in non-HOCM myocardium obtained from patients with aortic stenosis (AS, 182.1 +/- 22.7 and 8.0 +/- 1.2 ng/g tissue, respectively; n = 5), stable angina (SA, 117.4 +/- 20.9 and 7.5 +/- 2.7 ng/g tissue, respectively; n = 5), and transplanted hearts (TM, 166.3 +/- 30.1 and 6.4 +/- 1.2 ng/g tissue, respectively; n = 5). Maximal and high-affinity binding sites for IGF-I receptor in the HOCM were greater (P < 0.01 and P < 0.05) than the levels in AS, SA, and TM. The maximal receptor binding sites for TGF-beta 1 in HOCM were greater (P < 0.05) than those for SA and TM. Immunohistochemistry demonstrated that IGF-I and TGF-beta 1 receptors were located on the cardiomyocytes and TGF-beta 1 receptors were located on the fibroblasts. CONCLUSIONS: Increased IGF-I and TGF-beta 1 gene expression previously observed in HOCM myocardium results in elevated protein levels. IGF-I and TGF-beta 1 signals may be further amplified by increased receptor numbers on cardiomyocytes and fibroblasts. The data suggest a possible autocrine mechanism of IGF-I-stimulated cardiomyocyte hypertrophy and a paracrine mechanism of TGF-beta 1-stimulated extracellular matrix overproduction in HOCM.

Optimal myocardial preconditioning in a human model of ischemia and reperfusion.

BACKGROUND: Adenosine (ADE) may mediate the protective effects of preconditioning (PC). However, human data are lacking, and the optimal method of ADE administration and the mechanism of protection remain unresolved. METHODS AND RESULTS: We have developed a model of simulated "ischemia" (I) and "reperfusion" (R) in quiescent human ventricular cardiomyocytes. Cellular injury and metabolic parameters were assessed after various interventions: Cells were preconditioned with anoxia (PC0), hypoxia (PC16), anoxic supernatant (SUP0), or hypoxic supernatant (SUP16) with or without the ADE receptor antagonist (SPT) or ADE deaminase (ADA). ADE was applied before, during, or after I or continuously with and without SPT. Cells were treated with the PKC agonist PMA. PC cells were incubated with the protein kinase-C (PKC) antagonist Calphostin-C (Cal-C). PKC translocation and PKC activity were assessed. PC0 was most protective. Protection was transferable via SUP0, which produced the highest concentrations of ADE. Protection was lost with SPT or ADA. Intracellular ATP fell after PC and prolonged I and R. Exogenous ADE was most protective when administered before I at 50 mumol. ADE during I was partially protective. No additional protection was provided with continuous ADE treatment. ADE prevented ATP degradation but increased lactate immediately after its administration. SPT abolished the protective effects of ADE. PMA conferred protection, which was abolished with Cal-C. ADE stimulated PKC translocation and PKC activity in the absence of SPT. CONCLUSIONS: Maximal I confers maximal PC. The degree of I is reflected in supernatant ADE concentrations. The initial ATP fall with PC may account for a lack of ATP preservation after I and R. ADE reproduces the protective effects of PC, preserves ATP, and increases lactate production, perhaps by stimulating glycolysis. Clinical trials of ADE administered during cardiac surgery are necessary to further define its beneficial effects in humans.

Myocardial aerobic metabolism is impaired in a cell culture model of cyanotic heart disease.

A human pediatric cardiomyocyte cell culture model of chronic cyanosis was used to assess the effects of low oxygen tension on mitochondrial enzyme activity to address the postoperative increase in lactate and decreased ATP in the myocardium and the high incidence of low-output failure with restoration of normal oxygen tension, after technically successful corrective cardiac surgery. Chronically hypoxic cells (PO2 = 40 mmHg for 7 days) exhibited significantly reduced activities for pyruvate dehydrogenase, cytochrome-c oxidase, succinate cytochrome c reductase, succinate dehydrogenase, and citrate synthase. The activity of NADH-cytochrome c reductase was unaffected. Lactate production and the lactate-to-pyruvate ratio were significantly greater in hypoxic cardiomyocytes. Western and Northern analysis demonstrated a decrease in the levels of various mRNA and corresponding polypeptides in hypoxic cells. Thus hypoxia influences mitochondrial metabolism through acute and chronic adaptive mechanisms, reflecting allosteric (posttranscriptional) and transcriptional modulation. Transcriptional downregulation of key mitochondrial enzyme systems can explain the insufficient myocardial aerobic metabolism and low-output failure in children with cyanotic heart disease after cardiac surgery.

Insulin stimulates pyruvate dehydrogenase and protects human ventricular cardiomyocytes from simulated ischemia.

UNLABELLED: Impaired myocardial metabolism after cardioplegic arrest results in persistent anaerobic lactate production. Insulin may protect the heart from ischemia and reperfusion by enhancing myocardial metabolic recovery. However, the stimulation of glycolysis during ischemia may be detrimental because of an accumulation of metabolic end-products. We examined the effect of insulin on quiescent human ventricular cardiomyocytes subjected to simulated cardioplegic ischemia and reperfusion. METHODS: Primary cardiomyocyte cultures were established from patients undergoing corrective repair of tetralogy of Fallot. Cells were exposed to varying concentrations of glucose and insulin during 30 minutes of stabilization in 10 mL of phosphate-buffered saline solution. Ischemia was simulated by exposing the cells to a low volume (1.5 mL) of deoxygenated phosphate-buffered saline solution for 90 minutes followed by 30 minutes of simulated reperfusion in 10 mL of normoxic phosphate-buffered saline solution. Cell viability was assessed by trypan blue exclusion. The activity of mitochondrial pyruvate dehydrogenase was measured in 3 states: stabilization, ischemia, and reperfusion. In addition intracellular lactate, adenine nucleotides, extracellular lactate, pyruvate, and acid release were measured. RESULTS: Higher ambient glucose concentrations resulted in greater cellular injury although insulin-treated cells displayed less injury after ischemia and reperfusion. Insulin increased the pyruvate dehydrogenase activity by 31% in cardiomyocytes and reduced extracellular lactate production by 40%. Intracellular adenosine triphosphate was improved by 75% in cells exposed to high glucose concentrations in the presence of insulin. CONCLUSIONS: Insulin protected human ventricular cardiomyocytes from ischemia and reperfusion. This protection may be due to a stimulation of pyruvate dehydrogenase activity which resulted in improved aerobic metabolism.

Preconditioning human cardiomyocytes and endothelial cells.

BACKGROUND: The effects of simulated "ischemia" and "reperfusion" were evaluated in cell cultures of human ventricular cardiomyocytes and human saphenous vein endothelial cells. METHODS: Myocyte and endothelial cell cultures were exposed to a low volume (1.5 ml) of either hypoxic (oxygen tension = 16 mm Hg) or anoxic (oxygen tension = 0 mm Hg) phosphate-buffered saline solution for 90 minutes ("ischemia") followed by 30 minutes of simulated "reperfusion." Cell injury was evaluated by trypan blue exclusion. Next, the effects of a preconditioning stimulus were evaluated by a brief (10 minute) exposure to hypoxic or anoxic ischemia and 10 minutes of reperfusion before prolonged (90 minutes) anoxic ischemia. Finally, the effects of anoxic preconditioning on intracellular lactate accumulation and extracellular lactate and acid release were assessed. RESULTS: "Ischemia" and "reperfusion" resulted in greater injury to endothelial cells than to cardiomyocytes. In both cell types, anoxic ischemia resulted in greater injury than hypoxic ischemia. Preconditioning reduced cell injury in myocytes but not in endothelial cells. Endothelial cells produced more lactate than cardiomyocytes under normoxic conditions. Ischemia increased lactate accumulation and release in cardiomyocytes but not endothelial cells. Preconditioning reduced lactate accumulation and release in cardiomyocytes but not endothelial cells. CONCLUSIONS: Endothelial cells were more susceptible to the same period of simulated ischemia than cardiomyocytes. Preconditioning protected cardiomyocytes but not endothelial cells from a subsequent prolonged period of ischemia and reperfusion.

Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue.

BACKGROUND: Fetal rat cardiomyocytes transplanted into left ventricular scar tissue of the adult rat heart limit scar expansion and improve heart function. This study determined morphologic changes of transplanted fetal rat cardiomyocytes in myocardial scar tissue. METHODS AND RESULTS: The left ventricles of 500-g Sprague-Dawley rats were cryodamaged. At 4 weeks after myocardial injury, a transmural scar (54+/-11 mm2) (mean+/-1 SDak) formed at the apex (n=6). Cardiomyocytes freshly isolated from 18-day-gestation Sprague-Dawley rat hearts were transfected with plasmid containing the beta-galactosidase and then injected into the 4-week-old scar tissue. Cell culture medium was injected into the scar tissue of control animals. At 4 weeks posttransplantation, the cardiomyocytes had formed cardiac tissue (20.7+/-6.9 mm2, n=14), which stained positively for beta-galactosidase activity in the scar (90.4+/-25 mm2, n=14). The transplanted cardiomyocytes formed sarcomeres and were linked by junctions composed of desmosomes and fascia adherens. Lymphocyte infiltration occurred despite use of cyclosporin A. No myocardial tissue was found in the scar tissue of the control animals (n=14). More arterioles and venules were found (P<.01) in the cardiomyocyte grafts (1.2+/-0.6 vessel/0.8 mm2; n=14) than in the control scar tissue (0.1+/-0.1 vessels/0.8 mm2; n=14). At 20 weeks post-transplantation, the transplant tissue size (6+/-6 mm2; n=7) was smaller (P=.007) than 4-week old transplant, and the scar (162+/-46 mm2; n=7) was larger (P=.005) than 4-week-old scar. Lymphocyte infiltration was still present among the remaining transplanted cells. CONCLUSIONS: This study demonstrated that cardiac tissue formed by transplanted fetal cardiomyocytes in the myocardial scar tissue decreased in size with time probably secondary to rejection.

The human mitochondrial elongation factor tu (EF-Tu) gene: cDNA sequence, genomic localization, genomic structure, and identification of a pseudogene.

The human mitochondrial elongation factor Tu (EF-Tu) is nuclear-encoded and functions in the translational apparatus of mitochondria. The complete human EF-Tu cDNA sequence of 1677 base pairs (bp) with a 101 bp 5'-untranslated region, a 1368 bp coding region, and a 207 bp 3'-untranslated region, has been determined and updated. The predicted protein from this cDNA sequence is approximately 49.8 kDa in size and is composed of 455 amino acids (aa) with a putative N-terminal mitochondrial leader sequence of approximately 50 aa residues. The predicted amino acid sequence shows high similarity to other EF-Tu protein sequences from ox, yeast, and bacteria, and also shows limited similarity to human cystolic elongation factor 1 alpha. The complete size of this cDNA (1677 bp) obtained by cloning and sequencing was confirmed by Northern blot analysis, which showed a single transcript (mRNA) of approximately 1.7 kb in human liver. The genomic structure of this EF-Tu gene has been determined for the first time. This gene contains nine introns with a predicted size of approximately 3.6 kilobases (kb) and has been mapped to chromosome 16p11.2. In addition, an intronless pseudogene of approximately 1.7 kb with 92.6% nucleotide sequence similarity to the EF-Tu gene has also been identified and mapped to chromosome 17q11.2.

Overexpression of transforming growth factor-beta1 and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomyopathy.

BACKGROUND: Idiopathic hypertrophic cardiomyopathy (HCM) is characterized by regional myocardial hypertrophy. To investigate involvement of growth factors on myocardial hypertrophy in HCM patients, we evaluated gene expression and cellular localization of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factors (IGF-I and IGF-II), and platelet-derived growth factor-B (PDGF-B) in ventricular biopsies obtained from patients with HCM (n=8), aortic stenosis (AS) (n=8), or stable angina (SA) (n=8) and from explanted hearts with ischemic cardiomyopathy (TM) (n=7). METHODS AND RESULTS: Levels of TGF-beta1, IGF-I, IGF-II, and PDGF-B transcripts were quantified with the use of multiplex RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard. Antibodies against TGF-beta and IGF-I were used to localize their peptides within the myocardium. Antisense and sense (control) cRNA probes of TGF-beta1 and IGF-I, labeled with digoxigenin, were used to localize the growth factor transcripts by in situ hybridization. mRNA levels (densitometric ratio of growth factor/glyceraldehyde-3-phosphate dehydrogenase) of TGF-beta1 and IGF-I in HCM (0.75+/-0.05 and 0.85+/-0.15, respectively; mean+/-1 SEM) were significantly (P<.01 for all groups) elevated in comparison with non-HCM myocardium (AS: 0.38+/-0.07, 0.29+/-0.06; SA: 0.32+/-0.04, 0.18+/-0.05; TM: 0.25+/-0.03, 0.15+/-0.03). mRNA levels of TGF-beta1 and IGF-I in the hypertrophic AS myocardium were greater (P=.02, P=.05) than those in the explanted myocardium (TM). Immunohistochemical and in situ hybridization studies showed increased expression of TGF-beta1 and IGF-I in the HCM cardiomyocytes. CONCLUSIONS: Gene expression of TGF-beta1 and IGF-I was enhanced in idiopathic hypertrophic cardiomyopathy and may be associated with its development.

Chromosomal localization of the human liver form cytochrome c oxidase subunit VIIa gene.

The chromosomal loci corresponding to human cytochrome c oxidase (COX) subunit VIIa Liver (VIIa-L) isoform genes were determined utilizing a combined approach of genomic cloning, in situ hybridization, and somatic hybrid genetics. In contrast to the proposal of E. Arnaudo et al. (Gene (Amst.), 119: 299-305, 1992) that COX VIIa-L sequences are located on chromosomes 4 and 14, we found that COX VIIa-L related sequences reside on chromosome 6, while an additional COX VIIa-L cross-reacting sequence psi-gene) was located on chromosome 4.

Cloning, characterization, and chromosomal localization of human liver form cytochrome c oxidase subunit VIa related genes.

The chromosomal location of human cytochrome c oxidase (COX) subunit VIa Liver (VIa-L) isoform related sequences has been determined by a combination of in situ hybridization and analysis of human-hamster somatic cell hybrid panels. COX VIa-L related sequences were present on chromosomes 6 and 12. It has been verified that at least two COX VIa-L genes are on chromosome 6, one of which is a pseudogene. In total, four COX VIa-L related sequences have been cloned and their nucleotide sequences analyzed. At least three of the sequences represent pseudogenes; their relatedness to the COX VIa-L cDNA is discussed.

Normal mitochondrial DNA and respiratory chain activity in familial dysautonomia fibroblasts.

Familial dysautonomia (FD), an autosomal recessive disease mapped to chromosome 9q31, is a sensory and autonomic neuropathy of unknown etiology. We have previously reported light microscopic pleiomorphic changes in cells suggestive of altered plasma membranes, an increase in globotriaosylceramide (Gb3), reflected by an increase in Gb3 on the surface of the plasma membrane, and a decrease in the rate and amount of ganglioside synthesized. In unrelated studies, we demonstrated that storage of glycospingolipids (GSL) is deleterious to mitochondrial function. Recently, mitochondrial dysfunction has been associated with neurodegenerative disease, superimposed on an autosomal inheritance pattern. We have now probed Southern blots of total FD fibroblast DNA, digested with BamHI, EcoRII, and/or PvuII, with purified placental 32P-labeled mitochondrial DNA. The sizes of all FD mitochondrial DNAs were normal (16,569 bp), some containing previously identified BamHI polymorphisms. Lactate/pyruvate ratios, and activities of Complexes II and III, matched those of control cells. Electron microscopy revealed morphologically normal mitochondria, in conjunction with a normal oxidative state, determined using the redox dyes Mito Tracker CMXR and CMXR-H2 and fluorescence microscopy. We conclude that mitochondrial dysfunction, due to GSL accumulation, changes in mitochondrial DNA, or mutation of a chromosome 9q gene involved in mitochondrial function, is neither a primary nor a secondary cause of FD, as determined by a study of FD fibroblasts.

Familial cardiomyopathy with cataracts and lactic acidosis: a defect in complex I (NADH-dehydrogenase) of the mitochondria respiratory chain.

Four patients in one generation of a multiply consanguineous pedigree died with cardiomyopathy, cataracts, and lactic acidemia. Postmortem heart and skeletal muscle tissues from one patient were analyzed. A low (12% control) activity of NADH-CoQ reductase (complex I) in heart and normal activity in skeletal muscle mitochondria was found. Cultured skin fibroblasts obtained from two individuals in the pedigree showed elevated lactate to pyruvate ratios in the range of 2 to 3.5 times normal and decreased complex I + III activity (42 and 54% of control activities) in isolated mitochondria. Western blot analysis and enzymatic assay showed normal levels of CuZn-superoxide dismutase, but grossly elevated levels of the mitochondrial Mn-superoxide dismutase. Southern blot analysis in heart muscle cells from the patient tested revealed multiple mitochondrial DNA deletions which indicate free oxygen radical damage. We hypothesize that a nuclear-encoded defect in the respiratory chain is responsible for excessive free oxygen radical production in these infants which contributes to the prenatal onset of cardiomyopathy and cataracts.

An additional mitochondrial tRNA(Ile) point mutation (A-to-G at nucleotide 4295) causing hypertrophic cardiomyopathy.

A third point mutation in the mitochondrial tRNA(Ile) gene associated with hypertrophic cardiomyopathy and respiratory chain dysfunction in heart is reported. An A-to-G transition at nucleotide position 4295 was shown to be highly evolutionarily conserved, never present in control individuals, and to segregate with the disease. A PCR-based diagnostic test and endomyocardial biopsies were used to detect both the biochemical deficiency and the level of heteroplasmy in heart. The implications of this new mitochondrial DNA point mutation are discussed.