ACTN3/ACE genotypes and mitochondrial genome in professional soccer players’ performance.

Two nuclear genes, ACTN3, encoding for the α-actinin skeletal muscle isoform 3, and ACE encoding the angiotensin-converting enzyme, have both been associated with quantitative physical performance traits in the general population. The purpose of our study was to assess the association between the two nuclear gene variants, R577X (rs1815739) in ACTN3 and I/D (rs4340) in ACE, with elite athletes’ performance and the effect of training on the mitochondrial DNA (mtDNA) content in peripheral blood. We evaluated the genotypes and frequencies of ACTN3 R577X and ACE I/D polymorphisms between soccer players (n = 43) and healthy non-athletic controls (n = 128). Total DNA was extracted from peripheral blood samples using the standard procedure. The genotypes were assessed by PCR-RFLP analysis and mtDNA cellular content by RT-PCR. The soccer players showed a tendency to a prevalence of ACTN3RR and ACEDD genotypes both independently and in co-occurrence. The effect of physical training on the mitochondrial DNA content in the athletic population was reflected strikingly in its increase in peripheral blood. Based on our results, we suggest that the analysis of ACTN3 and ACE genotypes could predict talent in the soccer field and that knowledge of the genetic variants could determine types and training times for soccer players. In addition, the novelty of this work, never before described in the sports literature, is that the increase of mitochondrial content can be correlated with the training load, suggesting that the mtDNA copy number may be considered a viable bioenergetics biomarker.

Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I.

A comparison of the clinical presentation, disease course and results of laboratory and imaging studies of all patients so far published with a NDUFS4 mutation are presented. This reveals marked clinical heterogeneity, even in patients with the same genotype.

Complex I and the cAMP cascade in human physiopathology.

A cAMP-dependent protein kinase (PKA) is localized in mammalian mitochondria with the catalytic site at the matrix side of the membrane where it phosphorylates a number of proteins. One of these is the 18 kDa(IP) subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene. Mitochondria have a Ca(2+)-inhibited phosphatase, which dephosphorylates the 18 kDa phosphoprotein of complex I. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18 kDa protein is associated with stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome.

Mitochondrial disease mimicking polymyositis: a case report.

The authors report on a 34-year-old woman who had developed severe weakness and reduction in grip strength in both upper and lower limbs. Laboratory blood tests revealed increased levels of muscle enzyme. The presence of progressive bilateral ptosis and external ophthalmoplegia raised the suspicion of a mitochondrial disease, subsequently confirmed by deltoid biopsy and genetic analysis of mitochondrial DNA that showed a deletion indicative of Kearns-Sayre syndrome. In this report we emphasise the need for a differential diagnosis between myositis and other myopathies, particularly the mitochondrial ones.

The NADH: ubiquinone oxidoreductase (complex I) of the mammalian respiratory chain and the cAMP cascade.

Recent work has revealed cAMP-dependent phosphorylation of the 18-kDa IP subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene (chromosome 5). Phosphorylation of this protein has been shown to take place in fibroblast cultures in vivo, as well as in isolated mitochondria, which in addition to the cytosol also contain, in the inner-membrane matrix fraction, a cAMP-dependent protein kinase. Mitochondria appear to have a Ca2+-inhibited phosphatase, which dephosphorylates the 18-kDa phosphoprotein. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18-kDa protein is associated with potent stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome. In one case consisting of a 5 bp duplication, which destroyed the phosphorylation site, cAMP-dependent activation of complex I was abolished in the patient's fibroblast cultures. In another case consisting of a nonsense mutation, leading to termination of the protein after only 14 residues of the putative mitochondria targeting peptide, a defect in the assembly of complex I was found in fibroblast cultures.

A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome.

Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.

Alteration of mitochondrial DNA and RNA level in human fibroblasts with impaired vitamin B12 coenzyme synthesis.

Alterations of mitochondrial (mt) nucleic acid metabolism in methylmalonic aciduria (MMA) were studied in two cell lines from skin fibroblasts of patients with mitochondrial (GM00595) or cytosolic (GM10011) defects in the biosynthesis pathways of cobalamin coenzymes. The mtDNA level increased two-fold in GM00595 cells, which carry a mt defect in the adenosylcobalamin synthesis, whereas no appreciable change was found in GM10011 cells. The content of the two rRNAs 16S and 12S mtRNAs, normalized for the mtDNA copy number, decreased by 70% and 50% in GM00595 and GM10011, respectively. The normalized content of ND1, ND2 and CO I mRNAs decreased in GM00595, but was unchanged in GM10011. Respiratory chain complex activities measured in these two cell lines were not different from control activities. These data suggest that the maintenance of the mt function is due to doubling of mtDNA and that this compensatory response takes place only in those cells in which the greater reduction of the level of rRNA might have brought the content of these transcripts below the threshold value for optimal expression of the mt genome.

A novel insertion mutation (A169i) in the CLN1 gene is associated with infantile neuronal ceroid lipofuscinosis in an Italian patient.

Infantile neuronal ceroid lipofuscinosis (INCL) is a progressive encephalopathy characterized by psychomotor deterioration, early visual loss, and an evanishing EEG. Mutations in the CLN1 gene encoding palmitoyl-protein thioesterase (ppt) have been reported in all Finnish INCL patients and in several non-Finnish North European patients. No cases have been contributed from the Mediterranean area thus far. We identified a single adenine insertion at nucleotide position 169 (A169i) in the CLN1 gene in a family in which the proband suffered from an INCL-like syndrome. The novel mutation was homozygous in blood from the proband, heterozygous in his healthy parents, and not found in control alleles. The mutation leads to an early stop codon resulting in an abnormal and truncated ppt protein. Our observations provide the first molecular characterization of an Italian INCL patient and expand the list of the known defects in INCL.

Identification and characterization of human cDNAs specific to BCS1, PET112, SCO1, COX15, and COX11, five genes involved in the formation and function of the mitochondrial respiratory chain.

We have successfully applied a strategy based on the "cyberscreening" of the expressed sequence tags database using yeast protein sequences as "probes" to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes. In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-c oxidase. Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides. Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains. Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by an in vitro import and protease-protection assay. These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues. Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease. However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes.

Disorders of nuclear-mitochondrial intergenomic signalling.

In addition to sporadic or maternally-inherited mutations of the mitochondrial genome, abnormalities of mtDNA can be transmitted as mendelian traits. The latter are believed to be caused by mutations in still unknown nuclear genes, which deleteriously interact with the mitochondrial genome. Two groups of mtDNA-related mendelian disorders are known: those associated with mtDNA large-scale rearrangements and those characterized by severe reduction of the mtDNA copy number. The most frequent presentation of the first group of disorders is an adult-onset encephalomyopathy, defined clinically by the syndrome of progressive external ophthalmoplegia "plus", genetically by autosomal dominant transmission of the trait, and molecularly by the presence of multiple deletions of mtDNA. The second group of disorders comprises early-onset, organ-specific syndromes, associated with mtDNA depletion, that are presumably transmitted as autosomal recessive traits. Linkage analysis and search for candidate genes are two complementary strategies to clarify the molecular basis of these disorders of the nuclear-mitochondrial intergenomic signalling.

Decrease of D-loop frequency in heart and cerebral hemispheres mitochondrial DNA of aged rat.

A quantitative analysis of the frequency of the supercoiled mitochondrial DNA molecules containing the D-loop in rat heart and cerebral hemispheres, at different ages, is presented. Both tissues of aged animals exhibit a remarkable reduction in the content of super-coiled D-loop containing molecules compared to the adults. This alteration could be responsible for the age-dependent reduction of mitochondrial DNA transcription previously observed in rat brain and heart.

Extremely high levels of mutant mtDNAs co-localize with cytochrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243.

A single mtDNA point mutation at nt 3243 has been associated with two different clinical phenotypes: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes ('MELAS3243') and progressive external ophthalmoplegia ('PEO3243'). It has been shown that there is a much higher proportion of ragged-red fibers (RRF) with cytochrome c oxidase (COX) deficiency in PEO3243 than in MELAS3243. Using PCR/RFLP analysis of isolated individual skeletal muscle fibers from patients with both syndromes, we found a direct correlation between the localized concentration of the nt 3243 mutation and impairment of COX function at the single muscle fiber level: we found relatively low levels of mutant mtDNAs (56 +/- 21%) in 'normal' fibers; high levels (90 +/- 6%) in COX-positive RRF; and an almost complete segregation of mutant mtDNAs (95 +/- 3%) in COX-negative RRF. Thus, the differential distribution of fibers with extremely high concentrations of mutant mtDNAs characterizes, and probably distinguishes, the skeletal muscle of PEO and MELAS patients harboring the same nt-3243 mutation.

Lipid composition in synaptic and nonsynaptic mitochondria from rat brains and effect of aging.

The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.

Molecular analysis of the muscle pathology associated with mitochondrial DNA deletions.

Large-scale deletions of mitochondrial DNA (mtDNA) are associated with a subgroup of mitochondrial encephalomyopathies. We studied seven patients with Kearns-Sayre syndrome or isolated ocular myopathy who harboured a sub-population of partially-deleted mitochondrial genomes in skeletal muscle. Variable cytochrome c oxidase (COX) deficiencies and reduction of mitochondrially-encoded polypeptides were found in affected muscle fibres, but while many COX-deficient fibres had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. Our results suggest that a specific ratio between mutant and wild-type mitochondrial genomes is the most important determinant of a focal respiratory chain deficiency, even though absolute copy numbers may vary widely.