一个2型糖尿病家系中新发现的线粒体DNA G7444A 突变分析

应用PCR-RFLP和测序对一个2型糖尿病家系的线粒体DNA G7444A的突变进行检测, 并分析其临床资料的特点。结果发现, 27例家系成员中, 11例母系亲属均存在线粒体DNA G7444A突变, 而配偶及父系亲属中未发现该突变。11例突变者中确诊为2型糖尿病患者5例, 糖耐量受损1例, 均表现为乳酸和血糖增高。因此, 线粒体DNA G7444A突变是该家系中糖尿病的遗传易感因素, 是导致2型糖尿病的一个新的突变位点。     

A novel MT-CO1 m.6498C>A variation associated with the m.7444G>A mutation in the mitochondrial COI/tRNASer(UCN) genes in a patient with hearing impairment,diabetes and congenital visual loss

Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. Sensorineural hearing loss (SNHL) has been described in association to different mitochondrial multisystem syndromes, often involving the central nervous system, neuromuscular, or endocrine organs. In this study, we described a Tunisian young girl with hearing impairment, congenital visual loss and maternally inherited diabetes. No mutation was found in the mitochondrial tRNA^Leu(UUR) and the 12S rRNA genes. However, we detected the m.7444G>A mutation in the mitochondrial COI/tRNA^Ser(UCN) genes. This mutation eliminates the termination codon of the MT-CO1 gene and extends the COI polypeptide by three amino acids (Lys–Gln–Lys) to the C-terminal. The whole mitochondrial genome screening revealed the presence of a novel mutation m.6498C>A (L199I) in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX). This “probably damaging” transversion affects a highly conserved domain and it was absent in 200 Tunisian controls. The studied patient was classified under the haplogroup H2a.     

The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12811, 13637, and 13967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.     

Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.     

A novel mtDNA C11777A mutation in Leigh syndrome

A novel mitochondrial DNA point mutation, a C-to-A mutation at nucleotide position (np) 11,777, was identified in two unrelated patients out of 100 with Leigh syndrome. This mutation converted a highly evolutionary conserved arginine to a serine at codon 340 in ND4 gene. This codon was also converted by a G-to-A mutation at np 11,778, the most common mutation associated with Leber's hereditary optic neuropathy (LHON), but the amino acid replacement was different (R340S vs. R340H). Cybrid study revealed that the percentage of heteroplasmy was correlated with complex I function and that the novel mutation caused a much more deleterious effect than the np 11,778 LHON mutation in complex I activity.     

Sequence analysis of the complete mitochondrial genome in patients with Leber's hereditary optic neuropathy lacking the three most common pathogenic DNA mutations

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by central vision loss in young adults. The majority of LHON cases around the world are associated with mutations in the mitochondrial genome at nucleotide positions (np) 3460, 11,778, and 14,484. Usually, these three mutations are screened in suspected LHON patients. The result is important not only in respect to the diagnosis but also as different LHON mutations lead to variations in expression, severity, and recovery of the disease. There are, however, a significant number of patients without any of these primary mutations. In these situations, genetic counselling of a patient and his family can be difficult. We sequenced the complete mitochondrial DNA (mtDNA) in 14 LHON patients with the typical clinical features but without a primary mtDNA mutation to evaluate the potential of extensive mutation screening for clinical purposes. Our results suggest to include the mutation at np 15,257 in a routine screening as well as the ND6 gene, a hot spot for LHON mutations. Screening for the secondary LHON mutations at np 4216 and np 13,708 may also help in making the diagnosis of LHON as these seem to modify the expression of LHON mutations. Although they do not allow to prove the clinical diagnosis, their presence increases the probability of LHON. Sequencing the complete mitochondrial genome can reveal novel and known rare disease causing mutations. However, considering the effort it adds little value for routine screening.     

Confirmation of the mitochondrial ND1 gene mutation G3635A as a primary LHON mutation

We report the clinical and genetic characterization of two Chinese LHON families who do not carry the primary LHON-mutations. Mitochondrial genome sequence analysis revealed the presence of a homoplasmic ND1 G3635A mutation in both families. In Family LHON-001, 31 other variants belonging to the East Asian haplogroup R11a were identified and in Family LHON-019, 37 other variants belonging to the East Asian haplogroup D4g were determined. The ND1 G3635A mutation changes the conversed serine¹¹⁰ residue to asparagine. This mutation has been previously described in a single Russian LHON family and has been suggested to contribute to increased LHON expressivity. In addition, a mutation in cytochrome c oxidase subunit II at C7868T (COII/L95F) may act in synergy with G3635A, increasing LHON expressivity in Family LHON-001, which had a higher level of LHON penetrance than Family LHON-019. In summary, the G3635A mutation is confirmed as a rare primary pathogenic mutation for LHON.     

Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.     

Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.     

Analysis of mtDNA variation in African populations reveals the most ancient of all human continent-specific haplogroups.

mtDNA sequence variation was examined in 140 Africans, including Pygmies from Zaire and Central African Republic (C.A.R.) and Mandenkalu, Wolof, and Pular from Senegal. More than 76% of the African mtDNAs (100% of the Pygmies and 67.3% of the Senegalese) formed one major mtDNA cluster (haplogroup L) defined by an African-specific HpaI site gain at nucleotide pair (np) 3592. Additional mutations subdivided haplogroup L into two subhaplogroups, each encompassing both Pygmy and Senegalese mtDNAs. A novel 12-bp homoplasmic insertion in the intergenic region between tRNA(Tyr) and cytochrome oxidase I (COI) genes was also observed in 17.6% of the Pygmies from C.A.R. This insertion is one of the largest observed in human mtDNAs. Another 25% of the Pygmy mtDNAs harbored a 9-bp deletion between the cytochrome oxidase II (COII) and tRNA(Lys) genes, a length polymorphism previously reported in non-African populations. In addition to haplogroup L, other haplogroups were observed in the Senegalese. These haplogroups were more similar to those observed in Europeans and Asians than to haplogroup L mtDNAs, suggesting that the African mtDNAs without the HpaI np 3592 site could be the ancestral types from which European and Asian mtDNAs were derived. Comparison of the intrapopulation sequence divergence in African and non-African populations confirms that African populations exhibit the largest extent of mtDNA variation, a result that further supports the hypothesis that Africans represent the most ancient human group and that all modern humans have a common and recent African origin. The age of the total African variation was estimated to be 101,000-133,000 years before present (YBP), while the age of haplogroup L was estimated at 98,000-130,000 YBP. These values substantially exceed the ages of all Asian- and European-specific mtDNA haplogroups.     

Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.     

A cryptic, intergeneric cytochrome c oxidase I pseudogene in tyrant flycatchers (family: Tyrannidae)

Nuclear mitochondrial pseudogenes, or "numts", are nonfunctional copies of mitochondrial genes that have been translocated to the nuclear genome. Numts have been used to study differences in mutation rates between the nuclear and mitochondrial genomes, but have also been implicated as troublesome for phylogenetic studies and DNA-based species identification (i.e., DNA barcoding). In this study, a suspected numt discovered during a study of mitochondrial cytochrome c oxidase I (COI) diversity in North American birds was targeted and sequenced from tyrant flycatchers (family: Tyrannidae). In total, the numt was found in five taxa representing two genera. Substitution rates were compared between COI and numt sequences. None of the numt sequences harboured stop codons nor frameshift mutations, but phylogenetic analysis revealed they had accumulated more amino acid substitutions than the mitochondrial COI sequences. Mitochondrial COI appeared to be preferentially amplified in most cases, but methods for numt detection are discussed for cases like this where sequences lack obvious features for identification. Because of its persistence across a broad taxonomic lineage, this numt could form a valuable model system for studying evolution in numts. The full size of the numt and its location within the nuclear genome are yet to be determined.     

Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction

Mitochondria isolated from the skeletal muscle of an infant with mitochondrial myopathy and renal dysfunction were analyzed. Activities of NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase were severely decreased. Cytochromes aa3 and b were not detected in patient mitochondria, and the cytochrome c+c1 content was 14% of control. Immunoblotting demonstrated that the amount of cytochrome c oxidase subunits were markedly decreased in patient mitochondria. The polypeptide profile of patient mitochondria was quite different from that of control mitochondria. These results suggest that deterioration of mitochondria in a severe case of mitochondrial myopathy involves not only cytochrome c oxidase but also other mitochondrial proteins.     

Secondary Post-Geniculate Involvement in Leber’s Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is characterized by retinal ganglion cell (RGC) degeneration with the preferential involvement of those forming the papillomacular bundle. The optic nerve is considered the main pathological target for LHON. Our aim was to investigate the possible involvement of the post-geniculate visual pathway in LHON patients. We used diffusion-weighted imaging for in vivo evaluation. Mean diffusivity maps from 22 LHON visually impaired, 11 unaffected LHON mutation carriers and 22 healthy subjects were generated and compared at level of optic radiation (OR). Prefrontal and cerebellar white matter were also analyzed as internal controls. Furthermore, we studied the optic nerve and the lateral geniculate nucleus (LGN) in post-mortem specimens obtained from a severe case of LHON compared to an age-matched control. Mean diffusivity values of affected patients were higher than unaffected mutation carriers (P<0.05) and healthy subjects (P<0.01) in OR and not in the other brain regions. Increased OR diffusivity was associated with both disease duration (B = 0.002; P<0.05) and lack of recovery of visual acuity (B = 0.060; P<0.01). Post-mortem investigation detected atrophy (41.9% decrease of neuron soma size in the magnocellular layers and 44.7% decrease in the parvocellular layers) and, to a lesser extent, degeneration (28.5% decrease of neuron density in the magnocellular layers and 28.7% decrease in the parvocellular layers) in the LHON LGN associated with extremely severe axonal loss (99%) in the optic nerve. The post-geniculate involvement in LHON patients is a downstream post-synaptic secondary phenomenon, reflecting de-afferentation rather than a primary neurodegeneration due to mitochondrial dysfunction of LGN neurons.     

Leber's hereditary optic neuropathy: no significant evidence for primary or secondary pathogenicity of the 15257 mutation

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease of the optic nerves associated with various mitochondrial DNA (mtDNA) mutations. Four of these mutations, at nucleotide positions (np) 3460, 11778, 14484 and 15257, have been postulated to be of primary pathogenetical importance. Previously, we described the molecular and clinical findings in patients with the 11778 and 14484 mutations. Here we describe the molecular and clinical findings of patients in eight pedigrees with the 3460 mutation and in three pedigrees with the 15 257 mutation. In all three 15257 positive pedigrees the 3460, the 11778 or the 14484 mutation was also found. The first combination has not been reported before. We compared the clinical findings in these pedigrees with those of the 3460, 11778 and 14484 positive pedigrees that lack the 15257 mutation. No significant differences were found with respect to the age of onset, visual outcome or the probability of developing LHON. We conclude that there is no evidence that the 15257 mutation, which has been reported in normal controls, has primary causal significance, because it may coincide with the 3460, 11778 and 14484 mutations. We presume that the 15257 mutation has no secondary pathogenic importance, since it has no clear contribution to the degree or the probability of phenotypic expression.     

A novel mtDNA ND6 gene mutation associated with LHON in a Caucasian family

Leber's hereditary optic neuropathy (LHON) is a frequent cause of inherited blindness. A routine screening for common mtDNA mutations constitutes an important first in its diagnosis. However, a substantial number of LHON patients do not harbor known variants, both pointing to the genetic heterogeneity of LHON and bringing into question its genetic diagnosis. We report a familial case that exhibited typical features of LHON but lacked any of the common mutations. Genetic analysis revealed a novel pathogenic defect in the ND6 gene at 14279A that was not detected in any haplogroup-matched controls screened for it, nor has it been previously reported. This mutation causes a substantial conformational change in the secondary structure of the polypeptide matrix coil and may explain the LHON expression. Thus, it expands the spectrum of deleterious changes affecting ND6-encoding subunit and further highlights the functional significance of this gene, providing additional clues to the disease pathogenesis.     

A stop-codon mutation in the human mtDNA cytochrome c oxidase I gene disrupts the functional structure of complex IV.

We have identified a novel stop-codon mutation in the mtDNA of a young woman with a multisystem mitochondrial disorder. Histochemical analysis of a muscle-biopsy sample showed virtually absent cytochrome c oxidase (COX) stain, and biochemical studies confirmed an isolated reduction of COX activity. Sequence analysis of the mitochondrial-encoded COX-subunit genes identified a heteroplasmic G-->A transition at nucleotide position 6930 in the gene for subunit I (COX I). The mutation changes a glycine codon to a stop codon, resulting in a predicted loss of the last 170 amino acids (33%) of the polypeptide. The mutation was present in the patient's muscle, myoblasts, and blood and was not detected in normal or disease controls. It was not detected in mtDNA from leukocytes of the patient's mother, sister, and four maternal aunts. We studied the genetic, biochemical, and morphological characteristics of transmitochondrial cybrid cell lines, obtained by fusing of platelets from the patient with human cells lacking endogenous mtDNA (rho0 cells). There was a direct relationship between the proportion of mutant mtDNA and the biochemical defect. We also observed that the threshold for the phenotypic expression of this mutation was lower than that reported in mutations involving tRNA genes. We suggest that the G6930A mutation causes a disruption in the assembly of the respiratory-chain complex IV.     

Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides

With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species.