Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.     

Quantification of mitochondrial DNA mutation load

Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.     

Maternally transmitted diabetes mellitus associated with the mitochondrial tRNA(Leu(UUR)) A3243G mutation in a four-generation Han Chinese family

We report here the characterization of a four-generation Han Chinese family with maternally transmitted diabetes mellitus. Six (two males/four females) of eight matrilineal relatives in this family exhibited diabetes. The age of onset in diabetes varies from 15 years to 33 years, with an average of 26 years. Two of affected matrilineal relatives also exhibited hearing impairment. Molecular analysis of mitochondrial DNA (mtDNA) showed the presence of heteroplasmic tRNA(Lue(UUR)) A3243G mutation, ranging from 35% to 58% of mutations in blood cells of matrilineal relatives. The levels of heteroplasmic A3243G mutation seem to be correlated with the severity and age-at-onset of diabetes in this family. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the A3243G mutation and 38 other variants belonging to the Eastern Asian haplogroup M7C. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, the A3243G mutation is the sole pathogenic mtDNA mutation associated with diabetes in this Chinese family.     

人线粒体tRNALeu(UUR)基因A3243G点突变对其亮氨酰化活性的影响

化学法合成人线粒体野生型与A3243G点突变型tRNALeu(UUR)基因,体外转录生成相应的tRNALeu(UUR),表达并纯化人线粒体亮氨酰tRNA合成酶(mtLeuRS),用mtLeuRS催化野生型与突变型tRNALeu(UUR)与亮氨酸结合,分别检测两种类型tRNALeu(UUR)的氨酰化动力学常数。结果表明,野生型tRNALeu(UUR)的Km/Kcat仅为突变型tRNALeu(UUR)的63.9%,A3243G点突变使tRNALeu(UUR)接受亮氨酸的能力明显下降,提示此为A3243G点突变致病机制之一。 Abstract:The wild-type and mutant-type human mitochondrial tRNALeu(UUR) genes were synthesized and transcribed in vitro with T7 RNA polymerase.The kinetic parameters of human mitochondrial leucyl-tRNA synthetase(mtLeuRS) were determined with wild-type and mutant-type human mitochondrial tRNALeu(UUR) respectively.The results show that the value of Km/Kcat of mtLeuRS for the mutant-type tRNALeu(UUR) is 63.9% as compared with the wild-type.Human mitochondrial tRNALeu(UUR) gene A3243G point mutant can remarkably reduce it′s aminoacylation activity,suggesting it would be one of the mechanisms that the mutation could produce such clinical phenotypes.     

Ultraviolet radiation exposure accelerates the accumulation of the aging-dependent T414G mitochondrial DNA mutation in human skin

The accumulation of mitochondrial DNA (mtDNA) mutations has been proposed as an underlying cause of the aging process. Such mutations are thought to be generated principally through mechanisms involving oxidative stress. Skin is frequently exposed to a potent mutagen in the form of ultraviolet (UV) radiation and mtDNA deletion mutations have previously been shown to accumulate with photoaging. Here we report that the age-related T414G point mutation originally identified in skin fibroblasts from donors over 65 years also accumulates with age in skin tissue. Moreover, there is a significantly greater incidence of this mutation in skin from sun-exposed sites (chi(2)= 6.8, P < 0.01). Identification and quantification of the T414G mutation in dermal skin tissue from 108 donors ranging from 8 to 97 years demonstrated both increased occurrence with photoaging as well as an increase in the proportion of molecules affected. In addition, we have discovered frequent genetic linkage between a common photoaging-associated mtDNA deletion and the T414G mutation. This linkage indicates that mtDNA mutations such as these are unlikely to be distributed equally across the mtDNA population within the skin tissue, increasing their likelihood of exerting focal effects at the cellular level. Taken together, these data significantly contribute to our understanding of the DNA damaging effects of UV exposure and how resultant mutations may ultimately contribute towards premature aging.     

Investigation of the A1555G mutation in mitochondrial DNA (MT-RNR1) in groups of Brazilian individuals with nonsyndromic deafness and normal-hearing

BACKGROUND:

Mutations of mitochondrial DNA were described into two genes: The mitochondrially encoded 12S RNA (MT-RNR1) and the mitochondrially encoded tRNA serine^ucn (MT-TS1). The A1555G mutation in MT-RNR1 gene is a frequent cause of deafness in different countries.

AIM:

The aim of this work was to investigate the frequency of the A1555G mutation in the MT-RNR1 gene in the mitochondrial DNA in Brazilians individuals with nonsyndromic deafness, and listeners.

MATERIALS AND METHODS:

DNA samples were submitted to polymerase chain reaction and to posterior digestion with the Hae III enzyme.

RESULTS:

Seventy eight (78) DNA samples of deaf individuals were analyzed; 75 showed normality in the region investigated, two samples (2.5%) showed the T1291C substitution, which is not related to the cause of deafness, and one sample (1.3%) showed the A1555G mutation. Among the 70 non-impaired individuals no A1555G mutation or T1291C substitution was found.

CONCLUSION:

We can affirm that A1555G mutation is not prevalent, or it must be very rare in normal-hearing subjects in the State of Paraná, the south region of Brazil. The A1555G mutation frequency (1.3%) found in individual with nonsyndromic deafness is similar to those found in other populations, with nonsyndromic deafness. Consequently, it should be examined in deafness diagnosis. The investigation of the A1555G mutation can contribute towards the determination of the nonsyndromic deafness etiology, hence, contributing to the correct genetic counseling process.     

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.     

A novel mitochondrial DNA missense mutation at G3421A in a family with maternally inherited diabetes and deafness

OBJECTIVE: Mutations in mtDNA are thought to be responsible for the pathogenesis of maternally inherited diabetes. Here, we report a family with maternally inherited diabetes and deafness whose members did not harbour the mtDNA A3243G mutation, the most frequent point mutation in mitochondrial diabetic patients. This study aimed to investigate a possible other mtDNA mutation and its prevalence in type 2 diabetic patients. METHODS: Height, body weight, waistline, and hip circumference were measured and serum biochemical marks determined in all members of the family. In addition, a 75 g oral glucose tolerance test and electric listening test were conducted in these members. Genomic DNA was prepared from peripheral leukocytes. Direct sequencing of PCR products was used to detect the mtDNA mutation in this family. The prevalence of mtDNA G3421A nucleotide substitutions was investigated by restriction fragment length polymorphism analysis in 1350 unrelated type 2 diabetic patients recruited by random cluster sampling from the central city area of Shanghai, China. RESULTS: (1) A new missense homoplasmic mutation of mtDNA G3421A was found in a maternally inherited diabetic family and existed neither in 1350 unrelated type 2 diabetic patients nor in 50 non-diabetic individuals. (2) The mode of mutation and diabetes transmission was typical maternal inheritance in this family. (3) All diabetic family members were found to have an onset at 35-42 years of age, accompanied by deafness of varying degrees. CONCLUSION: mtDNA G3421A (Val39Ile) found in a family with maternally inherited diabetes and deafness is a novel missense mutation. Whether this is a diabetogenic mutation and its effect on mitochondrial function needs to be further studied.     

Clinical evaluation and mitochondrial DNA sequence analysis in three Chinese families with Leber's hereditary optic neuropathy

We report here the clinical, genetic, and molecular characterization of three Chinese families (WZ4, WZ5, and WZ6) with Leber's hereditary optic neuropathy (LHON). Clinical and genetic evaluations revealed the variable severity and age-of-onset in visual impairment in these families. Penetrances of visual impairment in these Chinese families were 33.3%, 35.7%, and 35.5%, respectively, with an average 34.8%. Furthermore, the average age-at-onset in those Chinese families was 17, 20, and 18 years. In addition, the ratios between affected male and female matrilineal relatives in these Chinese families were 3:0, 1:1, and 1.2:1, respectively. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical G11778A mutation associated with LHON in many families. The fact that mtDNA of those pedigrees belonged to different haplogroups F1, D4, and M10 suggested that the G11778A mutation occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these Chinese families. The I187T mutation in the ND1, the S99A mutation in the A6, the V254I in CO3, and I58V in ND6 mutation, showing high evolutional conservation, may contribute to the phenotypic expression of the G11778A mutation in the WZ6 pedigree. By contrast, none of mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence in WZ4 and WZ5 pedigrees. Apparently, these variants do not have a potential modifying role in the development of visual impairment associated with G11778A mutation in those two families. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and expressivity of LHON in these three Chinese families carrying the G11778A mutation.     

Only male matrilineal relatives with Leber's hereditary optic neuropathy in a large Chinese family carrying the mitochondrial DNA G11778A mutation

We report here the characterization of a five-generation large Chinese family with Leber's hereditary optic neuropathy (LHON). Very strikingly, six affected individuals of 38 matrilineal relatives (17 females/21 males) are exclusively males in this Chinese family. These matrilineal relatives in this family exhibited late-onset/progressive visual impairment with a wide range of severity, ranging from blindness to normal vision. The age of onset in visual impairment varies from 17 to 30 years. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the G11778A mutation in ND4 gene and 29 other variants. This mitochondrial genome belongs to the Southern Chinese haplogroup B5b. We showed that the G11778A mutation is present at near homoplasmy in matrilineal relatives of this Chinese family but not in 164 Chinese controls. Incomplete penetrance of LHON in this family indicates the involvement of modulatory factors in the phenotypic expression of visual dysfunction associated with the G11778A mutation. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and phenotypic variability of LHON in this Chinese family carrying the G11778A mutation.     

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.     

Leber's hereditary optic neuropathy is associated with the mitochondrial ND4 G11696A mutation in five Chinese families

We report here the clinical, genetic, and molecular characterization of five Chinese families with Leber's hereditary optic neuropathy (LHON). Clinical and genetic evaluations revealed the variable severity and age-of-onset in visual impairment in these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical ND4 G11696A mutation associated with LHON. Indeed, this mutation is present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families. In fact, the occurrence of the G11696A mutation in these several genetically unrelated subjects affected by visual impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. Furthermore, the N405D in the ND5 and G5820A in the tRNA(Cys), showing high evolutional conservation, may contribute to the phenotypic expression of G11696A mutation in the WZ10 pedigree. However, there was the absence of functionally significant mtDNA mutations in other four Chinese pedigrees carrying the G11696A mutation. Therefore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic expression of the LHON-associated G11696A mutation in these Chinese pedigrees.     

A novel m.3395A > G missense mutation in the mitochondrial ND1 gene associated with the new tRNA m.4316A > G mutation in a patient with hypertrophic cardiomyopathy and profound hearing loss

Mitochondria are essential for early cardiac development and impaired regulation of mitochondrial function was implicated in congenital heart diseases. We described a newborn girl with hypertrophic cardiomyopathy and profound hearing loss. The mtDNA mutational analysis revealed the presence of known polymorphisms associated to cardiomyopathy and/or hearing loss, and 2 novel heteroplasmic mutations: m.3395A > G (Y30C) occurring in a highly conserved aminoacid of the ND1 gene and the m.4316A > G located in the residue A54 of the tRNA^Ile gene. These 2 novel variations were absent in 150 controls. All these variants may act synergistically and exert a cumulative negative effect on heart function to generate the cardiomyopathy.