A Novel Amplification-Refractory Mutation System-PCR Strategy to Screen MT-TL1 Pathogenic Variants in Patient Repositories.

Aim: Pathogenic variants within mitochondrial tRNA and rRNA genes negatively affect protein synthesis function and cause oxidative phosphorylation defects. The majority of mitochondrial cytopathies are caused by pathogenic point variants within the mitochondrial tRNA gene for leucine (MT-TL1). This study was designed to evaluate a novel amplification-refractory mutation system (ARMS)-PCR based assay to screen patient samples with a clinical diagnosis of mitochondrial cytopathies. Methods: Tissue DNA samples from 219 affected individuals were screened for the pathogenic variants m.3271T>C, m.3291Ty >C, m.3303C>T, m.3256C>T, and m.3260A>G along with the most frequent m.3243A>G mutation in the MT-TL1 gene. The assay included a "High Resolution Melt curve analysis" to enhance detection limits. The precision of the assay was verified using synthetic controls with variant heteroplasmy ratios. Results: The screening identified the second reported m.3303C>T case as well as two patients with m.3243A>G variants and a rare variant exhibiting m.3290T>C. Conclusion: ARMS-PCR is superior to Sanger sequencing for the detection of variations exhibiting low heteroplasmy. These results provide "proof of concepts" for the implementation of this application for future screening of rare mtDNA variations in sample repositories.

Investigation on mitochondrial tRNA(Leu/Lys), NDI and ATPase 6/8 in Iranian multiple sclerosis patients.

As with chromosomal DNA, the mitochondrial DNA (mtDNA) can contain mutations that are highly pathogenic . In fact, many diseases of the central nervous system are known to be caused by mutations in mtDNA. Dysfunction of the mitochondrial Respiratory Chain (RC) has been shown in patients with neurological disease including Alzheimer's disease (AD), Parkinson's disease (PD) and Multiple sclerosis (MS). MS is a demyelinating disease of central nervous system characterized by morphological hallmarks of inflammation, demyelination and axonal loss. Considering this importance, we decided to investigate several highly mutative parts of mtDNA for point mutations as MT-LTI (tRNA(Leucine1(UUA/G))), MT-NDI (NADH Dehydrogenase subunit 1), MT-COII (Cytochrome c oxidase subunit II), MT-TK (tRNA(Lysine)), MT-ATP8 (ATP synthase subunit F0 8) and MT-ATP6 (ATP synthase subunit F0 6) in 20 Iranian MS patients and 80 age-matched control subjects by PCR and automated DNA sequencing to evaluate any probable point mutations. Our results revealed that 15 (75%) out of 20 MS patients had point mutations. Some of point mutations were newly found in this study. This study suggested that point mutation occurred in mtDNA might be involved in pathogenesis of MS.

Links between plant and rhizoplane bacterial communities in grassland soils, characterized using molecular techniques.

Molecular analysis of grassland rhizosphere soil has demonstrated complex and diverse bacterial communities, with resultant difficulties in detecting links between plant and bacterial communities. These studies have, however, analyzed "bulk" rhizosphere soil, rather than rhizoplane communities, which interact most closely with plants through utilization of root exudates. The aim of this study was to test the hypothesis that plant species was a major driver for bacterial rhizoplane community composition on individual plant roots. DNA extracted from individual roots was used to determine plant identity, by analysis of the plastid tRNA leucine (trnL) UAA gene intron, and plant-related bacterial communities. Bacterial communities were characterized by analysis of PCR-amplified 16S rRNA genes using two fingerprinting methods: terminal restriction fragment length polymorphisms (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Links between plant and bacterial rhizoplane communities could not be detected by visual examination of T-RFLP patterns or DGGE banding profiles. Statistical analysis of fingerprint patterns did not reveal a relationship between bacterial community composition and plant species but did demonstrate an influence of plant community composition. The data also indicated that topography and other, uncharacterized, environmental factors are important in driving bacterial community composition in grassland soils. T-RFLP had greater potential resolving power than DGGE, but findings from the two methods were not significantly different.

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes.

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.

Increased risk of stroke in patients with the A12308G polymorphism in mitochondria.

Factors which increase the risk of stroke in patients with the A3243G (mitochondrial encephalomyopathy, lactic acidosis, and stroke [MELAS]) mutation in human mitochondrial DNA are unclear. Previous work on lung-cancer cells with an A3243G mutation showed that a mutation in the mitochondrial transfer gene for leucine tRNA(Leu(CUN)) was able to ameliorate the A3243G-induced biochemical phenotype. We analysed the tRNA(Leu(CUN)) gene in 48 unrelated A3243G cases. We showed that a polymorphism, A12308G, in tRNA(Leu(CUN)) increases the risk of developing stroke in patients with the A3243G mutation (relative risk=2.17). This may have implications for genetic counselling.

Mitochondrial tRNALeu isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation.

We have investigated the representation of structural isoforms of the two mitochondrial leucyl tRNAs in lung carcinoma cybrid cell lines containing the np 3243 (MELAS) mtDNA mutation, alone or in combination with the np 12300 suppressor mutation. The mutant tRNALeu(UUR) is aminoacylated very poorly or not at all, whereas the suppressor tRNALeu(CUN) is efficiently aminoacylated. Deacylated mitochondrial tRNALeu(CUN) is present, in all human cells tested, in two structural isoforms that are separable on denaturing gels, indicating a difference in primary structure. The ratio of the two isoforms differs between cell types and is strongly biased towards one isoform in lung carcinoma cybrids containing high levels of the np 3243 mutation, compared with control cybrids. We propose that structural modification of tRNALeu(CUN) could be a natural suppression mechanism for the np 3243 and other mitochondrial tRNALeu(UUR) mutations and could underlie some of the phenotypic variability of np 3243 disease.

A tRNA suppressor mutation in human mitochondria.

Mitochondrial mutations are associated with a wide spectrum of human diseases. A common class of point mutations affects tRNA genes, and mutations in the tRNA-leu(UUR) gene (MTTL1) are the most frequently detected. In earlier studies, we showed that lung carcinoma cybrid cells containing high levels (greater than 95%) of mutated mtDNA from a patient with the pathological nucleotide pair (np) 3243 tRNA-leu(UUR) mutation can remain genotypically stable over time, and exhibit severe defects in mitochondrial respiratory metabolism. From such a cybrid containing 99% mutated mtDNA, we have isolated a spontaneous derivative that retains mutant mtDNA at this level but which has nevertheless reverted to the wild-type phenotype, based on studies of respiration, growth in selective media, mitochondrial protein synthesis and biogenesis of mitochondrial membrane complexes. The cells are heteroplasmic for a novel anticodon mutation in tRNA-leu(CUN) at np 12300, predicted to generate a suppressor tRNA capable of decoding UUR leucine codons. The suppressor mutation represents approximately 10% of the total mtDNA, but was undetectable in a muscle biopsy sample taken from the original patient or in the parental cybrid. These results indicate that the primary biochemical defect in cells with high levels of np 3243 mutated mtDNA is the inability to translate UUR leucine codons.

Gestational diabetes mellitus and gene mutations which affect insulin secretion.

We investigated whether genetic mutations known to impair insulin secretion and glucose tolerance are operative in a group of American women with gestational diabetes mellitus. Study groups were comprised of elderly non-diabetic controls (n = 55) with normal glucose tolerance and patients with gestational diabetes (n = 50), together with one family with maturity-onset diabetes of the young (three controls and three affected). No mutations were detected in any exon of the human glucokinase gene or the mitochondrial tRNA[Leu](UUR) gene by single strand conformational analysis and direct exon sequencing. Also, chi2 analysis showed no significant association with gestational diabetes for a polymorphism at position -30 (G --> A) of the beta-cell-specific glucokinase gene promoter. We have determined that glucokinase and mitochondrial tRNA[Leu](UUR) gene mutations, which are known to impair insulin secretion are relatively uncommon and do not constitute a large component of genetic risk for gestational diabetes in the study population.

Variable distribution of mutant mitochondrial DNAs (tRNA(Leu[3243])) in tissues of symptomatic relatives with MELAS: the role of mitotic segregation.

We studied multiple different postmortem tissue samples from a woman and two of her daughters with the MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) tRNA(Leu(UUR)) mutation at nucleotide 3243 in mitochondrial DNA (mtDNA). All tissues examined were heteroplasmic for the mutation. The mean proportion of mutant mtDNAs in the mother's tissues (0.30 +/- 0.10) was significantly lower than that of each of her daughters' (0.76 +/- 0.11, p < 0.03, and 0.72 +/- 0.13, p < 0.001); there was no difference in the fraction of mutant mtDNAs between the daughters (p < 0.71). This difference in the mean proportion of mtDNA mutants between family members correlates with their clinical profiles; the mother had the latest onset of disease and lived longest, while the two daughters had a strikingly similar clinical course. In individual patients, the mean proportion of mutant mtDNAs was not different in tissues deriving from ectodermal, mesodermal, and endodermal germ layers. Variance in the mutant:wild-type mtDNA ratio was normally distributed about the mean, both when all tissues were considered together and when different regions of the CNS were considered separately. Thus, the proportion of mtDNAs carrying the tRNA(Leu(3243)) mutation was not uniform in members of this pedigree and did not undergo rapid mitotic segregation along germ-layer divisions. These findings are consistent with the hypothesis that the overall proportion of mtDNAs carrying this mutation is primarily determined by segregation during oogenesis or early embryologic development and that random replicative (mitotic) segregation, subsequent to the establishment of primary germ layers, is responsible for the variation between tissues.

Correlation between clinical and molecular features in two MELAS families.

We describe the clinical, morphological, biochemical presentation in two MELAS families, and correlate it with the distribution and proportion of mitochondrial DNA carrying the A to G transition at nt 3243. Family A was characterized by late onset MELAS in two members, CPEO in one, and mild CNS involvement in another. 20-61% of mtDNA of affected and unaffected individuals was mutated in muscle, 2-18% in blood. There was no obvious correlation between clinical picture and proportion of mutated mtDNA. In family B full MELAS syndrome appeared only in the third generation, but the mutation was also detected in muscle of asymptomatic individuals of the first and second generation. The proportion of mutated mtDNA in blood, and to a lesser extent in muscle, correlated with the severity of the clinical presentation. The MELAS mutation is consistently detected in all asymptomatic maternal relatives of MELAS patients. We conclude that different clinical presentations of mitochondrial encephalomyopathy may coexist in the same family, and correlation between clinical severity and molecular abnormality is not always recognizable. Presence of the MELAS mutation in muscle and blood is a necessary but not sufficient condition for the expression of the typical MELAS phenotype.

Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNA(Leu)(UUR).

Different point mutations of the mitochondrial genome, which all affect the ability of mitochondria to translate their own genes and lead to partial defects of mtDNA-dependent respiratory complexes, are related to distinct clinical mitochondrial disorders. A new maternally inherited disorder, characterised by a combination of adult-onset myopathy and cardiomyopathy, with no clinical involvement of the nervous system, was found in members of a single large pedigree. A heteroplasmic new mutation was identified in the mtDNA gene specifying tRNA(Leu)(UUR). This mutation segregated specifically with the disorder, and there were significant correlations between the proportion of the mtDNA that was of the mutant form and the activities (normalised for citrate synthase activity) of the two mtDNA-dependent respiratory enzymes (complex I, r = -0.71, p less than 0.005: complex IV r = -0.77, p less than 0.005) and the maximum oxygen consumption (r = -0.82, p less than 0.005), a physiological index of aerobic metabolism. These findings strongly suggest that the tRNA(Leu)(UUR) mutation is the genetic cause of this disorder, and that lesions of mtDNA should be considered in the differential diagnosis of the hereditary cardiomyopathies.

[A study of the conformation of tRNA(IAGLeu) from the cow mammary gland using chemical modification methods]. / Issledovanie élementov prostranstvennoi struktury tRNK(IAGLeu) molochnoi zhelezy korov metodami khimicheskoi modifikatsii.

The nucleosides of tRNA(IAGLeu) (with a long variable loop) from the cow mammary gland included in formation of the three-dimensional structure have been analysed by the chemical modification methods. Exposed guanosine and cytidine residues were detected by means of dimethylsulfate, whereas diethylpyrocarbonate was used to detect exposed adenosine residues. The low level of the modification was characteristic of guanosine residues in positions 10 (m2G), 13, 15, 23, 24, 29, 30, 47 H, 51, 52, 53, 57; of cytidine residues in positions 48 (m5C), 56 and those involved in Watson--Crick pairing; of adenosine residues in positions 14, 22, 31, 42, 59, 64. Most bases of tRNA(IAGLeu) thus detected are similarly located in the yeast tRNA(Phe) molecule, which suggests a common role of these bases in the formation of the spacial structure of both tRNAs.