Distributions of five common point mutants in the human tracheal-bronchial epithelium

The mutations C742T, G746T, G747T in the TP53 gene and G35T in the KRAS gene have been repeatedly found in sectors of human tumors by direct DNA sequencing. The mutation G508A in the HPRT1 gene has been repeatedly found among peripheral T lymphocytes by clonal expansion under selective conditions. To discover if these mutations also occur frequently in normal tissues from which tumors arise, we have developed and validated allele-specific mismatch amplification mutation assays (MAMA) for each mutation. Reconstruction experiments demonstrated linearity in the range of 9-3000 mutant alleles among 3 x 10(6) wild-type alleles. The cumulative distributions of all negative controls established robust detection limits (P<0.05) of 34-125 mutants per 10(6) copies assayed depending on the mutation. One hundred and seventy-seven micro-anatomical samples of approximately (0.5-6)x10(6) tracheal-bronchial epithelial cells from nine non-smokers were assayed representing en toto the equivalent of approximately 1.6 human bronchial trees to the fifth bifurcation. Statistically significant mutant copy numbers were found in 257 of 463 assays. Clusters of mutant copies ranged from 10 to 1000 in 239/257 positive samples. As all five point mutations were detected at mutant fractions of >10(-5) in two or more lungs, we infer that they are mutational hotspots generated in lung epithelial stem cells. As the cancer-associated mutations did not differ in cluster size distribution from the HPRT1 mutation, we infer that none of the mutations conferred a growth advantage to somatic heterozygous clusters or maintenance turnover units. Specific mutants appeared in very large copy numbers, 1000-35,000, in 18/257 positive assays. Various hypotheses to account for the observed cluster size distributions are offered.     

Clustering of mutant mitochondrial DNA copies suggests stem cells are common in human bronchial epithelium

Tissue maintenance stem cells, as opposed to transition and/or terminal cells in the epithelium, are possible progenitor cells for human tumors, but little is known about their frequency in human tissues. It occurred to us that the colonies of mutants that should be created when a stem cell mutates and transmits the rare mutation to its descendent transition and terminal cells should, given a quantitative mutation assay, define the average number of cells in a maintenance turnover unit and permit calculation of stem cell number. To test this concept we used a combination of high fidelity PCR and constant denaturant capillary electrophoresis to enumerate mitochondrial point mutations and define their number and distribution among multiple small samples of approximately one million cells containing about 400 million copies of mitochondrial DNA. The bulk of the data were best explained by a model in which most stem cells, defined here as long-lived cells, give rise to colonies of approximately 8-128 cells. In addition, we found that about 1.5% of colonies contained hundreds or even thousands of homoplasmic mutant cells. These expanded turnover units suggest the bronchial epithelium may contain large clusters of cells with mutations, and possibly phenotypic alterations as well.     

Decreased CCA-addition in human mitochondrial tRNAs bearing a pathogenic A4317G or A10044G mutation

Pathogenic point mutations in mitochondrial tRNA genes are known to cause a variety of human mitochondrial diseases. Reports have associated an A4317G mutation in the mitochondrial tRNA(Ile) gene with fatal infantile cardiomyopathy and an A10044G mutation in the mitochondrial tRNA(Gly) gene with sudden infant death syndrome. Here we demonstrate that both mutations inhibit in vitro CCA-addition to the respective tRNA by the human mitochondrial CCA-adding enzyme. Structures of these two mutant tRNAs were examined by nuclease probing. In the case of the A4317G tRNA(Ile) mutant, structural rearrangement of the T-arm region, conferring an aberrantly stable T-arm structure and an increased T(m) value, was clearly observed. In the case of the A10044G tRNA(Gly) mutant, high nuclease sensitivity in both the T- and D-loops suggested a weakened interaction between the loops. These are the first reported instances of inefficient CCA-addition being one of the apparent molecular pathogeneses caused by pathogenic point mutations in human mitochondrial tRNA genes.     

Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.     

ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization

The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F1F0-ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F1F0-ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F1 and F0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F1F0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F1F0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.     

In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase

MERRF (myoclonic epilepsy with ragged-red fibers) is a severe, multisystem disorder characterized by myoclonus, seizures, progressive cerebellar syndrome, muscle weakness, and the presence of ragged-red fibers in the muscle biopsy. MERRF is associated with heteroplasmic point mutations, either A8344G or T8356C, in the gene encoding the mitochondrial tRNALys. The human ro cell system was utilized to examine the phenotypic consequences of these mutations, and to investigate their molecular genetic causes. Wild-type and mutant transmitochondrial cell lines harboring a pathogenic point mutation at either A8344G or T8356C in the human mitochondrial tRNALys gene were isolated and examined. Mitochondrial transformants containing 100% mutated mitochondrial DNAs (mtDNAs) exhibited severe defects in respiratory chain activity, in the rates of protein synthesis, and in the steady-state levels of mitochondrial translation products as compared with mitochondrial transformants containing 100% wild-type mtDNAs. In addition, both mutant cell lines exhibited the presence of aberrant mitochondrial translation products. These results demonstrate that two different mtDNA point mutations in tRNALys result in fundamentally identical defects at the cellular level, and that these specific protein synthesis abnormalities contribute to the pathogenesis of MERRF. (Mol Cell Biochem 174: 215–219, 1997)     

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing

Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.     

Patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinson's disease and mitochondrial encephalomyopathy

Comparison of total mitochondrial DNA sequences of patients with idiopathic (deleted or hypertrophic) cardiomyopathy with those of patients with Parkinson's disease and mitochondrial encephalomyopathies revealed distinct clustering of point mutations among patients. Furthermore, an inverse relation was found between the total number of base-substitution and life span of the patients. Among point mutations found in each patient, sequentially diverged six clusters consisting of 14, 10, 7, 1, 2, and 3 mutations, respectively, were detected. Five sub-clusters consisting of 2, 2, 11, 1, and 1 mutations, respectively, were detected. From each cluster, the patient's unique mutations were diverged with three types of the mutations specific for the disease. The divergence allowed construction of a phylogenetic tree which clearly indicated that patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinson's disease and mitochondrial encephalomyopathies.     

Detection of eight frequently encountered point mutations in mitochondria in Chinese patients suggestive of mitochondrial encephalomyopathies

To evaluate eight frequently encountered mitochondrial DNA (mtDNA) point mutations (A3243G, T8993G/C, A8344G, A1555G, G11778A, G3460A and T14484C) in Chinese, we recruited 1559 sporadic patients suspected of mitochondrial diseases and 206 family members. In suspected patients, 158 cases were detected with one of these eight mtDNA mutations (10.1%). A3243G was the most common mtDNA mutation both in suspected patients (9.4%) and in the relatives (34.2%). In addition, the ratios of A3243G (mutant/wild-type) and A8344G were significantly correlated with the patients’ age of examination. Moreover, in 76 unrelated probands, the ratio of A3243G was correlated well with their seizures and myopathies.     

Participation of the intracellular enzymes in the control of mutational processes

Summary The influence of UV-specific endonuclease and medium composition on the frequency and spectrum of genic mutations in Escherichia coli KI2 uvr ⁺ (with normal repair enzymes) and urv A6 (defective in UV-specific endonuclease) was studied. Mutations at the locus glu (gene controlling assimilation of glucose) were induced by ultra-violet irradiation and hydroxylamine treatment. To identify mutant colonies, triphenyl tetrazolium chloride (TTC) was added to the medium since it coloured the mutant colonies bright crimson and readily permitted distinction between pure mutant clones (complete mutations) and mixed clones (mosaic or sector mutations).A maximum mutation frequency after UV-irradiation was observed in E. coli uvr ⁺ cells but not in the E. coli uvr A6 strain. The curve of mutagenesis with a maximum was found in both studied strains after treatment by hydroxylamine which did not cause DNA damage recognized by UV-specific endonuclease.The highest frequency of mutations (at the point of maximum) in the series of experiments with enriched growth medium was almost 10 times higher than in the series of the experiments with poor medium.It was established that in bacteria with normal repair enzymes the frequency of complete mutations was higher than the frequency of mosaic mutations. It was also observed that the rate of UV-mutagenesis was higher in the case of E. coli uvr ⁺.The study of the distribution of mosaic mutant sectors in experiments with bacteria suspended in either a nutrient broth or a buffer during UV-irradiation revealed that the size of mutant sectors was rather variable and that the differences in the number of nucleoids per cell did not always determine the distribution of mutant sector sizes.Abbreviations HA Hydroxylamine hydrochloride - TTC Triphenyl tetrazolium chloride - TCA Trichloroacetic acid Other papers of this series are: Soyfer 1972; Soyfer et al. 1977; Soyfer and Kartel 1978     

The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu

We have taken advantage of the similarity between human and yeast (Saccharomyces cerevisiae) mitochondrial tRNA^Leu(UUR), and of the possibility of transforming yeast mitochondria, to construct yeast mitochondrial mutations in the gene encoding tRNA^Leu(UUR) equivalent to the human A3243G, C3256T and T3291C mutations that have been found in patients with the neurodegenerative disease MELAS (for mitochondrial 'myopathy, encephalopathy, lactic acidosis and stroke-like episodes'). The resulting yeast cells (bearing the equivalent mutations A14G, C26T and T69C) were defective for growth on respiratory substrates, exhibited an abnormal mitochondrial morphology, and accumulated mitochondrial DNA deletions at a very high rate, a trait characteristic of severe mitochondrial defects in protein synthesis. This effect was specific at least in the pathogenic mutation T69C, because when we introduced A or G instead of C, the respiratory defect was absent or very mild. All defective phenotypes returned to normal when the mutant cells were transformed by multicopy plasmids carrying the gene encoding the mitochondrial elongation factor EF-Tu. The ability to create and analyse such mutated strains and to select correcting genes should make yeast a good model for the study of tRNAs and their interacting partners and a practical tool for the study of pathological mutations and of tRNA sequence polymorphisms.     

Mapping 136 pathogenic mutations into functional modules in human DNA polymerase γ establishes predictive genotype–phenotype correlations for the complete spectrum of POLG syndromes

We establish the genotype–phenotype correlations for the complete spectrum of POLG syndromes by refining our previously described protocol for mapping pathogenic mutations in the human POLG gene to functional clusters in the catalytic core of the mitochondrial replicase, Pol γ (1). We assigned 136 mutations to five clusters and identify segments of primary sequence that can be used to delimit the boundaries of each cluster. We report that compound heterozygotes with two mutations from different clusters manifested more severe, earlier-onset POLG syndromes, whereas two mutations from the same cluster are less common and generally are associated with less severe, later onset POLG syndromes. We also show that specific cluster combinations are more severe than others and have a higher likelihood to manifest at an earlier age. Our clustering method provides a powerful tool to predict the pathogenic potential and predicted disease phenotype of novel variants and mutations in POLG, the most common nuclear gene underlying mitochondrial disorders. We propose that such a prediction tool would be useful for routine diagnostics for mitochondrial disorders. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.     

Ligation of high-melting-temperature ‘clamp’ sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10 000 and 5/100 000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 × 10^–4. Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10^–6 using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (~100 bp) juxtaposed with a ‘clamp’, a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent ~9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 × 10^–5 in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 × 10⁴ turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10⁵ persons.     

Screening of common mitochondrial mutations in Chinese patients with mitochondrial encephalomyopathies

To investigate the spectrum of common mitochondrial mutations in Northern China during the years of 2000-2005, 552 patients of mitochondrial encephalomyopathies clinically diagnosed as MELAS, MERRF or Leigh's syndrome, 14 cases of LHON and 46 cases of aminoglycoside induced deafness along with their family members, accepted routine point mutation tests at nucleotide positions 3243, 8344, 8993, 11778 or 1555 in mitochondrial genome. PCR-RFLP analysis, site-specific PCR and PCR-sequencing methods were used to identify the mutations. Fifty-seven cases with A3243G mutation, 4 cases with A8344G, 2 cases with T8993C and 1 case with T8993G were identified from the 552 encephalomyopathy patients. In addition, one case with G11778A was found from the 14 cases of LHON, and 5 cases with A1555G from the 46 cases of aminoglycoside ototoxicity patients. Additional screening for T8356G and T3271C merely had limited significance for the diagnosis of MERRF and MELAS. Differential diagnosis among mitochondrial encephalomyopathies was often complicated due to many similar clinical manifestations. For A3243G mutation, the proportion of mutant mtDNA was not related to severity of the disease but to the age of onset.     

Mitochondrial DNA defects in Saccharomyces cerevisiae caused by functional interactions between DNA polymerase gamma mutations associated with disease in human

The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and Ery(R) mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1(G651S) (hG848S) mutant the high mtDNA instability and increased frequency of point Ery(R) mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1(A692T-E900G) (hA889T-hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1(H734Y)/mip1(G807R) (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1(E900G) (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.     

Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions. Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing. We find that the B-domain is essential for gp32's stimulation of these reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.