Detection of heteroplasmic mitochondrial DNA in single mitochondria.

BACKGROUND: Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases. METHODOLOGY: This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion. SIGNIFICANCE: Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.

Advances in Huntington's disease diagnostics: development of a standard reference material.

Huntington's disease (HD) is a neurodegenerative disease that affects four to seven individuals per 100,000. The onset of symptoms usually begins in middle age, although approximately 5% become symptomatic as juveniles. Death occurs approximately 15 years following the onset of symptoms, which include choreic movements, cognitive decline and psychiatric changes. HD is an autosomal dominant inherited disease that is associated with an expansion of a trinucleotide (CAG) repeat located on chromosome 4. Physicians rely on a positive family history, and diagnostic and genetic tests to detect the expansion in the number of CAG trinucleotide repeats in the HD gene to confirm the diagnosis. More than 99% of HD patients have 40 or more CAG triplet repeats and, therefore, targeted mutational analysis is greater than 99% sensitive. Individuals with 26 triplet repeats or less are normal, and while those with 27-35 repeats may not demonstrate symptoms themselves, their offspring may have the disease. Individuals with 36-39 repeats may or may not exhibit symptoms. The College of American Pathology/American College of Medical Genetics Biochemical and Molecular Genetics Resource Committee has emphasized the need to standardize the methodology for the determination of the accurate number of CAG repeats. This will prevent false-positive or -negative results when conducting predictive or prenatal testing of at-risk individuals. The National Institute of Standards and Technology is developing a standard reference material to provide these positive and negative controls needed by clinical testing laboratories. The use of a HD standard reference material will provide the quality control and assurance that data from different laboratories are both comparable and accurate.

Profiling of length heteroplasmies in the human mitochondrial DNA control regions from blood cells in the Korean population.

The length heteroplasmies in the hypervariable (HV) regions of mitochondrial DNA (mtDNA) from blood cells were examined in 57 healthy Korean donors. Interestingly, all the healthy Korean subjects displayed length heteroplasmies in both the HV1 and HV2 regions. Closer examination of the HV2 length heteroplasmies indicated that most of these donors (84%) exhibited a minimal 303-315 homopolymeric C (poly-C) tract frameshift of 1 bp (mixture of one major and minor mtDNA type). Sixteen percent of the donors however had poly-C tract frameshifts of 2 bp or more. The donor group with major length variants (two or more frameshifts) had about a two-fold decrease in mtDNA copy number compared with the group exhibiting only a 1 bp frameshift. This result supports the possibility that a severe frameshift in the 303-315 poly-C tract may also cause the impairment of mtDNA replication in hematopoietic tissue.

The common deletion found in patient reexamined after 33 years and comparison with complete mtDNA sequences of maternal relatives.

In 1966, a male (17 years old) was clinically examined at the National Institutes of Health (NIH) and diagnosed with Idiopathic Progressive External Ophthalmoplegia (IPEO). A muscle biopsy showing ragged-red fibers implicated mitochondrial involvement. Since the sequence of human mitochondrial DNA (mtDNA) was not determined until 1981, no genetic confirmation of the disease was possible at that time. In 1999, clinical reexamination and sequencing the entire mtDNA of the patient and living maternal relatives (mother and brother) indicated a progressive mitochondrial myopathy and the presence of the 4977 base pair (bp) deletion (the common deletion) in the patient.

Genetically characterized positive control cell lines derived from residual clinical blood samples.

BACKGROUND: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications. METHODS: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing. RESULTS: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications. CONCLUSIONS: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Mitochondrial DNA spectra of single human CD34+ cells, T cells, B cells, and granulocytes.

Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors-37.9% +/- 3.6% heterogeneity in circulating CD34+ cells, 36.4% +/- 14.1% in T cells, 36.0% +/- 10.7% in B cells, and 47.7% +/- 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% +/- 5.7% in CD34+ cells, 15.2% +/- 9.0% in T cells, 15.4% +/- 6.7% in B cells, and 32.3% +/- 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells.

A Standard Reference Material to determine the sensitivity of techniques for detecting low-frequency mutations, SNPs, and heteroplasmies in mitochondrial DNA.

Human mitochondrial DNA (mtDNA) mutations are important for forensic identifications and mitochondrial disease diagnostics. Low-frequency mutations, heteroplasmies, or SNPs scattered throughout the DNA in the presence of a majority of mtDNA with the Cambridge Reference Sequence (CRS) are almost impossible to detect. Therefore, the National Institute of Science and Technology has developed heteroplasmic human mtDNA Standard Reference Material (SRM) 2394 to allow scientists to determine their sensitivity in detecting such differences. SRM 2394 is composed of mixtures ranging from 1/99 to 50/50 of two 285-bp PCR products from two cell lines that differ at one nucleotide position. Twelve laboratories using various mutation detection methods participated in a blind interlaboratory evaluation of a prototype of SRM 2394. Most of these procedures were unable to detect the mutation when present below 20%, an indication that, in many real-life cases, low-frequency mutations remain undetected and that more sensitive mutation detection techniques are urgently needed.

Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes.

We have reported marked mitochondrial DNA (mtDNA) sequence heterogeneity among individual CD34 clones from adult bone marrow (BM) and the age-dependent accumulation of mtDNA mutations in this mitotically active tissue. Here, we show direct evidence of clonal expansion of cells containing mtDNA mutations and that the mtDNA sequence may be easily determined by using peripheral blood (PB) as a CD34 cell source. Analysis of 594 circulating CD34 clones showed that 150 (25%) had mtDNA sequences different from the same donor's corresponding aggregate sequence. Examination of single granulocytes indicated that 103 (29%) from the same 6 individuals showed mtDNA heterogeneity, with sequences distinct from the corresponding aggregate tissue sequence and from the sequences of other single granulocytes. Circulating and BM CD34 cells showed virtually identical patterns of mtDNA heterogeneity, and the same changes were seen in progeny granulocytes as in their progenitors, indicating that blood sampling could be used in studies to determine whether mtDNA reflects an individual's cumulative or recent exposure to mutagens; as a marker of individual hematopoietic progenitors, stem cells, and their expansion; and for the detection of minimal residual disease in hematologic malignancies of CD34 cell origin.

Marked mitochondrial DNA sequence heterogeneity in single CD34+ cell clones from normal adult bone marrow.

Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in postmitotic tissues but have been postulated to be diluted and lost in continually proliferating tissues such as bone marrow (BM). Having observed marked sequence variation among healthy adult individuals' total BM cell mtDNA, we undertook analysis of the mtDNA control region in a total of 611 individual CD34+ clones from 6 adult BM donors and comparison of these results with the sequences from 580 CD34+ clones from 5 umbilical cord blood (CB) samples. On average, 25% (range, 11% to 50%) of individual CD34+ clones from adult BM showed mtDNA heterogeneity, or sequence differences from the aggregate mtDNA sequence of total BM cells of the same individual. In contrast, only 1.6% of single CD34+ clones from CB showed mtDNA sequence variation from the aggregate pattern. Thus, age-dependent accumulation of mtDNA mutations appears relatively common in a mitotically active human tissue and may provide a method to approximate the mutation rate in mammalian cells, to assess the contribution of reactive oxygen species to genomic instability, and for natural "marking" of hematopoietic stem cells; our data also have important implications for the aging process, forensic identifications, and anthropologic conclusions dependent on the mtDNA sequence.

Characterization of surface organic components of human hair by on-line supercritical fluid extraction-gas chromatography/mass spectrometry: a feasibility study and comparison with human identification using mitochondrial DNA sequences.

This paper discusses results of a supercritical fluid extraction-gas chromatography/mass spectrometry (SFE-GC/MS) study of small samples ( 100 microg to 1 mg) of human scalp hair. The method offers a number of benefits including greater sensitivity than liquid extraction methods because the entire extractable mass is transferred to the analytical system, compared with only a few percent from a conventional liquid extraction/injection. The project's goals were to determine if SFE-GC/MS analyses of the surface-extractable components of an individual's hair yield consistent chemical profiles and to investigate if the profiles are sufficiently different to distinguish them from those of other individuals. In addition, the mtDNA sequences from ten of the same individuals used in the SFE-GC/MS study from four family units were determined, and, while the families were distinguishable, the maternal relations yielded identical sequences. In tandem, SFE-GC/MS and mtDNA techniques may provide valuable complementary data from forensic hair samples.

Mitochondrial DNA mutations in patients with myelodysplastic syndromes.

We undertook to systematically analyze the entire mitochondrial genome by gene amplification and direct sequencing in 10 patients with myelodysplasia; results were compared with concomitantly studied 8 healthy volunteers as well as mtDNA sequences in a standard database. Nucleotide changes that were present in our healthy controls as well as those in published databases were counted as polymorphisms. Overall, there was no increase in the number of mtDNA genes harboring polymorphisms or "new" mutations between our patients and healthy controls, although there were a few more mtDNA changes resulting in amino acid changes in myelodysplasia (9 in 8 controls versus 16 in 10 patients). Thirty new mutations, all nucleotide substitutions, were found among the 10 patients, distributed throughout the mitochondrial genome; 5 mutations resulted in amino acid changes. None of the mutations in controls produced amino acid changes. We were not able to confirm previously described mutations in sideroblastic anemia or "hot spots" in the cytochrome c oxidase I and II genes. Our data do not support a major role for mitochondrial genomic instability in myelodysplasia, and they fail to reproduce previous reports of significant or widespread mitochondrial mutations in this disease. Modest changes in mutation numbers and mitochondrial microsatellites may be evidence of increased mutagenesis in mtDNA, or, more likely, a reflection of limited clonality among hematopoietic stem cells in this bone marrow failure syndrome.

Comparison of the complete mtDNA genome sequences of human cell lines--HL-60 and GM10742A--from individuals with pro-myelocytic leukemia and leber hereditary optic neuropathy, respectively, and the inclusion of HL-60 in the NIST human mitochondrial DNA standard reference material--SRM 2392-I.

Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and assurance that their results from sequencing unknown samples are correct. Therefore, the mitochondrial DNA (mtDNA) genome of HL-60, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology (NIST) SRM 2392-I. NIST human mtDNA SRM 2392 will continue to be available and includes the DNA from two apparently healthy individuals. Both SRM 2392 and 2392-I contain all the information (e.g. the sequences of 58 unique primer sets) needed to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392, the HL-60 mtDNA in SRM 2392-I has two tRNA differences and more polymorphisms resulting in amino acid changes. Four of these HL-60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM10742A) from an individual with LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset, J2, and may only be indicative that HL-60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European descent or may mean that haplogroup J is more prone to LHON. Both these mtDNA SRMs will provide enhanced quality control in forensic identification, medical diagnosis, and single nucleotide polymorphism detection.

Inter- and intragenerational transmission of a human mitochondrial DNA heteroplasmy among 13 maternally-related individuals and differences between and within tissues in two family members.

The transmission of a C16,291C/T heteroplasmy in the HV1 region of human mitochondrial DNA (mtDNA) was examined in buccal cells from 13 maternally-related individuals across three generations and in additional tissues (hair, blood, or finger nails) from three members of this family. The ratio of C:T at nucleotide position (np) 16,291 showed wide intra- and intergenerational variation as well as tissue variation within individuals. Our results demonstrate that one or two sequence differences between samples in the mtDNA does not warrant an exclusion. To avoid false exclusions especially when comparing mtDNA from hair samples, we recommend the analysis of as many samples as possible in order to minimize the possibility that the detection of a rare polymorphism in a single sample would be considered an exclusion when it is really a match. The observation that the transmission of a mtDNA heteroplasmy from one individual to her offspring is likely to differ among the first-generation offspring and between that generation and subsequent generations lends further credence to the bottleneck theory of inheritance of human mtDNA.

Design and use of a peptide nucleic acid for detection of the heteroplasmic low-frequency mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) mutation in human mitochondrial DNA.

BACKGROUND: Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G, that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood. METHODS: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing. RESULTS: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the wild-type sequence. CONCLUSION: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods.

MitoAnalyzer, a computer program and interactive web site to determine the effects of single nucleotide polymorphisms and mutations in human mitochondrial DNA.

MitoAnalyzer provides information about the effects of single nucleotide polymorphisms (SNPs) and mutations in human mitochondrial DNA (mtDNA). This program determines if a single base pair (bp) change is in the non-coding or coding region, in the first, second or third bp of the codon, in a rRNA, tRNA or a protein, causes an amino acid (aa) change, the nature of that change, the position of the aa change in the protein, and the new aa sequence of the changed protein. Mutations associated with published mitochondrial diseases are noted. This program, thus, facilitates rapid analysis and evaluation of SNPs and mutations in human mtDNA.

The Development of a Standard Reference Material for Calibration of the University of Pittsburgh Smoke Toxicity Method for Assessing the Acute Inhalation Toxicity of Combustion Products.

A standard reference material (SRM 1049) has been developed for the University of Pittsburgh smoke toxicity method. SRM 1049 is a nylon 6/6 and has the molecular structure of [-NH(CH2)6NHCO(CH2)4CO-] n . This SRM is for calibrating the apparatus and providing confidence that the method is being conducted in a correct manner and that the equipment is functioning properly. The certified figure of merit is a LC50 value plus its 95% prediction interval which were calculated and found to be 4.4 + 3.4 g. The 95% prediction interval indicates the range in which the next determined LC50 value would be expected to fall. Thus, if an investigator were to test this SRM under their laboratory conditions according to the specifications of the University of Pittsburgh test procedure and found the LC50 value fell within the certified 95% prediction interval, the probability is good that the test is being conducted correctly.

A Standard Reference Material for Calibration of the Cup Furnace Smoke Toxicity Method for Assessing the Acute Inhalation Toxicity of Combustion Products.

A standard reference material (SRM 1048) has been developed for use with the cup furnace smoke toxicity method. This SRM is to be used to calibrate the apparatus and to enable the user to have confidence that the method is being conducted in a correct manner and that the equipment is functioning properly. The toxicological results from this SRM should not be used to compare with those of other materials (i.e., to determine if the combustion products of a test material are more or less toxic than those from this SRM). SRM 1048 is an acrylonitrile-butadiene-styrene (ABS) and is the same as SRM 1007B which is used for calibrating the flaming mode of the Smoke Density Chamber test method (ASTM E-662 and NFPA 258). For the purposes of calibrating the cup furnace smoke toxicity method, LC50 and N-Gas values plus their respective 95% confidence limits have been determined and certified for two combustion modes (flaming and nonflaming) and two observation periods (for the 30 min exposure only and for the 30 min exposure plus a 14 d post-exposure period). The certified LC50 values plus 95% confidence intervals (in g/m3) are 27 ± 3 (30 min, flaming); 25 ± 3 (30 min+ 14 d, flaming); 58 ± 15 (30 min, nonflaming); and 53 + 12 (30 min+ 14 d, nonflaming). The certified N-Gas values plus 95% confidence intervals are 1.4 ± 0.2 (30 min, flaming); 1.5 ± 0.2 (30 min+ 14 d, flaming); 1.2 ± 0.2 (30 min, nonflaming); and 1.4 ± 0.2 (30 min+ 14 d, nonflaming). It is recommended that this SRM be used with the N-Gas approach to calibrate the cup furnace smoke toxicity method rather than to determine the complete LC50 values. The N-Gas approach has the advantage of providing information on the gases responsible for the lethalities as well as the toxic potency of the smoke. In addition, the N-Gas approach reduces the number of experimental animals, the time necessary to complete the calibration, and the expense.