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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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(CH)NHCO(CH)CO-] . 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.

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.