Nonradioactive characterization of low-level heteroplasmic mitochondrial DNA mutations by SSCP-PCR enrichment.

Mitochondrial DNA (mtDNA) mutations have been implicated in an increasing number of human diseases. Many of these mutations are heteroplasmic and are only present at low levels in readily accessible human tissue such as blood. The technique of single-stranded conformational polymorphism (SSCP) allows the detection of mtDNA variants from peripheral blood, but characterization of these variants by automated sequencing is hampered by the low level of heteroplasmy. We have therefore developed a technique for the enrichment of mtDNA mutations that allows reliable sequence data to be obtained even if the variant mtDNA represents only 1% of the total mtDNA. The procedure involves the excision, purification and subsequent PCR amplification of selected DNA fragments from SSCP gels. The techniques can be applied to other heterogeneous mutations such as mosaic mutations in skin biopsies or somatic oncogene mutations in tumor tissue.

Differential expression of mRNA in human thyroid cells depleted of mitochondrial DNA by ethidium bromide treatment.

A wide variety of human diseases have been associated with defects in mitochondrial DNA (mtDNA). The exact mechanism by which specific mtDNA mutations cause disease is unknown and, although the disparate phenotypes might be explained on the basis of impaired mitochondrial gene function alone, the role of altered nuclear gene expression must also be considered. In recent years, the experimental technique of depleting cells of mtDNA by culturing them with ethidium bromide has become a popular method of studying mitochondrial disorders. However, apart from depleting mtDNA, ethidium bromide may have many other intracellular and nuclear effects. The aim of the present study was to investigate the effects of ethidium bromide treatment on nuclear gene expression. A simian-virus-40-transformed human thyroid cell line was depleted of mtDNA by culture in ethidium bromide, and differential display reverse transcriptase-PCR (DDRT-PCR) was then employed to compare mRNA expression between wild-type, mtDNA-replete (rho(+)) and ethidium bromide-treated, mtDNA-depleted (rho(0)) cells. Expression of the majority of nuclear-encoded genes, including those for subunits involved in oxidative phosphorylation, remained unaffected by the treatment. Seven clones were found to be underexpressed; three of the clones showed significant similarity with sequences of the human genes encoding RNase L inhibitor, human tissue factor and ARCN1 (archain vesicle transport protein 1), a highly conserved species which is related to vesicle structure and trafficking proteins. We conclude that the effects of ethidium bromide treatment on nuclear gene expression are not simply limited to changes in pathways directly associated with known mitochondrial function. Further studies will be required to elucidate which of these changes are due to mtDNA depletion, ATP deficiency or other disparate effects of ethidium bromide exposure. Given that most genes appear unaffected, the results suggest that depleting cells of mtDNA by ethidium bromide treatment is a valuable approach for the study of mitochondrial mutations by cybrid techniques.

Mitochondrial DNA variations in patients with Type 2 (non-insulin dependent) diabetes mellitus and a Welsh control population. Mutation in brief no. 239. Online.

Type 2 (non-insulin dependent) diabetes mellitus may be inherited along the maternal line and a variety of mitochondrial DNA (mtDNA) variants have been implicated in the pathogenesis. We have previously reported mutations in five regions of the mitochondrial genome which encompass 11 of the 22 tRNA genes. Now we employ the technique of single stranded conformational polymorphism (SSCP) analysis to investigate a further 6 regions of the mitochondrial genome, covering the remaining 11 tRNA genes in 40 patients with Type 2 diabetes and 30 racially-matched normal controls. A variety of homoplasmic mutations were detected in patients with diabetes and these will be of value in further population association studies.

Genetic linkage study of a major susceptibility locus (D2S125) in a British population of non-insulin dependent diabetic sib-pairs using a simple non-isotopic screening method.

A polymorphic microsatellite marker (D2S125) was recently reported to show significant linkage to non-insulin dependent diabetes mellitus (NIDDM) in a population of Mexican-American affected sib-pairs. We have used a simple non-isotopic screening technique employing the polymerase chain reaction (PCR) with a biotinylated primer to study the genetic linkage and allele frequency distribution of the D2S125 marker in a population of 109 British NIDDMs (62 possible affected sib-pairs). The analysis provided no evidence for linkage of the D2S125 marker in the British subjects (MLS = 0.029, P > 0.05). The PCR screening method used proved to be a convenient and reliable alternative to the radiolabelling of PCR products.

Analysis of a polycytosine tract and heteroplasmic length variation in the mitochondrial DNA D-loop of patients with diabetes, MELAS syndrome and race-matched controls.

AIM: The T to C substitution at position 16189 nt of the human mitochondrial genome has been associated with the development of heteroplasmic length variation in the control region of mtDNA. Previous reports have suggested that this defect may be associated with the development of other pathogenic mtDNA mutations, including the diabetogenic A to G mutation in the tRNALEU(UUR). Recently the 16189 nt variant has also been associated with insulin resistance in British adult men. In order to investigate these associations further we studied 23 patients with the 3243 nt mutation, 150 patients with Type 2 diabetes and 149 non-diabetic controls. METHODS: The region around 16189 nt was investigated by polymerase chain reaction-restriction fragment length polymorphism analysis and automated sequencing. RESULTS: We find that the T to C substitution at 16189 nt is associated with heteroplasmic length variation only when the resultant polycytosine tract is not interrupted by a second mutation. There are no significant differences in the prevalence of the 16189 nt variant or heteroplasmic length variation between patients with the 3243 nt mutation, patients with Type 2 diabetes or race-matched normal controls. CONCLUSIONS: We conclude that these variants are likely to represent normal polymorphisms and that previously reported associations should be treated with caution unless they can be replicated in other populations.