Diagnostic screening of mitochondrial DNA mutations in Australian adults 1990-2001.

BACKGROUND: Many diverse pathogenic mitochondrial DNA (mtDNA) mutations have been described since 1988. The Melbourne Neuromuscular Research Institute (MNRI) has undertaken diagnostic detection of selected mtDNA mutations since 1990. MtDNA mutations screened have included point mutations associated with Leber's hereditary optic neuropathy (LHON; G3460A, G11778A and T14484C), mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS; A3243G), myoclonus epilepsy and ragged red fibres (MERRF; A8344G) and Leigh's syndrome/neuropathy ataxia retinitis pigmentosa (LS/NARP; T8993C/G). Samples have also been screened for deletions/ rearrangements associated with Kearns-Sayre syndrome (KSS) and chronic progressive external ophthalmoplegia (CPEO). AIMS: To present an audit of the MNRI mtDNA diagnostic service between 1990 and 2001, encompassing 1725 referred patients. METHODS: The detection techniques carried out included polymerase chain reaction amplification of mtDNA combined with restriction fragment length polymorphism analysis for mtDNA point mutation detection, supplemented with selected sequence analysis and Southern blots for the detection of deletions/ rearrangements. Tissues tested included blood, hair and skeletal muscle. RESULTS: Of the 1184 patients screened for MELAS A3243G, 6.17% were positive for the mutation, whereas for MERRF A8344G, 2.21% carried the mutation and for LS/NARP T8993C/G, 0.32% carried the mutation. The outcomes for the LHON mutations were G11778A, 6.60%, T14484C, 5.76% and G3460A, 0.29%. Of the patients referred for KSS and CPEO, 17.72% had deletions/rearrangements. CONCLUSIONS: Overall, the detection rate of mtDNA point mutations was low. The protean clinical features of mitochondrial disorders and the frequency of partial phenotypes lead to requests for tests in many patients with a relatively low likelihood of mtDNA mutations. An improved algorithm could involve mutation screening appropriate to the phenotype using sequencing of selected mtDNA regions in patients with a high likelihood of mtDNA disease. Features increasing the likelihood of mtDNA mutations include the following: (i) a typical phenotype, (ii) a maternal inheritance pattern and (iii) histochemical evidence of mitochondrial abnormality in the muscle biopsy. Efficient laboratory diagnosis of mtDNA disease involves good communication between the physician and laboratory scientists, coupled with screening of the appropriate tissue.

Mitochondrial DNA in stroke and migraine with aura.

Patients presenting with thrombotic stroke of unexplained etiology and or migraine with aura were screened for mitochondrial (mt) DNA mutations associated with cytopathies given that both migraine and stroke-like episodes are recognised with certain mt DNA mutations. Mutations usually associated with either mitochondrial encephalopathy, lactic acidosis and stroke-like episode, myoclonic epilepsy with ragged red fibres, or those strongly linked to Leber's hereditary optic neuropathy (LHON) were not detected in patients or controls. However, increased levels of two of the secondary LHON mutations were found. The T-->C mutation at nucleotide 4216 was more common than expected in patients aged 35 years or less, as was the 13708 G-->A mutation in young stroke patients. This data lends support to the possibility that an accumulation of minor mt DNA mutations may contribute to the pathoaetiology of stroke and migraine with aura in some young patients.

Detection of MELAS A3243G point mutation in muscle, blood and hair follicles.

Polymerase chain reaction (PCR) based methods for the diagnosis and screening of the mitochondrial disorders have been well established. A number of tissues are routinely used. In this study, we compared the detection rate for MELAS A3243G point mutation in muscle, blood and hair follicles. Ten subjects were studied; mean age was 47 years, (SD 16, range 23-73). All ten subjects had the MELAS A3243G point mutation detected in muscle and hair follicles, but only five had the abnormality in blood samples. The rate of detection of the point mutation in blood samples was age dependent. MtDNA analysis on hair follicles is as sensitive as muscle in detecting this mutation. Analysis using blood samples is not as sensitive, particularly in older subjects. The absence of the mutation in blood samples suggests that there is a preferential selection process for normal (wild type) mtDNA over time. This may be related to the rate of cell division and energy requirements of each tissue.

The polymerase chain reaction in the study of mitochondrial genetics.

Since its development in the late 1980's, the polymerase chain reaction (PCR) has revolutionised molecular genetic studies. It has provided direct access to genetic material in quantities sufficient for meaningful analyses to be performed. Adaptations to the basic technique have resulted in a wide range of applications from basic gene amplification to the estimation of DNA species quantities within cells. The study of human mitochondrial genetics is but one of the many disciplines to benefit from the rapid ascension of PCR based technology. In this communication we outline several uses of the PCR technique in the detection, quantification and characterisation of human mitochondrial genetic defects. The data presented in this communication highlight the versatility and applicability of PCR not only to mitochondrial research but to other disciplines of medical research.