Date of Award

Winter 1994

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Sciences

Committee Director

Frank J. Castora

Committee Member

Christopher J. Osgood

Committee Member

Mark S. Elliot

Committee Member

Bruce W. Tedeschi


Mitochondrial dysfunction resulting from mutations on mitochondrial DNA (mtDNA) is being recognized in a growing spectrum of diseases. These diseases, resulting from single base mutations, large deletions, or insertions, have been largely neuromuscular in origin. However, as an understanding of the effects of mtDNA mutations progresses, attention is now focusing on neurodegenerative diseases. Rett Syndrome (RS), a progressive neurodegenerative disease with predominantly female cases, demonstrates morphologic mitochondrial changes, mitochondrial enzyme deficiencies and maternal inheritance (characteristic of mtDNA diseases). No investigation of mtDNA involvement has been previously conducted and, to date, no biological marker exists for this disorder.

Our preliminary studies in mitochondrial myopathies indicated that mtDNA could be detected from limited amounts of blood, amniotic fluid, and single human lymphoblasts. Multiplex PCR, restriction digestion and single cell sequencing were used to identify the LHON (Leber's Hereditary Optic Neuropathy) mutation in control and LHON single lymphoblasts. We then examined the state of mtDNA heteroplasmy in the heteroplasmic disease MELAS (mitochondrial encephalomyopathy, lactic acidosis, strokelike symptoms) in single human lymphoblasts and identified an intercellular distribution of mutant and normal mtDNA. This differs from analyses performed in LHON and MERRF (myoclonic epilepsy, ragged red fibers) pedigrees which demonstrate a predominantly intracellular distribution. This may implicate MELAS as a homoplasmic-lethal mutation.

We then defined a procedure to identify single base mutations within the mitochondrial genome. Mitochondrial myopathy patient samples were used. The procedure employs DGGE (denaturing gradient gel electrophoresis) to define an area of interest within the genome. PCR amplification and SSCP (single-stranded conformational polymorphism) analysis were used to localize the mutation to a 200bp region which was then sequenced to identify the lesion.

Rett Syndrome demonstrates a multicomplex effect of the respiratory chain enzymes indicating a possible translational dysfunction. The above approach was employed in an analysis of the 22 mitochondrial tRNAs for mutations relating to RS. SSCP analysis identified a number of conformational changes which, following DNA sequencing, proved to be non-specific for the disease. Sequencing of additional regions of the mtDNA confirmed the SSCP results indicating that a mutation in the mitochondrial tRNAs is not associated with Rett Syndrome.


Dissertation Submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.