Neurological manifestations of mitochondrial diseases include ophthalmoplegia, seizures, sensorineural hearing loss, myoclonus, optic neuropathy, pigmentary retinopathy, myopathy, ataxia, dementia, and peripheral neuropathy ( 3). This threshold concentration also varies among different mtDNA point mutations.īecause mitochondrial disorders affect a variety of organ systems, a diverse group of systemic diseases have been associated with mitochondrial mutations. Accordingly, each tissue has a threshold concentration of mutant mtDNA that must be exceeded to cause respiratory chain dysfunction ( 4). The variable phenotypic expression of pathogenic mutations depends upon the degree of heteroplasmy and the energy requirements of the affected tissue. Usually, benign polymorphisms of mtDNA are homoplasmic and pathogenic mutations are heteroplasmic. A mutation in mtDNA may be present in all mtDNA copies (homoplasmy) or coexist with normal mtDNA copies (heteroplasmy). Because virtually all the mtDNA of a fertilized egg are derived from the oocyte, maternal inheritance of mtDNA defines the mode of transmission of mtDNA disorders. There are numerous mitochondria in each cell, usually hundreds to thousands, each having 2–10 copies of mitochondrial DNA (mtDNA) ( 3). 1 Therefore, genetic defects of mitochondrial function can be caused by mutations in either nuclear or mitochondrial genes. Importantly, mitochondrial biogenesis and maintenance also involves nuclear DNA (nDNA)-encoded proteins such that all essential mitochondrial processes such as replication, transcription, and translation require nDNA-encoded factors ( 1)( 2). It encodes 13 protein subunits of the complexes in the oxidative phosphorylation pathway, the 12S and 16S rRNA, and 22 tRNAs required for mitochondrial protein synthesis. The circular 16.6-kb human mitochondrial genome has been completely sequenced. The proton gradient generated by the stepwise transfer of electrons is used to phosphorylate ADP to form ATP by complex V, the ATP synthetase. Electrons pass from NADH and FADH 2 to complexes I through IV in the mitochondrial inner membrane. Mitochondria are eukaryotic cytoplasmic organelles where oxidative phosphorylation takes place. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. The system involved three pairs of primers to amplify mutation “hot spots” at tRNA leu(UUR), tRNA lys/ATPase, and ND 4 regions, followed by detection of point mutations with ASO probes. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA.
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