title: Qualitative and quantitative detection of mitochondrial heteroplasmy in cerebrospinal fluid using denaturing high-performance liquid chromatography.
abstract: Detecting and quantifying generalized mitochondrial heteroplasmy is essential if the field of mitochondrial genetics is to advance in the arena of complex genetic disorders. The majority of techniques used to detect and quantify mitochondrial heteroplasmy focus on a known mutation or polymorphism. The necessity of knowing the mitochondrial DNA (mtDNA) change beforehand means that non-specific heteroplasmy in general cannot be assessed. In this study, we assessed the extent that denaturing high-performance liquid chromatography (dHPLC) could detect and quantify mitochondrial heteroplasmy from cerebrospinal fluid (CSF). Although we used a known polymorphism to assess reliability and sensitivity of this technique, a distinct advantage to using dHPLC for heteroplasmy detection is that the entire fragment is screened for variability and any unique fragments will be detected regardless of the placement or type of change. Our results demonstrate that dHPLC can consistently and reliably detect mitochondrial heteroplasmy in a CSF sample down to 0.01%. In addition, the level of heteroplasmy was consistent with peak height for each homoduplex, giving a reliable method to quantify level of heteroplasmy.
authors: School of Nursing, University of Pittsburgh, 3500 Victoria Street, 440 Victoria Building, Pittsburgh, PA 15261, USA. yconley@pitt.edu
authors: Yvette P
authors: YP
authors: Conley
authors: Heather
authors: H
authors: Brockway
authors: Megan
authors: M
authors: Beatty
authors: Mary E
authors: ME
authors: Kerr
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000032:analysis / Q000134:cerebrospinal fluid; D004249:DNA Damage / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D015203:Reproducibility of Results
chemical_list: D004272:DNA, Mitochondrial; D004247:DNA