Comprehensive scanning of the entire mitochondrial genome for mutations.
BACKGROUND
Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome.


METHODS
We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis.


RESULTS
We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions.


CONCLUSIONS
Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.
Wong
Lee-Jun C
LJ
Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007, USA. wonglj@georgetown.edu
Liang
Min-Hui
MH
Kwon
Haeyoung
H
Park
John
J
Bai
Ren-Kui
RK
Tan
Duan-Jun
DJ
D002648:Child; D004272:DNA, Mitochondrial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008928:Mitochondria / Q000235:genetics*; D028361:Mitochondrial Diseases / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length
D004272:DNA, Mitochondrial