Next Generation Sequencing

Mitochondrial DNA Disorder

Purpose:

The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA–related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome.

Methods:

The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR–based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR–based Sanger sequencing using multiple pairs of primers.

Results:

Our results demonstrated the uniform coverage of the entire mitochondrial genome. Massively parallel sequencing of the single amplicon revealed the presence of single-nucleotide polymorphisms and nuclear homologs of mtDNA sequences that cause the erroneous and inaccurate variant calls when PCR/Sanger sequencing approach was used. This single amplicon massively parallel sequencing strategy provides an accurate quantification of mutation heteroplasmy as well as the detection and mapping of mitochondrial DNA deletions.

Conclusion:

The ability to quantitatively and qualitatively evaluate every single base of the entire mitochondrial genome is indispensible to the accurate molecular diagnosis and genetic counseling of mitochondrial DNA–related disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome.

Genet Med advance online publication 3 January 2013

Keywords:

 

long-range PCR; massively parallel sequencing; mitochondrial disorders; mitochondrial genome; mtDNA large deletions; nuclear mitochondrial DNA homologs (NUMTs)

It was previously believed that these mutations happened completely by chance, with no regard for their effects on the organisms. Recently, there have been studies suggesting that these mutations occur in response to environmental challenges. That is to say, they are more likely to occur when they are advantageous to the organism, rather than when they are neutral or disadvantageous. When cells were deprived of a certain amino acid, tryptophan, for prolonged periods of time, point mutations in trp operon reverted to tryptophan, leading to an advantageous result, more frequently than under normal conditions when the mutations were neutral. In addition, the tryptophan mutation rate was unaffected when the cells were deprived of another amino acid, cysteine, further suggesting that the mutation rate was specific to situations in which the mutation was advantageous.

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