Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Author and Reporter: Ritu Saxena, Ph.D.

On 5th of July, I  discussed a general overview of varied mitochondrial functions, diseases, diagnosis and the current research focused on treatment of mitochondrial diseases in a post titled “Mitochondria: More than just the powerhouse of the cell”. https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Current post talks about a new technique that has been introduced by the authors as a ‘Comprehensive 1-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing’. The technique was recently published in the Clinical Chemistry journal (2012) by Zhang et al.

One mitochondria may have multiple copies of mtDNA  and an interesting feature observed in mitochondria is the heteroplasmy, a phenomenon where mutant and wild-type mtDNA can co-exist. During cell division, the mutant and wild-type copies are distributed randomly in daughter cells. The impact is in the heterogeneity with respect to penetrance and expressivity along that has diverse manifestations in terms of organs being affected, age of onset and the rate of progression. With such variability, the diagnosis becomes even more challenging. Therefore, mutational analysis along with accurate heteroplasmy detection in the mtDNA is an important part of the diagnosis. Thus, there is need for accurate and faster mutation detection methods for patients that are suspected to carry a mitochondrial disease.

The current molecular diagnostic methods for the detection of mtDNA mutations involves several different and complimentary methods. The detection of mutations is approached by first screening for a panel of point mutations that have been commonly associated with the mitochondrial diseases, followed by the quantification of the mutant load. In case none of the point mutations show up in the screening, the whole genome sequencing of mtDNA is performed to identify rare or novel mutations that might be associated with the disease. Also, in order to analyze large deletions within the genome, a an additional step of Southern blotting needs to be performed. Zhang et al, however, developed a novel approach to analyze the mtDNA in “single” step.

The method employed for the 1-step technique is to first enrich the entire mtDNA using amplification by PCR followed by massively parallel sequencing to detect point mutations as well as large heteroplasmic deletions simultaneously. A total of 45 samples were analyzed for the evaluation of analytic sensitivity and specificity. As stated by the authors “Our analysis demonstrated 100% diagnostic sensitivity and specificity of base calls compared to the results from Sanger sequencing” and added ” the method also detected large deletions with the breakpoints mapped”. Apart from the fact that the 1-step technique is less complex, the detection of point mutations has been found to be more accurate compared to Sanger sequencing that doesn’t provide any quantitative information and falls short of detecting heteroplasmy lying below 15%.

Thus,  the 1-step technique developed by Zhang et al has been demonstrated to be better than the combination of methods currently utilized for the detection of mtDNA mutations in terms of simplicity, cost effectiveness and accuracy.

Sources:

http://www.ncbi.nlm.nih.gov/pubmed?term=Comprehensive%201-Step%20Molecular%20Analyses%20of%20Mitochondrial%20Genome%20by%20Massively%20Parallel%20Sequencing

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/