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mitochondria function and structure, the MitoCarta. In two articles by the group from the Broad Institute
using annotated genome sequences combined with tandem mass spectrometry and computation, the
proteome was estimated to consist of ~1,160 proteins [24,25] . Today, further work has increased sensitivity;
most think that the proteome contains about 1,500 proteins. Knowledge of these targeted gene products
increased the yield of detected nuclear genes involved in mitochondrial disease. However, the complete
compendium of the complete proteome remains unknown.

The next required factor in expanding the genetic detection of mitochondrial disease was commercial
payment of genetic testing by insurance companies. Reimbursement has allowed widespread clinical testing
and competitive pricing. NGS panels of genes became commercially available for suspected disease in 2010.
Within the next three years, whole exome sequencing (WES) entered the commercial landscape. Gene
panels began as limited spectrum of genes only involving nuclear- and mitochondrial-encoded ETC genes
[26]
known to induce disease . The effectiveness of this approach relied heavily on clinical acumen, analyte,
[27]
muscle testing, and neuroimaging . WES using NGS platforms soon moved into diagnostic testing. This
has led genetic testing outside the predesignated MitoCarta platforms into an unbiased non-targeted
“discovery” approach. Clinicians have now begun to use genetic testing to circumvent more expensive
and tedious multiple biochemical analyte and muscle biopsy procedures for diagnosis. This has switched
medical acumen to a “genetics first” approach . Findings may be clinically relevant if the known variant
[28]
occurs in a described disease gene, but, if the variant is of unknown significance, then functional validation
of the change is needed. However, the discovery of previously described pathogenic variants in healthy
[29]
individuals has muddied the waters on diagnosis . Here, functional validation is vital for affirmation of
pathogenicity, and a return to the past requiring skeletal muscle biopsy, analyte, and neuroimaging, with
integration of functional protein alteration, animal models, and rescue cell and animal model systems, are
needed for confirmation of variant pathogenicity . Hence, one can understand why prevalence numbers
[30]
for mitochondrial disease are low estimates.

Technology has now advanced to rapidly sequence the whole genome (WGS), which will expand diagnosis
further. The current expense and lack of commercial insurance payment has greatly limited this technology
from becoming mainstream. WGS can detect non-protein genetic factors that alter gene expression and
hence modify disease or primarily cause disease. However, currently, WGS is mostly used in research labs
and under certain circumstances, of urgent need. However, as technology improves, turnaround times
shorten, databases enlarge, and costs reduce, WGS will likely significantly enhance our detection of disease-
causing genetic alteration in the genome.

The rapid expansion of gene sequencing technology progressing from single gene, gene panels, whole
exome, to now whole genome sequencing has produced substantial variability in the level of evidence for
genotype to phenotype. The validation of each genetic variant within a gene supporting a gene-disease
relationship is beyond the scope of this paper. Standard guidelines have been developed to support a gene-
disease relationship and the subsequent framework to measure the strength of evidence of the gene-disease
relationship. The NIH-funded Clinical Genome Resource was developed to serve this purpose [30-32] . The
importance of this work to validate disease-causing gene changes is the high rate of variants of unclear
[32]
significance highlighting the need for clinical and research input . The culmination of this work is the
ability for the clinician to provide the patient and family an accurate and timely diagnosis and to hopefully
expand “precision” medicine treatments.

Nomenclature for mitochondrial diseases has been in flux due to changes in diagnosis and gene discovery.
The most logical approach to nosology has not yet been formulated, but the methodology needs to
include physiology, genetics, and clinical findings. The author leans toward the approach of physiological
functions: proteins that directly produce ATP; mtDNA replication and maintenance factors; tRNA and