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Page 176 Sulaiman et al. J Transl Genet Genom 2020;4:159-87 I https://doi.org/10.20517/jtgg.2020.27

genomics is an area of research of using multiple molecular data to interpret or understanding the impact
of a DNA sequence variant on complex biological processes in a cell. Typically, functional genomic utilizes
high-throughput data of multiple omics from a single patient or a disease, to evaluate the impact of these
genetic variants in transcription and protein translation [220,221] . After that, the validation of these variants is
achieved by the functional cell- and tissue-based assays, along with animal models to establish the genotype-
phenotypes association as evidence for the pathogenicity.

Typically, the first step of validation is via the confirmation of VUS pathogenicity in the mitochondrial
OXPHOS system, which can be done via tissue-based assays (e.g., skeletal muscle biopsy) or cell-based assays
(e.g., primary fibroblast culture) [222] . An example is the study of genetic myopathy patients, in which the
RNA-seq approach in the primary muscle samples was used to complement the DNA sequencing technology
to improve the identification of disease-causing mutations [207] . Importantly, in this study [207] , the pathogenicity
of these two splice site VUS were confirmed by analyzing the results with the tissue expression database
(Genotype-Tissue Expression (GTEx) Consortium [223] ). They found that these splice site VUS are observable
in the muscles but have very little presence in the cultured dermal fibroblasts [207] , thus indicating that a
correct sampling tissue type does matter to discover the relevant genetic defects. Since the GTEx database
is freely accessible online, for any mitochondrial study that lacked the tissue biopsy samples, a comparative
analysis via the proxy tissue sample data could further refine the findings. The confirmation of the protein
product expression and changes in the tissue and primary cells, via the biochemical assays and protein assays
such as SDS-PAGE or BN-PAGE, are also used to complement the DNA sequencing analysis [222] .

Once the pathogenicity is confirmed, most studies performed the additional assays to discover the disease
mechanism or molecular effects of the VUS in cell lines or animal models [222] . In most cases, the selected
genetic variant is introduced into a cell or animal model via the cell-directed mutagenesis or CRISPR-Cas9
(clustered regularly interspaced short palindromic repeats) technology [220,221] . In a study of patients with
mitochondrial respiratory chain complex deficiencies, the comprehensive analyses of genetic screening and
fibroblast biochemical analysis together with functional cell line assays were able to identify multiple three
novel causative variants, in which all of them were pathogenic based on functional cell-based investigation
assays [189] . This comprehensive evidence for the pathogenicity of the genetic variants is important to elucidate
the disease mechanism. Thus, most disease-specific genomic databases will have reported evidence of
pathogenicity to support the clinical significance of the variants found.

IMAGING TECHNOLOGIES
Another strategy is to complement the NGS data with magnetic resonance imaging (MRI) of the brain
or muscles to confirm the changes in the proteins and structures [224] . Most mitochondrial diseases are
heterogeneous in clinical presentation and symptoms, which are often mixed between diseases. Since many
of the patients exhibit neurological symptoms, the application of MRI can detect these changes. One example
is to use magnetic resonance spectroscopy (MRS) to evaluate brain chemistry for the detection of metabolic
and oxidative defects [224] . MRS is a non-invasive in vivo brain imaging to detect biochemical metabolites
such as N-acetyl aspartate (NAA), lactate, choline, creatine, and myoinositol [224] . Increased lactate levels are
a common feature in mitochondrial disease patients, and such lactate elevation has been observed in the
brain [225-227] , and muscle [228] of patients. However, the findings in the brain are more consistent compared to
the muscle [224] . Furthermore, this lactate elevation was evident in the early stage before any abnormalities
or lesion could be detected in the brain of the mitochondrial disease animal model [229] , indicating the
usefulness of this MRS technique to improve the diagnosis. Interestingly, the phenotypic changes at the brain
structures can complement the genetic screening analysis. In Leigh syndrome, the MRI scans of patients with
confirmed SURF1 and COX mutations (nuclear mutations) have T2-abnormalities in the brainstem nuclei,
whereas the caudate and putamen lesions are seen in patients with mtDNA mutations [230-232] . These findings
suggest that MRI scans can confirm the genotype-phenotype changes occurring in mitochondrial disease
and thereby improve diagnostic yield.