PGmito - Mitochondrial Genome Sequencing

Disorders of mitochondrial energy metabolism, or oxidative phosphorylation (OXPHOS) disorders, are characterized by reduced activity of one (isolated) or more (combined) mitochondrial respiratory chain complexes. Mitochondrial disorders are clinically heterogeneous, and phenotypes, which range in severity and progression, can involve single or multiple organ systems. As OXPHOS defects cripple the body’s ability to product adequate energy, organ systems with high metabolic demands (including the heart, brain, and muscle) are often most severely affected (Ghezzi and Zeviani. 2018. PubMed ID: 30030362). Lactic acidosis of the blood and/or cerebrospinal fluid is often a prevailing symptom (Chinnery. 2014. PubMed ID: 20301403). Disorders of mitochondrial energy metabolism may be caused by defects in either the mitochondrial DNA (mtDNA) genome, or by defects in the nuclear genome.

Distinct clinical presentations of OXPHOS disorders caused by pathogenic variants within the mtDNA genome may include but are not limited to: Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red fibers (MERRF), neurogenic weakness with ataxia and retinitis pigmentosa (NARP), or Leigh syndrome (LS) (Chinnery. 2014. PubMed ID: 20301403). Common phenotypes among these disorders may include ptosis, external opthalmoplegia, proximal myopathy and exercise intolerance, cardiomyopathy, sensorineural deafness, optic atrophy, pigmentary retinopathy, encephalopathy, seizures, migraine, stroke-like episodes, ataxia, spasticity, dementia, and/or diabetes mellitus (Chinnery. 2014. PubMed ID: 20301403). However, affected individuals may present with phenotypes that overlap with more than one syndrome, or they may present with isolated phenotypes (e.g., hearing loss). Prevalence of mitochondrial disease caused by mtDNA genome defects is challenging to determine, but is estimated to be ~10 per 100,000 adults (Gorman et al. 2015. PubMed ID: 25652200). 

Age of onset may also vary widely, ranging from infancy to adulthood. The exact reasons for variability in disease severity, clinical presentation, and age of onset, even among affected members of the same family, are not yet clear, but are likely to include the specific pathogenic variant in question, differential heteroplasmy levels in affected tissue/organ systems, nuclear-encoded modifier variants, and environmental factors including illness and medications (Saneto and Sedensky. 2013. PubMed ID: 23224691). 

At the present time, there are limited treatment options available to individuals with mitochondrial disorders. Management of these diseases is primarily supportive. A molecular diagnosis may allow for appropriate screening for potential future symptoms. Oral administration of certain supplements (e.g., riboflavin for complex I deficiency) has been shown to have some benefit in certain cases (Udhayabanu et al. 2017. PubMed ID: 28475111). Recurrence risk, with the possible exception of large deletion events, is often not possible to accurately estimate from the maternal side due to a mitochondrial bottleneck that restricts the number of mitochondrial genomes that are inherited (Stewart and Chinnery. 2015. PubMed ID: 26281784).

Pathogenic variants in over 250 genes, both nuclear and mitochondrial, have been described; however, more than 1,000 genes encode mitochondrial proteins, and it is likely that many of these will eventually be connected to disease (Chinnery. 2014. PubMed ID: 20301403; Craven et al. 2017. PubMed ID: 28415858). While the majority of reported genes encode for structural or assembly components of the mitochondrial respiratory complexes, causative variants in a number of genes involved in mitochondrial replication, transcription, or translation may also result in primary OXPHOS disorders, in addition to genes that function in cofactor biosynthesis or ubiquinone (coenzyme Q10) synthesis (Ghezzi and Zeviani. 2018. PubMed ID: 30030362). Secondary OXPHOS dysfunction may also result from defects in genes involved in fatty acid oxidation such as HADHA/HADHB or ECHS1, among others (Nsiah-Sefaa and McKenzie. 2016. PubMed ID: 26839416).

The majority of genes associated with mitochondrial disorders are nuclear-encoded, but 37 genes reside on the mitochondrial genome, an ~16.5 kb circular genome residing within the organelle. The mtDNA genome is present in multiple copies within a single cell, ranging from ~100 to 100,000 copies (Stewart and Chinnery. 2015. PubMed ID: 26281784). Copy number is primarily dependent on tissue/cell type, although individual differences may occur due to factors such as health and age. Within each cell, a variant can be present in either all of the mtDNA molecules (e.g., homoplasmy) or only a portion of the mtDNA molecules (e.g., heteroplasmy) (Stewart and Chinnery. 2015. PubMed ID: 26281784). Heteroplasmy level may vary between cells in the same tissue, or from tissue to tissue within the same individual. The heteroplasmy threshold (e.g., mutation load) for disease can vary widely, but is usually >70% in an affected tissue (Saneto and Sedensky. 2013. PubMed ID: 23224691).

Of the 37 genes that are encoded on the mitochondrial genome, 13 encode structural proteins of the oxidative phosphorylation complexes (Chinnery. 2014. PubMed ID: 20301403). MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, and MT-ND6 encode units of complex I; MT-CYB encodes a unit of complex III; MT-CO1, MT-CO2, and MT-CO3 encode units of complex IV; and MT-ATP6 and MT-ATP8 encode units of complex V. Two of the genes, MT-RNR1 and MT-RNR2, encode ribosomal RNAs. The remaining 22 genes (MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, and MT-TY) encode mitochondrial tRNAs.

Pathogenic variants in the mtDNA genome are maternally inherited, and confirmed disease-causing variants have been detected in most of the mtDNA genes to date (Lott et al. 2013. PubMed ID: 25489354; MITOMAP). Within protein-coding genes, the majority of reported variants are missense, although chain-terminating variants have also been reported.

Large deletions (1.1-10 kb) that encompass one or more genes of the mitochondrial genome also contribute to disease (primarily Kearns-Sayre syndrome, Pearson syndrome, and progressive external ophthalmoplegia), and are estimated to have a prevalence of ~1.2:100,000 in northern European populations (Chinnery. 2014. PubMed ID: 20301403; Goldstein and Falk. 2019. PubMed ID: 20301382). The most common deletions include the 4,977 bp common deletion (m.8470_13446del) and the ~7.4 kb deletion. The former harbors a deletion breakpoint defined by two perfect repeats, and removes four complex I genes, one complex IV gene, two complex V genes, and five tRNAs (Chen et al. 2011. PubMed ID: 21866113). In contrast to point pathogenic variants, which are more likely to be inherited, isolated mitochondrial deletions typically arise de novo, with an approximate recurrence risk of 1 in 24 (Chinnery. 2014. PubMed ID: 20301403). Large duplications or complex rearrangements are a very rare cause of disease. Note that single large mtDNA deletions or duplications are distinct from multiple mtDNA deletion/depletion syndrome, which is caused by defects in mtDNA replication and maintenance genes encoded by the nuclear genome (Suomalainen and Isohanni. 2020. PubMed ID: 20444604; El-Hattab and Scaglia. 2013. PubMed ID: 23385875).

Clinical sensitivity is very challenging to estimate because few large-scale studies have been published regarding mitochondrial disorders that include sequencing of both the mtDNA genome and the nuclear genome. Additionally, whole exome and whole genome sequencing continue to uncover novel candidate mitochondrial disease genes within the nuclear genome.

In one large study, 204 individuals were identified with a causative variant in the mtDNA genome; of these, 31 (~15%) harbored a single mtDNA deletion (Gorman et al. 2015. PubMed ID: 25652200). In the same study and over the same time period, 62 individuals with nuclear-associated mitochondrial DNA disease were identified.

Acceptable specimen types for this test are: whole blood, fresh/frozen tissue, or DNA extracted from whole blood or fresh/frozen tissue. 

This test utilizes long-range polymerase chain reaction followed by massively parallel, high-throughput sequencing (next generation sequencing) in order to accurately capture heteroplasmic mtDNA variants. To mitigate allele dropout, two separate PCR primer sets are used to amplify the entire mtDNA genome, resulting in two separate fragments for analysis. Next generation sequencing and subsequent pipeline analysis allows for accurate detection of low-level heteroplasmic (down to ~4%) variants in patient samples.

We sequence the entire mtDNA genome; however, a few regions are excluded from analysis (largely due to the presence of short tandem repeat tracts in non-coding regions). These are m.280-m.320, m.490-m.575, m.953-m.966, m.3106-m.3107, m.12418, and m.16150-m.16200. This report contains no information regarding variants in these regions.

Sanger confirmation is attempted on all reported sequence variants regardless of heteroplasmic load. However, due to technical limitations, variants <20% heteroplasmy may fall below the limit of detection for Sanger sequencing. If we are unable to confirm a variant in Sanger, the variant will be listed on reports with a limitation regarding the lack of confirmation with a second method. Testing an alternate tissue type may be necessary to clarify the finding.

Human Genome Variation Society (HGVS) recommendations are used to name sequence variants (Human Genome Variation Society). All differences from the reference sequences are assigned to one of five interpretation categories (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign and Benign) per ACMG Guidelines (Richards et al. 2015. PubMed ID: 25741868). mtDNA sequence variants are interpreted utilizing a combination of resources, including recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (Richards et al. 2015. PubMed ID: 25741868), which was primarily developed for interpretation of variants in nuclear genes; as well as recommendations from the MSeqDR consortium (McCormick et al. 2020. PubMed ID: 32906214) and Baylor College of Medicine (Wong et al. 2020. PubMed ID: 31965079), which were developed specifically for mtDNA variant interpretation. For protein-coding genes, rare variants and undocumented variants are nearly always classified as Likely Benign if there is no indication that they alter protein sequence. Benign and Likely Benign variants are not listed in reports, but are available upon request. Please note that we will only report known or possible primary causes of disease. Potential modifiers of disease (e.g., secondary LHON mutations) will not be reported as part of this test unless in the presence of a known primary cause. Additionally, this test has been designed to analyze the mitochondrial genome for variants relative to Mendelian disease only. Reports will not include any information relevant to pharmacogenomics.

Mitochondrial variants do not qualify for free family follow-up testing. In order to accurately quantify variant heteroplasic load in family members, next generation sequencing analysis is recommended.

Detection of gross deletions, duplications, or large rearrangements is not currently available for this test.