Mitochondrial Genome Test

This panel includes the 16.5-kb mitochondrial genome (mtDNA) containing 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes encode for polypeptides that form structural subunits of the respiratory chain (RC complexes I, III, IV, and V), which is functionally essential and evolutionarily constrained, and the RNA necessary for mtDNA translation, namely 2 rRNAs (MT-RNR1 and MT-RNR2, encoding 12S and 16S rRNA) and 22 transfer RNAs (tRNA, e.g. tRNALys), which are interspaced between the protein-encoding genes. The vast majority of over 1000 mitochondrial proteins are encoded by nuclear genes. There are several unique properties associated with the mitochondrial genome that are important in understanding the primary mitochondrial DNA disease: 1) there are multiple copies of mtDNA in each cell; 2) mtDNA is maternally inherited, 3) mutated mtDNA coexists with wild type mtDNA (heteroplasmy), 4) minimum critical proportion of mutated mtDNAs is necessary before tissue dysfunction comes apparent (threshold effect), 5) mutation load (heteroplasmy level) may vary among different tissues.Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. While some mitochondrial disorders only affect a single organ, many involve multiple organ systems. Patients’ symptoms can range from mild to severe and can occur at any age. Common clinical features of mitochondrial disease include fatigue, weakness, metabolic strokes, seizures, cardiomyopathy, arrhythmias, developmental or cognitive disabilities, diabetes mellitus, impairment of hearing, vision, growth, liver, gastrointestinal, or kidney function, and more. Typical early onset mitochondrial diseases include Leigh syndrome, depletions syndromes, Kearns-Sayre (KSS) and Pearson syndrome, whereas chronic progressive external ophthalmoplegia (CPEO), Leber’s hereditary optic neuropathy (LHON), neuropathy, ataxia, and retinitis pigmentosa (NARP), mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) are seen in later childhood or adult life. To date, close to 400 mutations have been reported, that are known to cause a spectrum of mitochondrial diseases.Most mtDNA alterations are neutral polymorphisms, which define different population haplogroups and have been used for example in tracking human migrations. Mitochondrial genetics differ considerably from Mendelian genetics. Uniparental inheritance, cellular polyploidy and a deviation from the standard genetic code are just some of the characteristics of mitochondrial genetics. Although mitochondrial variants are typically maternally inherited, they can be sporadic (de novo). There are currently no standard guidelines for reporting and classifying mtDNA variants. The nomenclature differs slightly from the standard HGVS nomenclature for nuclear genes. Mitochondrial variants are reported using gene name and m. numbering (e.g. m.8993T>C), and p. numbering for protein coding changes. The current accepted reference sequence is the Revised Cambridge Reference Sequence (rCRS) of the Human Mitochondrial DNA: GenBank sequence NC_012920 gi:251831106.4. The multi-copy nature of the mitochondrial genome leads to complicated genetics. MtDNA mutations can be either heteroplasmic (where both mutated and wild type mtDNA co-exist within the cell) or homoplasmic (only mutated species are present). Heteroplasmy percentages may vary in different tissue types from the sample tested; therefore, interpreting low heteroplasmic levels also must be done in the context of the tissue tested, and may be meaningful only in the affected tissue such as muscle. Lack of correlation between the heteroplasmy level and disease severity further complicate the interpretation of mtDNA variants. The genotype–phenotype correlation is not always clear, and most patients do not fit within any defined syndrome and even within a family the expressivity of the disease can be extremely variable.Diagnostics areas affected most: ​Cardiology (cardiomyopathy), ENT (hearing loss), Metabolic, Neurology, Ophthalmology

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding and regulatory variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. If the test includes the mitochondrial genome the target region gene list contains the mitochondrial genes. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases including, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as  SIFT, PolyPhen,MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with suboptimal coverage (<20X for nuclear genes and <1000X for mtDNA) if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists, and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the cornerstone of clinical interpretation and resulting patient management decisions. Our classifications follow the ACMG guideline 2015.

The final step in the analysis is orthogonal confirmation. Sequence and copy number variants classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing or by orthogonal methods such as qPCR/ddPCR when they do not meet our stringent NGS quality metrics for a true positive call.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes, and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene, and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts, and detailed information about related phenotypes. We also provide links to the references, abstracts, and variant databases used to help ordering providers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. Our laboratory is therefore well-positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering healthcare provider at no additional cost, according to our latest follow-up reporting policy.