Cristina Pereira, Raquel Neiva, Célia Nogueira, Lígia Almeida, Laura Vilarinho
Unidade de Rastreio Neonatal, Metabolismo e Genética, Departamento de Genética Humana, Instituto Nacional de Saúde
Doutor Ricardo Jorge, Porto.
e.mail: [email protected]
Mitochondrial diseases (MD) are an increasingly well-recognized, but notoriously heterogeneous, group of multisystemic energy deficiency disorders.
Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain
enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The mitochondrial DNA (mtDNA) is a compact
(16,569 bp), double-stranded (cytosine-rich light (L) and guanine-rich heavy (H) strands), circular genome which encodes 13 essential polypeptides of
the OXPHOS system and the necessary RNA machinery (2rRNAs and 22 tRNAs) for their translation within the organelle (Fig.1). The diagnosis of MD
is a great challenge complicated by mtDNA heteroplasmy, the lack of clear genotype:phenotype correlations in many patients and the complex
interactions between the nuclear and mitochondrial genome. More than 300 point mutations, deletions and duplications in mtDNA and over 110
nuclear genes are known to be mutated in these disorders. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial
variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations.
Mitochondrial DNA Analysis
For mtDNA analysis, if the clinical phenotype clearly points to a specific syndrome, targeted gene or mutation/deletion analysis could be performed,
otherwise the whole mitochondrial genome analysis should be considered. The mtDNA molecular investigation can yield confirmatory (15-30%) and/or
powerful diagnostic information. It has been postulated that mtDNA ‘background’ has an effect on expression or penetrance of MD, and ancient
adaptive mtDNA variants and somatic mutations are affecting individual predisposition to degenerative diseases, aging and cancer. The mtDNA may
hold relevant information even when a primary mutation is detected in nDNA since synergistic interactions between mtDNA and nuclear gene variants
may play a role in the pathogenecity of the disorders. Furthermore the information contained in the mtDNA may be of major relevance to the
understanding of many human diseases that may directly or indirectly cause secondary mitochondrial dysfunction.
Fig.2 - Work-flow used for the sequencing of mtDNA through next generation sequencing.
Fig.1 – Morbidity map of the human mitochondrial genome. Numbers in circles represent
number of mutations reported at the given site.
In our center we have been performing the whole mitochondrial genome analysis using the conventional Sanger sequencing. With the recent
advances in technology we are implementing the mitochondrial genome analysis through next generation sequencing which enables the quantification
of mtDNA point mutation at low levels of heteroplasmy and detection of deletions in a timely and cost-effective fashion (Fig. 2).
References
• Nogueira C, Coutinho M, Pereira C, Tessa A, Santorelli FM, Vilarinho L. 2011. Molecular Investigation of Pediatric Portuguese Patients with Sensorineural Hearing Loss. Genetics Research International.
doi:10.4061/2011/587602.
• Pereira C, Nogueira C, Barbot C, Santorelli FM, Vilarinho L. 2007. Identification of a new mtDNA mutation (14724G>A) associated with mitochondrial leukoencephalopathy. Biochem Biophys Res Commun.
354(4):937-41
• Nogueira C, Nunes J, Evangelista T, Pereira C, Santorelli FM, Vilarinho L. 2007.A new mtDNA-tRNA(Glu) mutation (14728T>C) presenting a late-onset mitochondrial encephalomyopathy. Mitochondrion.7(6):396-8
• Santorelli FM, Tanji K, Kulikova R, Shanske S, Vilarinho L, Hays AP et al.1997. Identification of a novel mutation in the mtDNA ND5 gene associated with MELAS. Biochem. Biophys. Res. Commun. 238: 326-28.