Cândida Mendes

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n=39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann-Whitney-U test: p=0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.

Frontotemporal dementia and mitochondrial DNA transitions

Frontotemporal dementia (FTD) is the second most common type of primary degenerative dementia. Some patients present an overlap between Alzheimers disease (AD) and FTD both in neuropathological and clinical aspects. This may suggest a similar overlap in physiopathology, namely an involvement of mitochondrial DNA (mtDNA) in FTD, as it has been associated to AD. To determine if mtDNA is involved in FTD, we performed a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, specific to mtDNA NADH Dehydrogenase subunit 1 (ND1) nucleotides 3337-3340, searching for mutations previously described in Parkinsons and AD patients. We could identify one FTD patient with two mtDNA transitions: one already known (3316 G-to-A) and another unreported (3337 G-to-A). Additionally, mitochondrial respiratory chain complex I activity was reduced in leukocytes of this patient (36% of the control mean activity). To our knowledge, this is the first report of mtDNA variants in FTD patients.

In silico analysis for predicting pathogenicity of five unclassified mitochondrial DNA mutations associated with mitochondrial cytopathies' phenotypes

Mitochondrial DNA (mtDNA) mutations have been assigned as a major cause of genetic disease. When a novel sequence variation is found, it is necessary to evaluate its functional impact, usually requiring functional molecular studies. Given the fact that this approach is difficult to put in practice in a routine basis, it is possible to take advantage of the in silico tools available and predict protein/RNA structure changes and therefore pathogenicity. Here, we describe the characterization of five undescribed mtDNA variants, upon detection of 23 unclassified alterations at Laboratory of Biochemical Genetics, from 2004 to 2014. Those five sequence variations are located in protein-coding genes, in five patients with a diverse range of mitochondrial respiratory chain disease phenotypes including encephalopathy, optic neuropathy, developmental delay, deafness and epilepsy. According to the prediction established by in silico analysis using tools to predict structure and function changes (ClustalW2®, PolyPhen-2®, SIFT®, MutationAssessor®, PredictProtein®, Provean®, I-TASSER®, Haplogrep®), from the 23 variants analyzed, the five described are potentially pathogenic. This approach is inexpensive and compatible with a rapid first line response to clinical demanding, contributing to a more rationale genetic diagnosis concerning novel mutations and to clarify the mtDNA involvement in these pathologies.

Identification of a novel deletion in SURF1 gene: Heterogeneity in Leigh syndrome with COX deficiency

Leigh syndrome (LS) is a rare, progressive neurodegenerative mitochondrial disorder of infancy. It is a genetically heterogeneous disease. The mutations in SURF1 gene are the most frequently known cause. Here two cases of LS likely caused by SURF1 gene variants are reported: a 39-year-old male patient with a novel homozygous deletion (c.-11_13del), and a case of a 6-year-old boy with the same deletion and a nonsense mutation (c.868dupT), both in heterozygosity. Blue native PAGE showed absence of assembled complex IV. This is the first report of a variant that may abolish the SURF1 gene initiation codon in two LS patients.

Metabolic Effects of Hypoxia in Colorectal Cancer by 13C NMR Isotopomer Analysis

13C NMR isotopomer analysis was used to characterize intermediary metabolism in three colorectal cancer cell lines (WiDr, LS1034, and C2BBe1) and determine the “metabolic remodeling” that occurs under hypoxia. Under normoxia, the three colorectal cancer cell lines present high rates of lactate production and can be seen as “Warburg” like cancer cells independently of substrate availability, since such profile was dominant at both high and low glucose media contents. The LS1034 was the less glycolytic of the three cell lines and was the most affected by the event of hypoxia, raising abruptly glucose consumption and lactate production. The other two colorectal cell lines, WiDr and C2BBe1, adapted better to hypoxia and were able to maintain their oxidative fluxes even at the very low levels of oxygen. These differential metabolic behaviors of the three colorectal cell lines show how important an adequate knowledge of the “metabolic remodeling” that follows a given cancer treatment is towards the correct (re)design of therapeutic strategies against cancer.

Disclosing the functional changes of two genetic alterations in a patient with Chronic Progressive External Ophthalmoplegia: Report of the novel mtDNA m.7486G>A variant

Highlights • Novel mt-tRNASer(UCN) (m.7486G>A) variant found in CPEO patient with 4,977 bp deletion.• The variant located in the anticodon loop meets the pathogenicity criteria.• Both genetic defects segregate with the biochemical phenotype in muscle.• Assembly impairment of MRC complexes was detected.• Mitochondrial translation defect and bioenergetic dysfunction were revealed.

Genetic Variation of MT-ND Genes in Frontotemporal Lobar Degeneration: Biochemical Phenotype-Genotype Correlation.

Background: Frontotemporal lobar degeneration (FTLD) is the second most common early-onset dementia. Over the last few decades, a growing number of evidence suggests mitochondrial involvement in neurodegeneration, namely modifications of mitochondrial DNA (mtDNA) contributing to energy impairment. Objective: To sequence the 7 mitochondrially encoded complex I (MT-ND) genes in 70 FTLD patients and investigate mitochondrial respiratory chain (MRC) complex I activity. Methods: A sample of 70 patients was studied (39 females and 31 males; age range: 38-82 years, mean ± SD: 63 ± 11 years) with a probable diagnosis of FTLD. Total DNA was extracted from peripheral blood, and sequencing analysis of 7 MT-NDn (1, 2, 3, 4L, 4, 5, 6) genes was performed. Variants identified were submitted to in silico study. Spectrophotometric evaluation of MRC activity in lymphocytes was performed, and results were compared with age-matched controls. Results: A total of 358 (161 different) alterations were found in 92.9% of patients. According to in silico analysis of nonsynonymous variants, only 5 variations are possibly or probably damaging. Complex I activity is significantly decreased in patients. Conclusion: To our knowledge, this is the first report of the complete sequence of the MT-ND genes in FTLD patients and correlation with MRC activity. The high number of mtDNA variations identified and a significant decrease in complex I activity suggest a possible involvement of mtDNA alterations in FTLD. Although the majority of these alterations are not primarily pathogenic, an interaction with other mutations may occur, leading to the disease, worsening its expression or influencing age of onset.