Maria João Santos

Mitochondrial-dependent apoptosis in Huntington's disease human cybrids

We investigated the involvement of mitochondrial-dependent apoptosis in Huntingtons disease (HD) vs. control (CTR) cybrids, obtained from the fusion of human platelets with mitochondrial DNA-depleted NT2 cells, and further exposed to 3-nitropropionic acid (3-NP) or staurosporine (STS). Untreated HD cybrids did not exhibit significant modifications in the activity of mitochondrial respiratory chain complexes I-IV or in mtDNA sequence variations suggestive of a primary role in mitochondrial susceptibility in the subpopulation of HD carriers studied. However, a slight decrease in mitochondrial membrane potential and increased formation of intracellular hydroperoxides was observed in HD cybrids under basal conditions. Furthermore, apoptotic nuclei morphology and a moderate increase in caspase-3 activation, as well as increased levels of superoxide ions and hydroperoxides were observed in HD cybrids upon 3-NP or STS treatment. 3-NP-evoked apoptosis in HD cybrids involved cytochrome c and AIF release from mitochondria, which was associated with mitochondrial Bax translocation. CTR cybrids subjected to 3-NP showed increased mitochondrial Bax and Bim levels and the release of AIF, but not cytochrome c, suggesting a different mode of cell death, linked to the loss of membrane integrity. Additionally, increased mitochondrial Bim and Bak levels, and a slight release of cytochrome c in untreated HD cybrids may help to explain their moderate susceptibility to mitochondrial-dependent apoptosis.

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.

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.

Factor Structure and Measurement Invariance of the Coimbra Neuropsychological Assessment Battery (BANC)

Objective This study aimed to investigate the factor structure of the Coimbra Neuropsychological Assessment Battery (BANC), which is an individually administered battery designed to assess a wide range of neurocognitive functions in children. Method Using the standardization sample of the BANC, a confirmatory factor analysis and a multiple-group analysis were conducted to examine the factor structure and the measurement invariance of three main domains (Memory, Language, and Attention/Executive Functions) in 833 children aged 7-15 years. Results Consistent with the BANCs conceptualization, the three-correlated-factor model demonstrated the most adequate fit to the data. The measurement invariance of the three-correlated-factor model across two age-groups (7-9 years and 10-15 years) was supported (configural, metric, and partial scalar invariance). Conclusion Overall, the BANC shows adequate psychometric properties and provides useful information regarding the childrens neuropsychological functioning.

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.

mtDNA copy number associated with age of onset in familial amyloid polyneuropathy

Background Transthyretin-related familial amyloid polyneuropathy (TTR-FAP Val30Met) shows a wide variation in age-at-onset (AO) between generations and genders, as in Portuguese families, where women display a later onset and a larger anticipation (>10 years). Mitochondrial DNA (mtDNA) copy number was assessed to clarify whether it has a modifier effect on AO variability in Portuguese patients. Methods The mtDNA copy number of 262 samples (175 Val30Met TTR carriers and 87 controls (proven Val30Val)) was quantified by quantitative real-time PCR. Statistical analysis was performed using IBM SPSS V.23 software. Results This study shows that Val30Met TTR carriers have a significantly higher (p<0.001) mean mtDNA copy number than controls. Furthermore, the highest mtDNA copy number mean was observed in early-onset patients (AO <40 years). Importantly, early-onset offspring showed a significant increase (p=0.002) in the mtDNA copy number, when compared with their late AO parents. Conclusions The present findings suggest, for the first time, that mtDNA copy number may be associated with earlier events and may therefore be further explored as a potential biomarker for follow-up of TTR-FAP Val30Met carriers.

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.

Molecular genetics investigation of 5HTR2A gene in frontotemporal lobar dementia

WCN 2013 No: 2324 Topic: 5 — Dementia Molecular genetics investigation of 5HTR2A gene in frontotemporal lobar dementia D. Luis, I. Santana, M.J. Santos, D. Duro, J. Pratas, P. Cerveira, F. Silva, M. Grazina. Faculty of Medicine, University of Coimbra, Portugal; CNC — Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; CHUC EPE — Neurology Service, Portugal; Centre of Ophthalmology and Vision Sciences, IBILI — Faculty of Medicine,