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| Pediatric Research: metabolic disorders |
| Luísa Diogo (CHC); Catarina Oliveira (CNC); Manuela Grazina (CNC) |
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Mitochondrial respiratory chain diseases (MRCD) are a diverse group of disorders with a broad spectrum of clinical manifestations, characterised by defects in mitochondrial energetic function. Inherited defects causing mitochondrial dysfunction can be due to mutations either in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Each mitochondrion contains its own DNA that codes for 13 peptides of the mitochondrial respiratory chain (MRC) system, where the oxidative phosphorylation (OXPHOS) occurs, plus the two structural rRNAs and 22 tRNAs necessary for mtDNA genes expression.
We have continued the approach implemented last year for the molecular differential analysis of mitochondrial cytopathies, as a high-throughput screening. The quantification of mtDNA mutation, by real time PCR, is being implemented and a collaboration with University of Newcastle upon Tyne has been established. The evaluation of complex I activity in cell suspensions, namely fibroblasts, and the study of Krebs cycle enzymes both in fibroblasts and peripheral blood leukocytes is now being applied in the differential diagnosis with metabolic disorders.
In 2006 the experimental work leading to the MSc Thesis intitled “Determination of the frequency of mtDNA haplogroups in patients suspected of Mitochondrial Cytopathy” was concluded. The study was performed in 350 subjects, including 83 subjects with definite diagnosis based on mtDNA analysis positive result (PDD), and 216 control subjects. A statistically significant difference was detected between haplogrup H1 frequencies of PDD and controls, (29% and 15%, respectively; p<0.008). The results suggest that macro-haplogrups JT, UK and L may influence clinical phenotype manifestation, but a higher phylogenetic resolution in a higher magnitude sample, is required to confirm these data. Accordingly, the accumulation of polymorphisms in the internal branches of human phylogeny could affect the fixation of potentially deleterious mtDNA mutations and influence the phenotype expression.
A PhD Thesis focused on the MRC activity and mtDNA investigation, during a period of 7 years (1997-2003), was also concluded. The study included the evaluation of 577 individuals, including 46 family members and 531 patients, followed at several Portuguese hospitals, mainly Paediatric Hospital (346) and Neurology Service of University Hospitals (160) of Coimbra. Our data show that MRC deficiency can be detected in leukocytes isolated from peripheral blood (LEU) with high sensibility, but with a low specificity, as compared to evaluation in other(s) tissue(s). Additionally, we have clearly confirmed that analysis of isolated mitochondria and fresh frozen muscle homogenate (HM2) provide different results concerning MRC analysis. These results highlight additional information contributing to a more accurate biochemical study of muscle tissue, taking into account the differences observed for the fractions obtained during mitochondria isolation. The genetic analysis of mtDNA has allowed the identification of mtDNA sequence variations in 120 individuals, including six new alterations probably pathogenic. These results have contributed to the definite diagnosis of MRC defect in 89 patients (17.5%), including 56 adults and 28 children. The estimated frequency for pathogenic mtDNA mutations in children and adults is 8.3:100,000 (1:12,048) and 2.8:100,000 (1:35,714), respectively, in the center region of Portugal.
As expected, we have found high heterogeneous results, concerning all data presented, both at genetic, or biochemical level, as well as clinical information. Nevertheless, a positive correlation, for the same tissue, for both biochemical and genetic data could be demonstrated in some cases. In subjects with multiple CRM deficiencies, diverse mtDNA alterations were found, essentially deletions (isolated point mutation was identified only in two individuals). In addition, a positive correlation was observed, taking into account mtDNA analysis and the CRM enzymatic activity evaluation.
Taking into consideration the total number of cases evaluated, both adults and children, it was possible to establish the diagnosis of a significant number of cases. According to demographic data for Centre Portugal region population results (CENSOS 2001; source: INE), a frequency of 1:20,000 for DCRM in the population of Centre Portugal could be estimated. |
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| PUBLICATIONS |
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| Grazina MMM (2006). Mitochondrial Genome and Energetic Deficiency in Diagnosis of Mitochondrial Respiratory chain Diseases (PhD Thesis). |
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| Correia C, Coutinho AM, Diogo L, Grazina M, Marques C, Miguel TS, Ataíde A, Borges L, Oliveira C, Oliveira G, Vicente AM (2006). High frequency of biochemical markers for mitochondrial dysfunction in autism: no association with the mitochondrial aspartate/glutamate carrier SLC25A12 gene. J Autism Dev Disord. 36(8): 1137-1140. |
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| Cordeiro M, Rodrigues D, Garcia P, Lopes da Silva S, Macário MC, Vilarinho L, Grazina M, Oliveira CR, Diogo L (2006). Sindrome de Leigh: revisão de oito casos. Sinapse, 6 (1): 118. |
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| Grazina MM, Diogo L, Garcia P, Silva E, Garci T, Robalo C, Oliveira C. Atypical presentation of Leber`s hereditary optic neuropathy associated to mtDNA 11778G > A point mutation – A case report. Eur J Paediatr Neurol (in Press). |
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