Reverse Translational Biomedical Research in Bigenomic Disorders and Personalized Medicine
Manuela Grazina
Biochemical genetics study in Metabolic and proliferation disorders
Manuela Grazina (FMUC, CNC), Luisa Diogo (CHUC, CNC), Catarina R. Oliveira (FMUC, CNC)
Collaborators: Carmo Macário, Paula Garcia, Guiomar Oliveira, Paulo Moura (CHUC); Lina Carvalho (FMUC, CHUC), Filipe Silva (IBILI)
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. The precise pathogenic mechanisms by which these biochemical abnormalities induce tissue dysfunction are not clearly understood and diagnosis of these disorders is complex, requiring specialised techniques and correlation between clinical and biochemical/ genetic data. The genetic causes of these complex disorders are located either in mtDNA or nuclear DNA, affecting the subunits of MRC system and all factors involved in mitochondrial biogenesis or mtDNA replication, transcription or stability.

The implementation of mtDNA copy number/mutation quantification by real time PCR was an important step for patients’ diagnostic workup, but also for translational research projects, and represents a major advance for our centre in this area. We have gathered the results of the first 18 months of studies and compared copy number with mtDNA pathogenic mutations findings in the same sample. We have found that depletion is 4-5 fold more frequent in children than point mutations, suggesting that the screening in paediatric samples should start by copy number investigation. Furthermore, we have found that about 40% of the depletion patients have mutations in the nuclear encoded gene DGUOK, which has an important role in mtDNA replication. Additionally, depletion in heart has not been characterized in detail. Given the high number (~30) of myocardium samples in LBG from patients remaining without definitive diagnosis, we have investigated it for depletion and we have found 3 cases with depletion in heart. These results are being gathered for publication.

A collaborative project is in progress with Dr. Fernando Scaglia and Prof. Lee-Jun Wong (Baylor College of Medicine, Houston, Texas, USA) for the study of MRCD and autism patients, for the study of complete mtDNA sequence and several nuclear genes affecting mtDNA biogenesis and maintenance. The results are being gathered for publication.

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Publications
Nobre S, Grazina M, Silva F, Pinto C, Gonçalves I, Diogo L. (2012) Neonatal liver failure due to deoxyguanosine kinase deficiency. BMJ Case Rep. doi:pii: bcr1220115317. 10.1136/bcr.12.2011.5317.

Henriques M, Diogo L, Garcia P, Pratas J, Simões M, Grazina M. (2012) Mitochondrial DNA 8993T>G mutation in a child with ornithine transcarbamylase deficiency and leigh syndrome: an unexpected association. J Child Neurol. (8):1059-61. doi: 10.1177/0883073811431015.

Grazina MM. (2012) Mitochondrial respiratory chain: biochemical analysis and criterion for deficiency in diagnosis. Methods Mol Biol. 2012;837:73-91. doi: 10.1007/978-1-61779-504-6_6.
Bigenomic investigation in Neurodegeneratiion
Manuela Grazina (FMUC, CNC), Isabel Santana (FMUC, CHUC, CNC), Catarina R. Oliveira FMUC, CNC)
Collaborators: Beatriz Santiago, Diana Duro (CHUC), Filipe Silva (IBILI)
Neurodegenerative disorders are complex and the mechanisms underlying the phenotypic expression of this group of diseases are not clearly understood. Finding genetic risk factors, either from nuclear or mitochondrial genome origin, will contribute to identify new tools for early diagnosis. Our aim is to search for genetic risk factors in our population and identify disease risk groups.
We have finished, in collaboration with Neurology Department of University Hospitals, a Research Project for Medical Students, concerning the evaluation of mtDNA ND1 sequence variations in a larger sample of FTD patients, following the evidences of the involvement of MRC complex I in FTD, reported in 2004 (Grazina M, Silva F, Santana I, Santiago B, Oliveira M, Cunha L, Oliveira C. Frontotemporal dementia and mitochondrial DNA transitions. Neurobiol. Dis. 2004; 15-2: 306-311). Our results point to the involvement of mtDNA and MRC in FTD. The role of mtDNA needs further examination, but our results support mitochondrial cascade hypothesis in FTD etiopathogeny.

One of the most complex neurodegenerative diseases is Multiple Sclerosis, and we aimed to investigate the role of mitocondrial respiratory chain (MRC) and mtDNA genetic variations, including haplogroups, in this disease and we have found that 48% of patients have MRC deficiency correlating with haplogroup J and with the presence of mtDNA sequence variations (3 fold higher).
Additionally we have continued the genetic characterization of dementias related to 5HTR2A. Accordingly, the project of the PhD student Daniela Luís entitled “Genetic Regulation of 5HT2A receptor in Frontotemporal Dementia”, assigned by FCT in 2008 (SFRH/BD/45387/2008), aiming to analyse the coding exons and the flanking intronic regions of 5HTR2A gene, in 92 samples from FTD patients was concluded. We have found 174 sequence variations, 3 of which are novel, 2 in the coding region (no aminoacid alteration) and 1 intronic (does not affect splicing), undergoing in silico characterization, to evaluating possible pathogenicity and selection for further functional studies.

Additionally, collaboration within CNC/UC has been started with the group of Sandra Cardoso for the analysis of mtDNA in Parkinson cybrids. The samples were extracted and sequencing of the 7 mtDNA-encoded ND genes has been initiated.

We have continued the genetic studies in eye disorders, namely Kjer type optic atrophy in collaboration with IBILI - FMUC and “Serviço de Oftalmologia” - CHUC.
Personalized Medicine
Manuela Grazina (FMUC, CNC), Carolina Ribeiro (CHUC), José Albuquerque
Collaborators: Ana Valentim, Ana Eufrásio, Teresa Lapa, Luís Rodrigues (CHUC), Filipe Silva (IBILI), Isabel Santana (FMUC, CHUC, CNC), Adrián Llerena, Eva Peñas-Lledó (Univ. Extremadura)
Since 2007, we have developed several projects aiming to identify genetic variants that will contribute for either identification of susceptibility factors or to support the development of more rationale therapies, including a pharmacogenetic approach.

We have concluded a pharmacogenomic project in Alzheimer’s disease, studying CYP2D6, which is involved in the oxidative metabolism of many different classes of commonly used drugs including donepezil.

The aim of this study was to investigate the association between four CYP2D6 alleles:* 2, *3,*4 and *10 in a group of 96 patients with probable diagnosis of Alzheimer’s disease and their clinical characteristics. Our results reveal a positive association with the age, age of onset and depression features with alleles *4 and *10. suggesting that genetic variations previously associated to decreased CYP2D6 activity may be a protective factor on the manifestation and progression of Alzheimer’s disease.

We have performed the evaluation of 40 DNA samples from women undergoing epidural after labouring, on the scope of a MSc study, for genetic analysis of CYP2D6 alleles * 2, *3,*4 and *10. We have found that profiles of poor metabolizers are more associated to higher pain scores. The results are being gathered for publication.

Other projects applying pharmacogenomics approaches in pain are in progress.
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