Manuela Grazina

University of Coimbra | UC · Faculty of Medicine & Biochemical Genetics Laboratory - CNC

Background Pharmacological oncological treatments interfere with the patient's quality of life on physical, psychological, and social dimensions. Besides all the care in hosting conditions, hospital clowns (HCs) use artistic sketches aiming to reduce distress, but studies analyzing their effects on biomarkers in association with psychological state...

Introduction: Leigh syndrome (LS) is clinically and genetically heterogeneous and presents defective mitochondrial bioenergetics. Patients present neurological symptoms and imagiological features that may result in early death [1]. The LS has been associated with mitochondrial DNA (mtDNA) variants, e.g., m.8993T>G (L156R) and m.8993T>C (L156P), in...

Introduction Mitochondrial diseases are rare, heterogeneous, incurable and complex to diagnose. Probably due to their rareness, there is still a lack of literacy in this area, especially in society, but also in schools and in general, health care services. Accordingly, tools that may bring advancement in science and health literacy are needed. Mit....

Leber's Hereditary Optic Neuropathy (LHON) has been mainly (90-95%) associated to one of three variants: m.3460G>A, m.11778G>A, m.14484T>C. Herein, a screening method was developed for its detection, supporting clinical/therapeutics decision. It relies on real-time PCR with High-Resolution Melting (HRM) analysis. Variant classification is made usin...

Optic atrophies, particularly Leber’s Hereditary Optic Neuropathy (LHON), have been extensively studied and many cases remain without a genetic cause identified1,2. The LHON diagnosis has been focused on 3 mitochondrial genome (mtDNA) variants (TOP3): m.3460G>A, m.11778G>A, and m.14484T>C. It presents with bilateral painless vision loss, usually se...

Introduction The diagnosis of Leber’s Hereditary Optic Neuropathy(LHON) is mainly associated to three mitochondrial genome variants: m.3460G>A,m.11778G>A & m.14484T>C (top-3), representing over 90% of the pathogenic variants found. It presents with bilateral painless vision loss, usually sequentially, within six weeks to six months. A fast diagnosi...

The MEF2C gene encodes a transcription factor known to play a crucial role in molecular pathways affecting neuronal development. MEF2C mutations were described as a genetic cause of developmental disease (MRD20), and several reports sustain its involvement in dementia-related conditions, such as Alzheimer’s disease and amyotrophic lateral sclerosis...

Mutations in the MPV17 gene are associated with hepatocerebral form of mitochondrial depletion syndrome. The mechanisms through which MPV17 mutations cause respiratory chain dysfunction and mtDNA depletion is still unclear. The MPV17 gene encodes an inner membrane mitochondrial protein that was recently described to function as a non-selective chan...

Introduction: Primary open-angle glaucoma is the most frequent subtype of glaucoma. Relatives of primary open-angle glaucoma patients have an increased risk of developing the disease, suggesting a genetic predisposition to the disease. MYOC was the first primary open-angle glaucoma-causing gene identified. This study aimed to identify sequence var...

We present the distribution of CYP2D6, CYP2C9 and CYP2C19 variants and predicted phenotypes in 33 native and admixed populations from Ibero‐America (n>6,000) in the context of genetic ancestry (n=3,387). Continental ancestries are the major determinants of frequencies of the increased‐activity allele CYP2C19*17 and CYP2C19 gUMs (negatively associat...

Pharmacogenetic variation in Latin Americans is understudied, which sets a barrier for the goal of global precision medicine. The RIBEF-CEIBA Network Consortium was established to characterize interindividual and between population variations in CYP2D6, CYP2C9, and CYP2C19 drug metabolizing enzyme genotypes, which were subsequently utilized to cata...

List of the 513 nuclear genes related to mitochondrial function and structure sequenced by Next Generation Sequencing (Mitome500 panel, Unpublished, Lee-Jun C. Wong).

List of the sequence variations detected in nuclear genes in patient's samples, after filtering for variant calling.

Glycolytic function measured for patient and controls' cells. Data are representative of the mean ± SEM. (A) Acidification profile; (B) Glycolysis; (C) Glycolytic Capacity; (D) Glycolytic Reserve.

Assessment of enzymatic activity of the mitochondrial respiratory chain complexes. Results are expressed as nmol of substrate per min per mg of protein normalized to Citrate Synthase. Patient values are presented as the mean of replicates. Control values are shown as mean ± SD.

Methods and results description.

Mitochondrial sequence variations detected by whole mitochondrial genome sequencing in the patient presenting CPEO.

Representation of mt-tRNASer(UCN) steady-state levels by high-resolution northern blot in skeletal muscle, without significant differences between patient and controls.

Objective: Prospective observational study to analyze CYP2D6 pharmacogenetics in 55 Portuguese adult parturients undergoing elective cesarean section and to investigate the association between CYP2D6 alleles and pain score. Methods: DNA was extracted from peripheral blood by standard methods. Genetic analysis included allelic discrimination (CYP...

Chronic Progressive External Ophthalmoplegia (CPEO) is characterized by ptosis and ophthalmoplegia and is usually caused by mitochondrial DNA (mtDNA) deletions or mt-tRNA mutations. The aim of the present work was to clarify the genetic defect in a patient presenting with CPEO and elucidate the underlying pathogenic mechanism. This 62-year-old fema...

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 e...

Background: Glutamine depletion is common in the critically-ill patients. Glutaminemia lower than 420 μmol/l has been considered as an independent predictive factor of mortality, but the indications for exogenous glutamine supplementation remain controversial. This study intends to determine the glutaminemia profile in critical surgical patients an...

Background: Glutamine depletion is common in the critically-ill patients. Glutaminemia lower than 420 μmol/l has been considered as an independent predictive factor of mortality, but the indications for exogenous glutamine supplementation remain controversial. This study intends to determine the glutaminemia profile in critical surgical patients a...

Background: Small-bowel dysfunction exerts a relevant prognostic impact in the critically ill patients. Citrullinemia has been used in the evaluation of the intestinal function and it is considered an objective parameter of the functional enterocyte mass. Present study proposes to determine the intestinal dysfunction prevalence and the citrullinem...

Background: CYP2D6 belongs to P450 superfamily, and is responsible for the metabolism of 25% of the drugs used clinically. Genetic variability of CYP2D6 affects individual drug or toxic response leading to differences in the drug outcome or toxicity mediating adverse drug effects. The different variant alleles are associated with increased, decrea...

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 advantag...

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),...

CYP2C9, CYP2C19 and CYP2D6 metabolize around 40 % of drugs and their genes vary across populations. The Costa Rican population has a trihybrid ancestry and its key geographic location turns it into a suitable scenario to evaluate interethnic differences across populations. This study aims to describe the diversity of CYP2C9, CYP2C19 and CYP2D6 poly...

Background Arginine is an amino acid determinant in the metabolic, immune and reparative responses to severe trauma. The present study aims to determine argininemia and plasma arginine bioavailability (PAB) in critical trauma patients and to analyze its correlation with prognosis. MethodsA prospective study of 23 critical trauma patients was undert...

Mitochondrial translation defects are important causes of early onset mitochondrial disease. Although the biochemical (combined respiratory chain deficiency) signature and neuroimaging are usually distinctive, they are not diagnostic as the genetic origin of mitochondrial translation defects is heterogeneous. We report a female child, born at term...

We evaluated the coenzyme Q₁₀ (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n = 72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary...

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. To sequence the 7 mitochondrially encoded complex I (MT-ND) gen...

Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4...

(13)C 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 c...

Aim: CYP2D6 codes for a protein that is vastly involved in the metabolism of various substances. This gene is highly polymorphic, which influences the enzymatic activity and contributes to the huge variability in the enzyme hydroxylation capacity. Different metabolic profiles determine the processing of xenobiotics and endobiotics, thereby influen...

Background: Citrullinemia is been reported as a quantitative parameter of the enterocyte mass and function. Aim: The objective of this research is to analyse the value of fasting and stimulated citrullinemias in the intestinal function evaluation. Methods: A case-control study was undertaken, including 11 patients with short bowel syndrome, 13...

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...

Background: Citrullinemia is been reported as a quantitative parameter of the enterocyte mass and function. Aim: The objective of this research is to analyse the value of fasting and stimulated citrullinemias in the intestinal function evaluation. Methods: A case-control study was undertaken, including 11 patients with short bowel syndrome, 13 pati...

Mitochondrial respiratory chain diseases are a heterogeneous group of pathologies caused by genetic alterations affecting mitochondrial energy production. Theoretically, this deficiency may lead to any symptoms, in any organ or tissue, at any age even before birth. The aim of our study was to identify the frequency and characterize antenatal manife...

Abnormal presence of autophagic vacuoles is evident in brains of patients with Parkinson's disease (PD), in contrast to the rare detection of autophagosomes in a normal brain. However, the actual cause and pathological significance of these observations remain unknown. Here, we demonstrate a role for mitochondrial metabolism in the regulation of th...

Nonketotic hyperglycinemia is a rare metabolic disorder with severe, frequently fatal, neurologic manifestations. Reliable and accurate diagnosis depends on careful interpretation of laboratory findings. The clinical suspicion should lead to determination of glycine in plasma and cerebrospinal fluid. Amino acid analysis presents diagnostic values f...

Deoxyguanosine kinase (dGK) deficiency, a rare severe cause of mitochondrial DNA (mtDNA) depletion, has two forms of presentation: hepatocerebral syndrome and isolated hepatic disease. The authors report three cases with neonatal liver failure due to dGK deficiency. Consanguinity was present in all patients. One patient had a brother who died with...

MC, female, is the third child of a nonconsanguineous Portuguese couple, born after an uneventful pregnancy and delivery. A positive family history of ornithine transcarbamylase deficiency, associated with the IVS8+1 G>A mutation in the ornithine transcarbamylase gene, prompted prenatal diagnosis with identification of the same mutation in the prob...

Spectrophotometric evaluation of mitochondrial respiratory chain (MRC) enzymatic complexes is the main approach to the biochemical investigation and diagnosis in oxidative phosphorylation disorders (also known as mitochondrial cytopathies). Regular dual beam spectrophotometers may be used, but we describe the protocols for double wavelength devices...

We investigated the involvement of mitochondrial-dependent apoptosis in Huntington's 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...

Mitochondrial respiratory chain (MRC) disorders have variable clinical manifestations which are mainly neurologic. Diagnosis in children is more complex than in adults because the classic phenotype, ragged red fibers, and mtDNA mutations are rarely seen in children. Moreover, clinical manifestations of disease in developing brains are less explicit...

Mitochondrial respiratory chain disorders (MRCD) are a heterogeneous group of diseases leading to an inadequate production of ATP. Brain and heart are among the most affected organs. Thus far, no specific relationship has been found between specific affected areas in the central nervous system and cardiac involvement. This study investigated the re...

The present study reviewed mitochondrial respiratory chain disorders diagnosed at the sole tertiary pediatric hospital in the Centro region of Portugal and estimated incidence and prevalence in this region. The Bernier criteria were used to retrospectively classify 200 children investigated from 1997 to 2006. A total of 78 patients were assigned wi...

Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder characterized by bilateral loss of central vision, most frequently found in young adult males. In most patients there are no other neurological manifestations and cerebral neuroimaging is normal, but some rare cases of "LHON plus" have been described. Classi...

In the present study we confirm the previously reported high frequency of biochemical markers of mitochondrial dysfunction, namely hyperlactacidemia and increased lactate/pyruvate ratio, in a significant fraction of 210 autistic patients. We further examine the involvement of the mitochondrial aspartate/glutamate carrier gene (SLC25A12) in mitochon...

Alzheimer's disease (AD) is the most common neurodegenerative disorder associated to dementia in late adulthood. Amyloid precursor protein, presenilin 1 and presenilin 2 genes have been identified as causative genes for familial AD, whereas apolipoprotein E epsilon4 allele has been associated to the risk for late onset AD. However, mutations on the...

A minority of cases of autism has been associated with several different organic conditions, including bioenergetic metabolism deficiency. In a population-based study, we screened associated medical conditions in a group of 120 children with autism (current age range 11y 5mo to 14y 4mo, mean age 12y 11mo [SD 9.6mo], male:female ratio 2.9:1). Childr...

Alzheimer's disease (AD) is the most common neurodegenerative disorder associated with dementia in late adulthood. Mitochondrial respiratory chain impairment has been detected in the brain, muscle, fibroblasts and platelets of AD patients, indicating a possible involvement of mitochondrial DNA (mtDNA) in the etiology of the disease. Several reports...

Frontotemporal dementia (FTD) is the second most common type of primary degenerative dementia. Some patients present an overlap between Alzheimer's 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 asso...

Envelhecer Vivendo pode confundir-se com uma “frase feita”, ou se quisermos com um “título feito”, espelhando a utopia do envelhecimento conduzir à vida, ou à necessidade de contrariar a universalidade e a “fatalidade” de envelhecer e morrer. Mas nunca se confundirá se estivermos atentos ao facto do envelhecimento em qualquer idade, mas especialmen...