Laura Guimarães

High Germinal Instability of the (CTG)n at the SCA8 Locus of Both Expanded and Normal Alleles

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of late-onset, neurodegenerative disorders for which 10 loci have been mapped (SCA1, SCA2, SCA4-SCA8, SCA10, MJD, and DRPLA). The mutant proteins have shown an expanded polyglutamine tract in SCA1, SCA2, MJD/SCA3, SCA6, SCA7, and DRPLA; a glycine-to-arginine substitution was found in SCA6 as well. Recently, an untranslated (CTG)n expansion on chromosome 13q was described as being the cause of SCA8. We have now (1) assessed the repeat size in a group of patients with ataxia and a large number of controls, (2) examined the intergenerational transmission of the repeat, and (3) estimated the instability of repeat size in the sperm of one patient and two healthy controls. Normal SCA8 chromosomes showed an apparently trimodal distribution, with classes of small (15-21 CTGs), intermediate (22-37 CTGs), and large (40-91 CTGs) alleles; large alleles accounted for only0.7% of all normal-size alleles. No expanded alleles (>/=100 CTGs) were found in controls. Expansion of the CTG tract was found in five families with ataxia; expanded alleles (all paternally transmitted) were characterized mostly by repeat-size contraction. There was a high germinal instability of both expanded and normal alleles: in one patient, the expanded allele (152 CTGs) had mostly contraction in size (often into the normal range); in the sperm of two normal controls, contractions were also more frequent, but occasional expansions into the upper limit of the normal size range were also seen. In conclusion, our results show (1) no overlapping between control (15-91) and pathogenic (100-152) alleles and (2) a high instability in spermatogenesis (both for expanded and normal alleles), suggesting a high mutational rate at the SCA8 locus.

Transient ischemic attacks in rural and urban northern Portugal: incidence and short-term prognosis.

Background and Purpose— There are no community-based studies on the incidence of transient ischemic attacks (TIAs) in Portugal. This study was designed to determine TIA incidence and the risk of stroke occurrence in rural and urban populations in northern Portugal. Methods— All suspected first-ever TIAs occurring between October 1998 and September 2000 in 18 677 residents in a rural municipality and 86 023 living in the city of Porto were entered into a population-based registry. Standard definitions and comprehensive sources of information were used for identification of patients who were followed up at 3 and 12 months after the TIA. Results— During a 24-month period, 141 patients with a first-ever TIA were registered, 36 in rural and 104 in urban areas. The vascular territory was carotid in 66.7% of the patients, vertebrobasilar in 29.1%, and undetermined in 4.3%. The overall crude annual incidence rate per 1000 was 0.67 (95% CI, 0.45 to 1.04), 0.61 (95% CI, 0.38 to 1.01) for rural, and 0.96 (95% CI, 0.43 to 2.33) for urban populations. The risk of stroke within the first 7 days of the index event was 12.8% (95% CI, 7.3 to 18.3), reaching 21.4% (95% CI, 14.6 to 28.1) at 1 year. Three factors were associated with stroke occurrence within 120 days after TIA: patients’ age ≥65 years and an episode in the carotid distribution lasting ≥3 hours. Conclusions— The incidence of TIA in northern Portugal, particularly in rural populations, ranks among the highest reported in community-based studies, following closely the stroke incidence trend (ACINrpc). Early recognition of TIA by patients and physicians is crucial for effective stroke prevention.

Yellow eel (Anguilla anguilla) development in NW Portuguese estuaries with different contamination levels.

The aims of the present study were to compare the health status of yellow eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different levels of pollution and their physiological responses to combined effects of environmental variation and pollution. For this, a field study was performed using a multi-parameter approach, including eels condition indexes and biomarkers, water quality variables and other environmental factors. Sixteen biological parameters were assessed, namely: hepatosomatic index (LSI), Fulton’s condition index (K), lipid peroxidation (LPO), total glutathione (TG), reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG, and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), sodium-potassium ATPase (Na+/K+-ATPase), ethoxyresorufin O-deethylase (EROD), glutathione S-transferases (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR). Ten environmental factors were also measured in water: temperature, salinity, pH, phosphates, nitrates, nitrites, ammonium, silica, phenol and hardness. Globally, the biomarkers indicate exposure and toxic effects of pollutants on eels living in contaminated estuaries. The relationships between biological and environmental variables were assessed through redundancy analysis. K and LSI indexes, AChE and Na+/K+-ATPase, total glutathione levels and the antioxidant enzymes CAT, GR, and SOD where the factors most discriminating reference (Minho River estuary) from contaminated estuaries (Lima and Douro Rivers estuaries). Moreover, the most striking outcomes of pollutants exposure on biological responses were observed during winter, probably due to a joint effect of cold weather and pollution stress. Altogether, the results indicate that the development of eels in the polluted estuaries of Lima and Douro rivers is interfering with physiological functions determinant for their survival and performance. This may increase the mortality rates during the continental life-phase of the species and decrease the percentage of animals able to successfully complete their oceanic migration and, thus, reduce the contribution of each generation to the next one.

Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal).

The health status of eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different types and levels of pollution was compared in relation to morphometric parameters, Fulton condition index (F index) and several biomarkers. Relatively to the reference population, glass eels from the Lima estuary had lower weight and length, cholinesterase (ChE) and lactate dehydrogenase (LDH) inhibition, and lower levels of some anti-oxidant parameters, while yellow eels also showed a decreased F index, and increased Na(+)/K(+)-ATPase and lipid peroxidation (LPO) levels. Relatively to the reference population, glass eels from the Douro estuary had increased Na(+)/K(+)-ATPase and glutathione-S-transferase activities and LDH inhibition, while yellow eels also had ChE inhibition and increased LPO. Overall, these results indicate that eels from polluted estuaries showed a poor health status than those from a reference estuary, and that adverse effects become more pronounced after spending several years in polluted estuaries.

The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

Health status of Pomatoschistus microps populations in relation to pollution and natural stressors: implications for ecological risk assessment

Effects induced on wild populations by recurrent environmental contamination may difficult the ecological risk assessment of punctual pollution events such as oil spills. Here, the issue was addressed by comparing the health status of Pomatoschistus microps populations from four NW Iberian estuaries, using an integrated chemical–biological monitoring. Despite high seasonal variability, the parameters measured discriminated estuaries with different contamination levels and associated biological effects with chemical and abiotic stress. The decreased health status of fish from polluted sites strengthens the need of considering pollution-induced background effects and seasonal variability when assessing impacts and risks of oil and other chemical spills.

Portuguese families with dentatorubropallidoluysian atrophy (DRPLA) share a common haplotype of Asian origin

Dentatorubropallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a variable combination of progressive ataxia, epilepsy, myoclonus, choreoathetosis and dementia. This disease is caused by a (CAG)n expansion in the DRPLA gene, on chromosome 12p13. DRPLA is prevalent in Japan, but several families of non-Japanese ancestry have already been published. To identify the origin of expanded alleles in Portuguese families with DRPLA, we studied two previously reported intragenic SNPs in introns 1 and 3, in addition to the CAG repeat of the DRPLA gene. The results showed that all four Portuguese DRPLA families shared the same haplotype, which is also common to that reported for Japanese DRPLA chromosomes. This haplotype is also the most frequent in Japanese normal alleles, whereas it was rare in Portuguese control chromosomes. Thus, our findings support that a founder DRPLA haplotype of Asian origin was introduced in Portugal, being responsible for the frequency of the disease in this country.

Genetic and developmental models for the neural control of breathing in vertebrates

The present paper reviews some of the possible mechanisms that may link gene function in the brainstem and breathing patterns in vertebrates. On one hand, adaptation and acclimatisation of mature breathing to environmental constraints such as hypoxia, involves complex regulation of the gene expression in precise cardiorespiratory sites of the brainstem. On the other hand, targeted inactivation of different genes suggests that postnatal respiratory variables at rest depend on genes controlling the prenatal development of the brainstem. During embryogenesis, neurotrophins (gdnf, bdnf) regulate the survival of specific cellular populations composing the respiratory neuronal network. The expression of developmental genes such as Hox and Krox-20 initiates hindbrain segmentation, the earliest sign of regionalisation in the brainstem. As shown in the chick embryo, segmental specifications allow the establishment of an active embryonic rhythmic network and later insertion of specific neuronal circuits increasing the primordial rhythm frequency to near mature values.

The CAG repeat at the Huntington disease gene in the Portuguese population: insights into its dynamics and to the origin of the mutation

AbstractHuntington disease (HD) is caused by an expansion of a CAG repeat. This repeat is a dynamic mutation that tends to undergo intergenerational instability. We report the analysis of the CAG repeat in a large population sample (2,000 chromosomes) covering all regions of Portugal, and a haplotype study of (CAG)n and (CCG)n repeats in 140 HD Portuguese families. Intermediate class 2 alleles represented 3.0% of the population; and two expanded alleles (36 and 40 repeats, 0.11%) were found. There was no evidence for geographical clustering of the intermediate or expanded alleles. The Portuguese families showed three different HD founder haplotypes associated with 7-, 9- or 10-CCG repeats, suggesting the possibility of different origins for the HD mutation among this population. The haplotype carrying the 7-CCG repeat was the most frequent, both in normal and in expanded alleles. In general, we propose that three mechanisms, occurring at different times, may lead to the evolution from normal CAGs to full expansion: first, a mutation bias towards larger alleles; then, a stepwise process that could explain the CAG distributions observed in the more recent haplotypes; and, finally, a pool of intermediate (class 2) alleles more prone to give rise to expanded HD alleles.

Molecular diagnosis of Huntington disease in Portugal: implications for genetic counselling and clinical practice.

Huntington disease (HD) is a neurodegenerative, autosomal dominant disorder of late-onset, caused by the expansion of a CAG repeat in the coding region of the gene. Ours is the reference laboratory for genetic testing in HD, in Portugal, since 1998; 90.1% of all 158 families known were identified for the first time, including patients with unusual presentation or without family history. A total of 338 genetic tests were performed: 234 for diagnosis, 96 for presymptomatic and four for prenatal testing (four were done for family studies). Most referring physicians were neurologists (90.6%); 82.8% of all clinical diagnosis were confirmed, while 83.1% of those sent for exclusion were in fact excluded. In presymptomatic testing, an excess of female subjects (59.4%) was again verified; 37.5% of the consultands were found to be carriers. None of the foetuses, in four prenatal tests, were mutation carriers. One juvenile case was inherited from her mother. Our patient population is very similar to others described so far, namely in terms of mean age at onset and (CAG)n distribution, except perhaps for a higher frequency of large normal (class 2) alleles (3.7%). We also identify cases posing particular problems for genetic counselling, such as, ‘homozygosity’ that can pose a serious ethical dilemma, carriers of large normal alleles, and ‘homoallelism’ for a normal gene, which will demand further procedures and may delay results in presymptomatic and prenatal testing.