M. Martínez de Pancorbo

Gene promoter hypermethylation in oral rinses of leukoplakia patients—a diagnostic and/or prognostic tool?

Leukoplakia is the most frequent oral precancerous lesion and shows a variable rate of malignant transformation. We hypothesised that the detection of molecular alterations, like the promoter hypermethylation of DNA, in oral cytological samples from patients at risk of developing primary or recurrent tumours could be a valuable diagnostic and prognostic tool in the management of these lesions. Two groups of patients with differing risks of developing oral squamous cell carcinoma (OSCC) were analysed. DNA was extracted from the oral rinse of each patient. The methylation status of the p16, p14 and MGMT gene promoters was determined using a methylation-specific polymerase chain reaction (MSP). Methylation of p16 and MGMT was observed in 44 and 56% of the oral samples, respectively. Only 12% of the cases showed p14 methylation. DNA hypermethylation was more frequent in patients with previous OSCC. DNA promoter hypermethylation is frequent during early oral carcinogenesis and even more so in the later stages. MSP using oral rinses is a non-invasive and highly sensitive technique which could be used to monitor patients with precancerous and cancerous oral lesions.

Cardiac sympathetic denervation precedes nigrostriatal loss in the E46K mutation of the α-synuclein gene (SNCA)

IntroductionHere we report the case of an asymptomatic carrier of the E46K substitution in α-synuclein gene where we have documented that cardiac sympathetic denervation precedes nigrostriatal dopaminergic loss.Material and methodsShe has been followed up regularly with standard neurological examination, UPDRS, neuropsychological formal testing, parkinson disease sleep scale-PDSS, Epworth scale, Hamilton-D scale, SCOPA Aut, orthostatic hypotension test, brief smell identification test, polysomnography, cerebral 123-I-FP-CIT SPECT, and, 123I-MIBG cardiac scintigraphy.ResultsShe shows no presence of orthostatic hypotension. Olfactory test results demonstrate normal limits. In the PSG the nocturnal sleep shows mild abnormalities although the sleep efficiency and stage proportion remain under normal limits. The 123-I-FP-CIT SPECT is normal; in contrast, the 123I-MIBG cardiac scintigraphy shows a complete lack of isotopic uptake compatible with a severe sympathetic myocardial denervation.ConclusionThis example of monogenic autosomal dominant parkinsonism due to an α-synuclein mutation favours the hypothesis that peripheral autonomous nervous system involvement occurs earlier than the CNS degeneration.

DNA typing of Diptera collected from human corpses in Portugal

Medico-legal entomology, one area in the broad field of entomology, is routinely used in forensic applications. Insects are often collected from a corpse during criminal information related to the body, but requires the fast and accurate identification of the species attracted to the remains. The local entomofauna in most cases is important for explaining entomological evidence. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different species on corpses. A morphological and DNA-based methods for species identification were used in this study. Thirty-two cases are reported from indoors and outdoors conditions. Specimens were collected from corpses during autopsy procedures in the National Institute of Legal Medicine, Portugal, and cases were summarized by sex, death local, month of discovery, probable cause of death, species found and number of analyzed specimens. Just eight species, mainly Calliphoridae together with one Sarcophagidae were reported from corpses. The DNA sequencing was performed to study the mitochondrial encoded subunit I of the cytochrome oxidase gene. The aim of this work was the beginning of a database of the cadaveric entomofauna in Portugal.

Population study of 3 STR loci in the Basque Country (northern Spain)

The tetrameric STRs, HUMTH01, HUMVWA31A and HUMFES/FPS, were studied in a population from the Basque Country (northern Spain) for their frequency distribution and applicability to identity and paternity testing. All systems conformed to Hardy-Weinberg equilibrium; pairwise comparisons demonstrated the allelic independence between loci, and furthermore, all systems seemed to be in agreement with expectations from the Stepwise Mutation Model (SMM) of the mutation-drift theory, which indicates the homogeneity of the population and suggests a replication slippage mechanism as a possible model for generating alleles. A comparison with other population groups appeared to indicate that frequencies are well conserved in Caucasians, but differ from other racial groups. The calculated parameters “a priori probability of exclusion” (PEX) and “index of discrimination” (ID), show the informativeness of these loci for the determination of identity and relatedness of individuals.

Somatic mosaicism in a case of apparently sporadic Creutzfeldt-Jakob disease carrying a de novo D178N mutation in the PRNP gene†‡

Transmissible spongiform encephalopathies (TSEs) are a group of rare fatal neurodegenerative disorders. Creutzfeldt‐Jakob disease (CJD) represents the most common form of TSE and can be classified into sporadic, genetic, iatrogenic and variant forms. Genetic cases are related to prion protein gene mutations but they only account for 10–20% of cases. Here we report an apparently sporadic CJD case with negative family history carrying a mutation at codon 178 of prion protein gene. This mutation is a de novo mutation as the parents of the case do not show it. Furthermore the presence of three different alleles (wild type 129M‐178D and 129V‐178D and mutated 129V‐178N), confirmed by different methods, indicates that this de novo mutation is a post‐zygotic mutation that produces somatic mosaicism. The proportion of mutated cells in peripheral blood cells and in brain tissue was similar and was estimated at approximately 97%, suggesting that the mutation occurred at an early stage of embryogenesis. Neuropathological examination disclosed spongiform change mainly involving the caudate and putamen, and the cerebral cortex, together with proteinase K‐resistant PrP globular deposits in the cerebrum and cerebellum. PrP typing was characterized by a lower band of 21u2009kDa. This is the first case of mosaicism described in prion diseases and illustrates a potential etiology for apparently sporadic neurodegenerative diseases. In light of this case, genetic counseling for inherited and sporadic forms of transmissible encephalopathies should take into account this possibility for genetic screening procedures.

High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma

Summary.u2002 To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4–8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki‐67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53‐positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell.

Distribution of Cepaea nemoralis according to climatic regions in Spain

121 Populations of Cepaea nemoralis were sampled in Spain. In an area which extends from the coast of the Bay of Biscay to 182 km inland, following the meridian 3° west of Greenwich, and which has a width of approximately 40 km. In this study, we analysed the relationships between the populations and some selective agents, such as predation by Turdus ericetorum and climatic factors. These latter are represented by indirect parameters, such as altitude and distance from the sea, and direct parameters such as rainfall or temperatures. Visual selection is unimportant in the sampled populations. It was observed that our area of study is subdivided into 3 zones: Zone 1. Transitional and Zone 2, which have very different phenotype frequencies. What is more, these Zones correspond to very different climatic regions. Zone 1 is located in the climatic region known as “Green Iberia”. The most frequent phenotypes of Cepaea nemoralis are the pale ones. Zone 2 is located in “Brown Iberia”, and the most frequent phenotypes are the dark ones. Therefore we believe that in the populations sampled, one or more climatic factors are operating, and presumably causing the subdivision of these populations into Zones 1 and 2.

A review of the collaborative exercises on DNA typing of the Spanish and Portuguese ISFH working group

Abstract Since 1992 the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Haemogenetics (ISFH) has been organizing collaborative exercises on DNA profiling with the aim of making progress on standardization and discussing technical and statistical problems in DNA analysis. A total of four exercises (GEP-92 to GEP-95) have been carried out until now. A consequence of these exercises was the creation of a quality control programme in Spain and Portugal in 1995 which was carried out simultaneously with the GEP-95 exercise. The number of participating laboratories increased from 10 in the first exercise (GEP-92) to 19 in the last exercise (GEP-95). Despite this increasing number of participating laboratories, results remained satisfactory. In the last exercises, all the laboratories used PCR-based DNA polymorphisms with an increasing number of markers obtaining good results. SLPs were used by only 30% of laboratories in the last two exercises but the results indicated a good level of expertise in most of these laboratories. The reasons for these successful results are the common use of the EDNAP protocol for SLP analysis and commercially available kits or common sequenced allelic ladders for PCR-based DNA polymorphisms.

Y-chromosomal STR haplotype diversity in males from Santa Catarina, Brazil.

One-hundred and nine unrelated and healthy males from Santa Catarina, Brazil were included in this study. Allele frequencies and gene diversities for the loci DYS456, DYS458 and DYS448 were calculated. A comparison between our population and others was performed.

p53, un gen supresor tumoral

Resumen Los genes supresores tumorales estan implicados en diversos procesos de division celular como la regulacion de la expresion genica, control del ciclo celular, programacion de la muerte celular y estabilidad del genoma. La perdida de actividad de estos genes provoca la incapacidad de respuesta a los mecanismos de control que regulan la division celular, de modo que se produce una proliferacion mas o menos incontrolada de la celula lo cual conduce en ocasiones al desarrollo de neoplasias y a la evolucion de las mismas hacia procesos tumorales mas agresivos. El gen p53 pertenece al grupo de genes implicados en el control del ciclo celular. Este gen tiene multiples funciones ya que aparece implicado no solo en el control del ciclo celular sino tambien en la integridad del ADN y la supervivencia de las celulas expuestas a agentes que danan el ADN. La alteracion del gen p53 confiere un riesgo muy elevado de desarrollar cancer y la mutacion del mismo es uno de los cambios genomicos mas frecuentes en el cancer humano.