Discovery of the Principal Cystic Fibrosis Mutation (F508del) in Ancient DNA from Iron Age Europeans

The most common, life-threatening autosomal recessive disease of Europeans and Euro-Americans, cystic fibrosis (CF), occurs predominately in patients with the F508del mutation.1 Although F508del is currently detectable as a single allele in 1/30-1/40 Europeans2-4 and Euro-Americans,5 it has not been determined what heterozygote selective advantage(s) might account for its relatively high prevalence. Indirect evidence6 suggests that this mutation was present in Brittany at least 3000 years ago, but no direct analyses of ancient DNA have been reported to identify F508del and clarify its frequency in prehistoric inhabitants of Europe. Here we show that F508del was present in 3 of 32 Iron Age inhabitants of Austria from whom DNA could be recovered from molar teeth using procedures that fulfill authenticity criteria.7 Because these individuals, who were buried in cemeteries along the Danube river, were shown by radiocarbon dating of isolated bone collagen to have lived there during 544-255 BC, this indicates that the F508del mutation is definitely more than 2000 years old and that CF (the disease) was present among them. More generally, the apparent enrichment of this Iron Age population in F508del suggests an evolutionary advantage in their environment that can be investigated by interdisciplinary strategies of paleoepidemiology

A new PBTK modelling method based on multi interaction paradigm

International audienceOne of the problems not clearly solved nowadays in food toxicology, is the evaluation of internal exposure to natural chemical contaminants. The worst solution is based on the concept that internal and external exposure are of the same level. One can go through this concept by considering biomarkers which can be used to calculate the inner contamination. Another solution is the use of a PBTK simulation method (Physiologically-Based Toxico-Kinetics). This method allows the knowledge of distribution of the contaminant over all the organs and their evolution in time. Unfortunately, the modeling and simulation processes are very hard to implement and use. The use of a technology issued from virtual modeling shows a good opportunity of developing a user interface easy to use for biologists. This interface is based on the multi interactions paradigm which is an evolution of the multi agent simulations

CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain

Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations

An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed

Approche moleculaire du gene de la mucoviscidose

Available from INIST (FR), Document Supply Service, under shelf-number : T 82573 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

La mucoviscidose (du gène à la correction des mutations ?)

La mucoviscidose est la maladie génétique la plus répandue dans les populations d origine caucasienne. En Bretagne, quatre mutations surie gène CFTR sont retrouvées préférentiellement : deltaF508, G551D, 1O78deIT et W846X(2), ainsi que le polymorphisme Il 027T associé en cis à la mutation deltaF5O8. L apparition de ces mutations dans la région a été calculée à partir des fréquences alléliques de microsatellites autour du gène CFTR. Nous estimons qu elles sont apparues il y a respectivement 3000, 1200, 1000 et 600 ans, et 600 ans pour le polymorphisme I1027T. Ces calculs permettent de situer l apparition de ces mutations dans un contexte historique. Chez un certain nombre de patients, les bases génétiques de la mucoviscidose restent indéterminées. Les domaines conservés du promoteur de CFTR et le domaine 3 -UTR ont été séquencés dans une cohorte présentant un génotype ((atypique . Cette analyse et celle des exons des gènes codant le canal sodique ENaC, n a permis d identifier que des SNPs déjà décrits dans la littérature. Les bases génétiques de la maladie chez ces patients restent donc inconnues. Depuis près de dix ans, des stratégies visant à corriger le défaut génétique sur le gène CFTR ont émergé. La technique SFHR a été utilisée pour corriger la mutation deltaF508 sur une lignée cellulaire épithéliale bronchjque humaine. Plus de huit semaines après la transfection, une correction de la mutation était encore observée, mais le taux de correction restait faible (<0,5%). Il est désormais indispensable de déterminer les mécanismes de ce système pour améliorer l efficacité de la correction génique par SFHR, nécessaire à la mise en place d une stratégie thérapeutique.Cystic fibrosis (CF) is the most common genetic disorder affecting children in Caucasian populations. In Brittany (western France), tour mutations within the CFTR gene are predominantly found: deltaF5O8, G551D, lO78delT and W846X(2), as well as the l1027T polymorphism, which is associated in cis with the deltaF5O8 mutation. The age of the most recent common ancestor for each mutation was estimated by studying allelic frequencies of microsatellites surrounding the gene. Our calculations indicated that the mutations respectively appeared 3,000, 1,200, 1,000 and 600 years ago; and 600 years ago for the polymorphism. These estimates give historical perspectives that can be associated with population migrations. In a number of patients, the genetic basis of CF are still unknown. Evolutionary conserved regions within the promoter region as well as the 3 -UTR were systematically sequenced in a cohort of atypical CF patients. This analysis, as well as the study of the exons within the genes encoding ENaC subunits only indicated previously reported SNPs. Genetic basis of the disease then remain unspecified in these patients. For about ten years, CFTR gene repair strategies have emerged. SFHR-mediated gene correction of the deItaF5O8-CFTR mutation was evaluated in a human bronchial epithelial cell line. Although conversion was observed up to eight weeks following the initial transfection, the repair percentage was relatively low (<0.5%). The precise mechanisms must now be precisely identified to be able to improve SFHR-medited gene repair and ultimately use it as a therapeutic approach for CF treatment.BREST-BU Médecine-Odontologie (290192102) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Analyse de la lignée neuronale hippocampique HT22 exposée aux ions métalliques

Les maladies neurodégénératives sont des maladies complexes dont la majorité des cas sont sporadiques et suggèrent une étiologie multifactorielle. Des études épidémiologiques ont montré que certains métaux pourraient représenter un facteur de risque. Ce travail présente deux objectifs principaux : - mesurer l entrée et l'accumulation cellulaire des métaux - évaluer leurs effets sur la production d espèces réactives de l oxygène et sur les variations de taux de transcrits de gènes d intérêts. Les métaux étudiés pénètrent dans les cellules HT22 et leur accumulation augmente globalement en fonction du temps et de la concentration d exposition. Les métaux les plus abondants dans l organisme (cuivre, fer, manganèse, zinc) semblent s accumuler en plus grande quantité dans les cellules HT22 que d autres moins communs (cobalt, nickel) et que des métaux non essentiels (aluminium, plomb et cadmium). Des études de co-exposition révèlent que le manganèse est capable de diminuer l accumulation du fer et du cuivre, indiquant l existence d une voie commune d entrée pour ces ions métalliques. De plus, parmi les métaux analysés, seul le manganèse induit une augmentation de la quantité d espèces réactives de l oxygène et provoque l augmentation du taux de transcrits de nombreux gènes, incluant des gènes de l homéostasie des métaux et en particulier ceux du fer, des gènes régulant le stress oxydatif comme les superoxydes dismutases 1 et 2 et la catalase, et même du gène codant l amyloïd precursor protein. Ces résultats corroborent ainsi d autres études qui mettent en évidence un lien entre le manganèse et le développement de certaines maladies neurodégénératives.The neurodegenerative diseases are complex diseases which majority of cases are sporadic and suggest multifactorial etiology. Epidemiologic studies have shown that some metals are potential risk factors. This work presents two main objectives: - to measure the cellular uptake and accumulation of metals - to assess their effects on reactive oxygen species production and on transcript level of genes of interest. All metals used in the study enter in HT22 cells except aluminiun and cadmium which seem not detectable with the probes employed here. Spectrometric measurements indicate that metals accumulation globally increases with time and concentration exposure. Most important metals for the organism (iron, zinc, copper, manganese) accumulate more than less common (cobalt, nickel) and non-essential metals (aluminium, lead, cadmium). Co-exposure experiments show that manganese inhibits iron and copper accumulation, indicating a common pathway for these metal ions. Moreover, among all metals tested, only the manganese induces an increase of the level of reactive oxygen species. Using these concentrations, particularly manganese induces an increase in transcript level of many genes, including genes of metal homeostasis, especially those of iron genes regulating the oxydative stress, and the gene coding the amyloïdal precursor protein. These results corroborate other studies showing a link between manganese and the development of some neurodegenerative diseases.BREST-BU Médecine-Odontologie (290192102) / SudocSudocFranceF

Optimisation et limites du transfert de gènes par voie intraveineuse à l'aide de vecteurs non viraux de type lipides cationiques chez la souris Swiss (application à une thérapie génique de la mucoviscidose (doctorat : sciences de la vie et de la santé))

BREST-BU Médecine-Odontologie (290192102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Expressions phénotypiques au cours du développement humain de deux pathologies (la mucoviscidose et les anomalies de migration neuronale)

L expression phénotypique de protéines impliquées dans la physiopathologie de maladies géniques a été étudiée dans deux types de pathologies l une non malformative, l autre malformative. Les anomalies morphologiques foetales et l expression de la protéine CFTR ont été recherchées dans la mucoviscidose. Au niveau broncho-pulmonaire, un retard d expression de 3 semaines, une intensité maximale et une topographie diffuse précoce puis une décroissance spatiale et en intensité de signal ont été trouvées. Des signes de destruction de l appareil excréteur génital male ont été mis en évidence parallèlement à l expression précoce de la protéine, persistant mais ne comportant pas de gradient d intensité à l inverse des tissus adultes. Il apparaît un renforcement apical de l expression parallèlement à la différentiation tissulaire épithéliale. L expression précoce et intense de la protéine CFTR, sa cinétique, sa localisation cytoplasmique puis avec renforcement apical évoquent un rôle particulier pendant lé développement. Les lissencéphalies de type 1 forment un groupe hétérogène d anomalies de migration neuronale. L expression des protéines de liaison du calcium (calrétinine, calbindine, parvalbumine) qui colocalisent avec l acide gamma-aminobutyrique (GABA) au niveau des interneurones a été étudiée dans trois syndromes lissencephaliques par atteinte des gènes LISI, DCX, ARX. Ceci a permis de mettre de comparer les anomalies de la migration tangentielle et de la migration radiaire confirmant l hypothèse d une atteinte différente des interneurones selon le gène incriminé. Ces altérations établissent un rôle majeur aux interneurones lors des étapes critiques du développement.The morphological analysis and the expression of several proteins have been performed in two different genetic disorders, the former without malformation the later with malformation. The pattern of expression of the CFTR protein has been analysed in the respiratory tract and in the male genital excretory tract. The CFTR expression appeared with a 3- week delay in the respiratory tract; had a different pattern of spatial expression according to the tissue differentiation. In the genital tract, the expression of die protein remained diffuse without any gradient of intensity conversely to the adult pattern. The early and diffuse localisation of the CFTR protein raises the question of its role during development. Neuronal migration anomalies form a large group in which type 1 lissencephaly group of disorders belongs. We studied the calcium binding proteins to analyse the GABAergic interneurons in three agyric/pachygyric syndromes dues to defects in the LISJ, DCX, ARX genes. GABAergic neurons migration was disturbed in a particular pattern in each case. These data argue for impairment in both the radial telencephalic migration and the tangential telencephalic migration in these different disorders.BREST-BU Médecine-Odontologie (290192102) / SudocSudocFranceF

Epidémiologie moléculaire de la mucoviscidose en Bretagne

BREST-BU Médecine-Odontologie (290192102) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF