Sudden death: ethical and legal problems of post-mortem forensic genetic testing for hereditary cardiac diseases.

Hereditary non-structural diseases such as catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT, and the Brugada syndrome as well as structural disease such as hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) cause a significant percentage of sudden cardiac deaths in the young. In these cases, genetic testing can be useful and does not require proxy consent if it is carried out at the request of judicial authorities as part of a forensic death investigation. Mutations in several genes are implicated in arrhythmic syndromes, including SCN5A, KCNQ1, KCNH2, RyR2, and genes causing HCM. If the victim's test is positive, this information is important for relatives who might be themselves at risk of carrying the disease-causing mutation. There is no consensus about how professionals should proceed in this context. This article discusses the ethical and legal arguments in favour of and against three options: genetic testing of the deceased victim only; counselling of relatives before testing the victim; counselling restricted to relatives of victims who tested positive for mutations of serious and preventable diseases. Legal cases are mentioned that pertain to the duty of geneticists and other physicians to warn relatives. Although the claim for a legal duty is tenuous, recent publications and guidelines suggest that geneticists and others involved in the multidisciplinary approach of sudden death (SD) cases may, nevertheless, have an ethical duty to inform relatives of SD victims. Several practical problems remain pertaining to the costs of testing, the counselling and to the need to obtain permission of judicial authorities.

A systematic and feasible method for computing nuclear contributions to electrical properties of polyatomic molecules

An analytic method to evaluate nuclear contributions to electrical properties of polyatomic molecules is presented. Such contributions control changes induced by an electric field on equilibrium geometry (nuclear relaxation contribution) and vibrational motion (vibrational contribution) of a molecular system. Expressions to compute the nuclear contributions have been derived from a power series expansion of the potential energy. These contributions to the electrical properties are given in terms of energy derivatives with respect to normal coordinates, electric field intensity or both. Only one calculation of such derivatives at the field-free equilibrium geometry is required. To show the useful efficiency of the analytical evaluation of electrical properties (the so-called AEEP method), results for calculations on water and pyridine at the SCF/TZ2P and the MP2/TZ2P levels of theory are reported. The results obtained are compared with previous theoretical calculations and with experimental values

Natural genetic variation of root system architecture from Arabidopsis to Brachypodium: towards adaptive value.

Root system architecture is a trait that displays considerable plasticity because of its sensitivity to environmental stimuli. Nevertheless, to a significant degree it is genetically constrained as suggested by surveys of its natural genetic variation. A few regulators of root system architecture have been isolated as quantitative trait loci through the natural variation approach in the dicotyledon model, Arabidopsis. This provides proof of principle that allelic variation for root system architecture traits exists, is genetically tractable, and might be exploited for crop breeding. Beyond Arabidopsis, Brachypodium could serve as both a credible and experimentally accessible model for root system architecture variation in monocotyledons, as suggested by first glimpses of the different root morphologies of Brachypodium accessions. Whether a direct knowledge transfer gained from molecular model system studies will work in practice remains unclear however, because of a lack of comprehensive understanding of root system physiology in the native context. For instance, apart from a few notable exceptions, the adaptive value of genetic variation in root system modulators is unknown. Future studies should thus aim at comprehensive characterization of the role of genetic players in root system architecture variation by taking into account the native environmental conditions, in particular soil characteristics.

Genetic and phenotypic population divergence on a microgeographic scale in brown trout.

Salmonid populations of many rivers are rapidly declining. One possible explanation is that habitat fragmentation increases genetic drift and reduces the populations' potential to adapt to changing environmental conditions. We measured the genetic and eco-morphological diversity of brown trout (Salmo trutta) in a Swiss stream system, using multivariate statistics and Bayesian clustering. We found large genetic and phenotypic variation within only 40 km of stream length. Eighty-eight percent of all pairwise F(ST) comparisons and 50% of the population comparisons in body shape were significant. High success rates of population assignment tests confirmed the distinctiveness of populations in both genotype and phenotype. Spatial analysis revealed that divergence increased with waterway distance, the number of weirs, and stretches of poor habitat between sampling locations, but effects of isolation-by-distance and habitat fragmentation could not be fully disentangled. Stocking intensity varied between streams but did not appear to erode genetic diversity within populations. A lack of association between phenotypic and genetic divergence points to a role of local adaptation or phenotypically plastic responses to habitat heterogeneity. Indeed, body shape could be largely explained by topographic stream slope, and variation in overall phenotype matched the flow regimes of the respective habitats.

Fishery-induced selection on an Alpine whitefish: quantifying genetic and environmental effects on individual growth rate

Size-selective fishing, environmental changes and reproductive strategies are expected to affect life-history traits such as the individual growth rate. The relative contribution of these factors is not clear, particularly whether size-selective fishing can have a substantial impact on the genetics and hence on the evolution of individual growth rates in wild populations. We analysed a 25-year monitoring survey of an isolated population of the Alpine whitefish Coregonus palaea. We determined the selection differentials on growth rate, the actual change of growth rate over time and indicators of reproductive strategies that may potentially change over time. The selection differential can be reliably estimated in our study population because almost all the fish are harvested within their first years of life, i.e. few fish escape fishing mortality. We found a marked decline in average adult growth rate over the 25 years and a significant selection differential for adult growth, but no evidence for any linear change in reproductive strategies over time. Assuming that the heritability of growth in this whitefish corresponds to what was found in other salmonids, about a third of the observed decline in growth rate would be linked to fishery-induced evolution. Size-selective fishing seems to affect substantially the genetics of individual growth in our study population.

Genetic structure of the genus Leptospira by multilocus enzyme electrophoresis

Thirty strains from the 11 species of the genus Leptospira were studied by multilocus enzyme electrophoresis at 12 enzyme loci, all of which were polymorphic. The mean number of alleles per locus was 6.5. Twenty-five electrophoretic types were distinguished. Grouping of the strains by cluster analysis was in general agreement with species delineation as determined by DNA-DNA hybridization, except for the strains of Leptospira meyeri and Leptospira inadai, which were scattered throughout the genus, reflecting previously recognized taxonomic uncertainties. Analysis of the clonality within Leptospira interrogans sensu stricto indicated that this population was relatively heterogeneous and a lack of gene linkage disequilibrium could not be excluded. There was a genetic discrimination between the pathogenic species and the saprophytic ones. The phenotypically intermediate species (L. inadai and Leptospira fainei) were also genetically separated and were probably closer to the saprophytes than to the pathogens.

Highly parallel genetic approaches in Mendelian and complex diseases

The recent advance in high-throughput sequencing and genotyping protocols allows rapid investigation of Mendelian and complex diseases on a scale not previously been possible. In my thesis research I took advantage of these modern techniques to study retinitis pigmentosa (RP), a rare inherited disease characterized by progressive loss of photoreceptors and leading to blindness; and hypertension, a common condition affecting 30% of the adult population. Firstly, I compared the performance of different next generation sequencing (NGS) platforms in the sequencing of the RP-linked gene PRPF31. The gene contained a mutation in an intronic repetitive element, which presented difficulties for both classic sequencing methods and NGS. We showed that all NGS platforms are powerful tools to identify rare and common DNA variants, also in case of more complex sequences. Moreover, we evaluated the features of different NGS platforms that are important in re-sequencing projects. The main focus of my thesis was then to investigate the involvement of pre-mRNA splicing factors in autosomal dominant RP (adRP). I screened 5 candidate genes in a large cohort of patients by using long-range PCR as enrichment step, followed by NGS. We tested two different approaches: in one, all target PCRs from all patients were pooled and sequenced as a single DNA library; in the other, PCRs from each patient were separated within the pool by DNA barcodes. The first solution was more cost-effective, while the second one allowed obtaining faster and more accurate results, but overall they both proved to be effective strategies for gene screenings in many samples. We could in fact identify novel missense mutations in the SNRNP200 gene, encoding an essential RNA helicase for splicing catalysis. Interestingly, one of these mutations showed incomplete penetrance in one family with adRP. Thus, we started to study the possible molecular causes underlying phenotypic differences between asymptomatic and affected members of this family. For the study of hypertension, I joined a European consortium to perform genome-wide association studies (GWAS). Thanks to the use of very informative genotyping arrays and of phenotipically well-characterized cohorts, we could identify a novel susceptibility locus for hypertension in the promoter region of the endothelial nitric oxide synthase gene (NOS3). Moreover, we have proven the direct causality of the associated SNP using three different methods: 1) targeted resequencing, 2) luciferase assay, and 3) population study. - Le récent progrès dans le Séquençage à haut Débit et les protocoles de génotypage a permis une plus vaste et rapide étude des maladies mendéliennes et multifactorielles à une échelle encore jamais atteinte. Durant ma thèse de recherche, j'ai utilisé ces nouvelles techniques de séquençage afin d'étudier la retinite pigmentale (RP), une maladie héréditaire rare caractérisée par une perte progressive des photorécepteurs de l'oeil qui entraine la cécité; et l'hypertension, une maladie commune touchant 30% de la population adulte. Tout d'abord, j'ai effectué une comparaison des performances de différentes plateformes de séquençage NGS (Next Generation Sequencing) lors du séquençage de PRPF31, un gène lié à RP. Ce gène contenait une mutation dans un élément répétable intronique, qui présentait des difficultés de séquençage avec la méthode classique et les NGS. Nous avons montré que les plateformes de NGS analysées sont des outils très puissants pour identifier des variations de l'ADN rares ou communes et aussi dans le cas de séquences complexes. De plus, nous avons exploré les caractéristiques des différentes plateformes NGS qui sont importantes dans les projets de re-séquençage. L'objectif principal de ma thèse a été ensuite d'examiner l'effet des facteurs d'épissage de pre-ARNm dans une forme autosomale dominante de RP (adRP). Un screening de 5 gènes candidats issus d'une large cohorte de patients a été effectué en utilisant la long-range PCR comme étape d'enrichissement, suivie par séquençage avec NGS. Nous avons testé deux approches différentes : dans la première, toutes les cibles PCRs de tous les patients ont été regroupées et séquencées comme une bibliothèque d'ADN unique; dans la seconde, les PCRs de chaque patient ont été séparées par code barres d'ADN. La première solution a été la plus économique, tandis que la seconde a permis d'obtenir des résultats plus rapides et précis. Dans l'ensemble, ces deux stratégies se sont démontrées efficaces pour le screening de gènes issus de divers échantillons. Nous avons pu identifier des nouvelles mutations faux-sens dans le gène SNRNP200, une hélicase ayant une fonction essentielle dans l'épissage. Il est intéressant de noter qu'une des ces mutations montre une pénétrance incomplète dans une famille atteinte d'adRP. Ainsi, nous avons commencé une étude sur les causes moléculaires entrainant des différences phénotypiques entre membres affectés et asymptomatiques de cette famille. Lors de l'étude de l'hypertension, j'ai rejoint un consortium européen pour réaliser une étude d'association Pangénomique ou genome-wide association study Grâce à l'utilisation de tableaux de génotypage très informatifs et de cohortes extrêmement bien caractérisées au niveau phénotypique, un nouveau locus lié à l'hypertension a été identifié dans la région promotrice du gène endothélial nitric oxide sinthase (NOS3). Par ailleurs, nous avons prouvé la cause directe du SNP associé au moyen de trois méthodes différentes: i) en reséquençant la cible avec NGS, ii) avec des essais à la luciférase et iii) une étude de population.

Anharmonicity contributions to the vibrational second hyperpolarizability of conjugated oligomers

Restricted Hartree-Fock 6-31G calculations of electrical and mechanical anharmonicity contributions to the longitudinal vibrational second hyperpolarizability have been carried out for eight homologous series of conjugated oligomers - polyacetylene, polyyne, polydiacetylene, polybutatriene, polycumulene, polysilane, polymethineimine, and polypyrrole. To draw conclusions about the limiting infinite polymer behavior, chains containing up to 12 heavy atoms along the conjugated backbone were considered. In general, the vibrational hyperpolarizabilities are substantial in comparison with their static electronic counterparts for the dc-Kerr and degenerate four-wave mixing processes (as well as for static fields) but not for electric field-induced second harmonic generation or third harmonic generation. Anharmonicity terms due to nuclear relaxation are important for the dc-Kerr effect (and for the static hyperpolarizability) in the σ-conjugated polymer, polysilane, as well as the nonplanar π systems polymethineimine and polypyrrole. Restricting polypyrrole to be planar, as it is in the crystal phase, causes these anharmonic terms to become negligible. When the same restriction is applied to polymethineimine the effect is reduced but remains quantitatively significant due to the first-order contribution. We conclude that anharmonicity associated with nuclear relaxation can be ignored, for semiquantitative purposes, in planar π-conjugated polymers. The role of zero-point vibrational averaging remains to be evaluated

The mapping of the local contributions of Fermi and Coulomb correlation into intracule and extracule density distributions

The contributions of the correlated and uncorrelated components of the electron-pair density to atomic and molecular intracule I(r) and extracule E(R) densities and its Laplacian functions ∇2I(r) and ∇2E(R) are analyzed at the Hartree-Fock (HF) and configuration interaction (CI) levels of theory. The topologies of the uncorrelated components of these functions can be rationalized in terms of the corresponding one-electron densities. In contrast, by analyzing the correlated components of I(r) and E(R), namely, IC(r) and EC(R), the effect of electron Fermi and Coulomb correlation can be assessed at the HF and CI levels of theory. Moreover, the contribution of Coulomb correlation can be isolated by means of difference maps between IC(r) and EC(R) distributions calculated at the two levels of theory. As application examples, the He, Ne, and Ar atomic series, the C2-2, N2, O2+2 molecular series, and the C2H4 molecule have been investigated. For these atoms and molecules, it is found that Fermi correlation accounts for the main characteristics of IC(r) and EC(R), with Coulomb correlation increasing slightly the locality of these functions at the CI level of theory. Furthermore, IC(r), EC(R), and the associated Laplacian functions, reveal the short-ranged nature and high isotropy of Fermi and Coulomb correlation in atoms and molecules

Simple finite field method for calculation of static and dynamic vibrational hyperpolarizabilities: curvature contributions

In the static field limit, the vibrational hyperpolarizability consists of two contributions due to: (1) the shift in the equilibrium geometry (known as nuclear relaxation), and (2) the change in the shape of the potential energy surface (known as curvature). Simple finite field methods have previously been developed for evaluating these static field contributions and also for determining the effect of nuclear relaxation on dynamic vibrational hyperpolarizabilities in the infinite frequency approximation. In this paper the finite field approach is extended to include, within the infinite frequency approximation, the effect of curvature on the major dynamic nonlinear optical processes

Density functional energy decomposition into one- and two-atom contributions

The present work provides a generalization of Mayer's energy decomposition for the density-functional theory (DFT) case. It is shown that one- and two-atom Hartree-Fock energy components in Mayer's approach can be represented as an action of a one-atom potential VA on a one-atom density ρ A or ρ B. To treat the exchange-correlation term in the DFT energy expression in a similar way, the exchange-correlation energy density per electron is expanded into a linear combination of basis functions. Calculations carried out for a number of density functionals demonstrate that the DFT and Hartree-Fock two-atom energies agree to a reasonable extent with each other. The two-atom energies for strong covalent bonds are within the range of typical bond dissociation energies and are therefore a convenient computational tool for assessment of individual bond strength in polyatomic molecules. For nonspecific nonbonding interactions, the two-atom energies are low. They can be either repulsive or slightly attractive, but the DFT results more frequently yield small attractive values compared to the Hartree-Fock case. The hydrogen bond in the water dimer is calculated to be between the strong covalent and nonbonding interactions on the energy scale

Electronic and vibrational contributions to first hyperpolarizability of donor-acceptor-substituted azobenzene

In this study we report on the electronic and vibrational (hyper)polarizabilities of donor–acceptorsubstituted azobenzene. It is observed that both electronic and vibrational contributions to the electricdipole first hyperpolarizability of investigated photoactive molecule substantially depend on the conformation. The contributions to the nuclear relaxation first hyperpolarizability are found to be quite important in the case of two considered isomers (cis and trans). Although the double-harmonic term is found to be the largest in terms of magnitude, it is shown that the total value of the nuclear relaxation contribution to vibrational first hyperpolarizability is a result of subtle interplay of higher-order contributions. As a part of the study, we also assess the performance of long-range-corrected densityfunctional theory in determining vibrational contributions to electric dipole (hyper)polarizabilities. In most cases, the applied long-range-corrected exchange correlation potentials amend the drawbacks of their conventional counterparts

Protective and Aggravating Effects of Nlrp3 Inflammasome Activation in IBD Models: Influence of Genetic and Environmental Factors.

Background: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1β and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3(-/-) mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3(-/-) mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. Methods: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we investigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3(-/-) mice, as well as by antibiotic treatment. Results: Nlrp3(-/-) mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3(-/-) mice in steady state conditions. Interestingly, after cohousing, Nlrp3(-/-) mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3(-/-) mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. Conclusions: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3(-/-) mice. This observation may - at least in part - explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD.

Low dispersing species : population genetic processes in space and time : the genus "Trochulus" (Gastropoda: Hygromiidae) as a case study

RÉSUMÉ Une espèce est rarement composée d'une population unique. Parce que les individus ont des capacités de dispersion limitées et que les paysages sont des mosaïques d'habitats, la plupart des espèces sont plutôt composées de sous-populations connectées par la migration. Cette variation spatiale influence directement la distribution de la variabilité génétique dans et entre les populations. Durant ce travail, nous avons abordé certains des processus populationnels qui ont joué un rôle supposé dans l'apparition de nouvelles espèces au sein du genre Trochulus. Plus précisément, nous avons tenté d'évaluer les impacts respectifs de l'isolement passé (facteurs historiques) et présent (facteurs locaux). Nous avons d'abord pu montrer que les faibles capacités de dispersion des escargots terrestres ont directement influencé leur histoire évolutive à toutes les échelles spatiales et temporelles. En réduisant l'effet homogénéisant de la migration, une faible dispersion maintient dans les populations les traces génétiques d'évènements passés. A l'échelle de la distribution globale de Trochulus villosus, ces traces ont permis de reconstruire une histoire faite d'isolements et d'expansions de populations. En combinant des données génétiques avec une modélisation de la niche climatique passée, il a été possible de proposer un scénario significativement meilleur que toutes les hypothèses alternatives que nous avons testées. A l'échelle locale par contre, l'héritage historique est difficile à distinguer de la dynamique actuelle. Ce fut le cas des lignées mitochondriales du complexe sericeus-hispidus : les deux principales lignées étaient phylogénétiquement éloignées, avaient eu des démographies passées différentes et corrélaient avec des différences morphologiques. D'un autre côté, le flux de gène nucléaire était fort, contredisant l'idée de deux espèces cryptiques isolées reproductivement. Pour pouvoir conclure à la présence ou non de deux espèces, il nous a manqué des informations locales sur la dynamique des populations et les conditions écologiques que l'on trouve dans la région d'étude. Enfin, nous avons pu souligner que la connectivité entre populations d'escargots est soumise à la qualité des habitats et à leur organisation spatiale. Les escargots sont dépendants d'un habitat et s'y adaptent, comme l'indiquent la présence de «poils » uniquement sur la coquille d'espèces vivant dans des habitats humides ou la corrélation entre morphologie et habitat au sein du complexe sericeus-hispidus. Logiquement donc, les escargots migrent préférentiellement au travers d'habitats favorables comme l'a montré la réduction de flux de gènes au travers des prairies chez T. villosus (une espèce forestière). De ces données, nous pouvons supposer que les populations d'escargots en particulier, et des espèces à faible dispersion en général, ont de fortes chances d'être affectées par les changements climatiques, avec de probables implications pour leurs histoires évolutives. SUMMARY : Species rarely consists in a single population. Because individuals have limited dispersal abilities, because landscapes are habitat patchworks, most species are made of several subpopulations connected by migration. This spatial variation has consequences on the distribution of genetic diversity within and between populations, creating a structure among the populations. During the present work, we investigated some of the population processes assumed to have played an important role on the speciation within the genus Trochulus. More specifically, we questioned the respective impacts of past (historical factors) or present (local factors) population isolations. We first could show that the poor dispersal abilities of land snails have had profound impacts on their evolutionary histories at all spatial and temporal scales. Low dispersal maintains a strong signature of past events in the populations by minimising the homogenising effects of geneflow. At the scale of Trochulus villosus global distribution, they allowed to retrieve the detailed history of this species population isolations and expansions. Combining a large genetic dataset with paleo-climatic niche modelling ended up with a historical scenario significantly better than all traditional alternatives we tested. At local scale on the contrary, past events become difficult to tease apart from ongoing processes. This was the case for the divergent mitochondria) lineages within the sericeus-hispidus complex: the two principal lineages appeared to be phylogenetically distant, to have experienced different demographic histories and to correlate with morphological differences. On the other hand, nuclear (present day) geneflow was high, contradicting the idea of two reproductively isolated cryptic species. Information on the local population dynamics and environmental conditions are lacking to be able to decide whether past isolation has indeed resulted here in new species. Finally, we emphasised the importance of the habitat types present in a landscape as well as their spatial organisation for the population connectivity of land snails. These species are tightly dependent on a habitat and adapt to it as shown by thé occurrence of hair-like structures only in species living in humid environments or by the correlation between shell morphology and habitat in the sericeus-hispidus complex. As a result, land snails preferentially migrate through favourable habitats: Trochulus villosus, a forest species, had its geneflow significantly reduced across meadows. From these data, we can hypothesise that the populations of land snails in particular and of low dispersing species in general are likely to be strongly affected by the ongoing climate changes, with potential major consequences on their evolutionary histories.