Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

RYR1 mutations are the most common cause of structural congenital myopathies and may exhibit both dominant and recessive inheritance. Histopathological findings are variable and include central cores, multi-minicores, type 1 predominance/ uniformity, fibre type disproportion, increased internal nucleation and fatty and connective tissue. Until recently, diagnostic RYR1 sequencing was limited to mutational hotspots due to the large size of the gene. Since the introduction of full RYR1 sequencing in 2007 we have detected pathogenic mutations in 77 families: 39 had dominant inheritance and 38 recessive inheritance. In some cases with presumably recessive inheritance, only one heterozygous mutation inherited from an asymptomatic parent was identified. Of 28 dominant mutations, 6 were novel; 37 of the 59 recessive mutations were also novel. Dominant mutations were more frequently in recognized hotspot regions, while recessive mutations were distributed throughout the coding sequence. Dominant mutations were predominantly missense, whereas recessive mutations included many nonsense and splice mutations expected to result in reduced RyR1 protein. There was wide clinical variability in patients with both dominant and recessive inheritance. As a group, those with dominant mutations were generally more mildly affected than those with recessive inheritance, who had earlier onset and were weaker with more functional limitations. Extraocular muscle involvement was almost exclusively observed in the recessive group. Bulbar involvement was also more prominent in this group, resulting in a larger number requiring gastrostomy insertion. In conclusion, genomic sequencing of the entire RYR1 leads to the detection of many novel mutations, but may miss large genetic rearrangements in some cases. Assigning pathogenicity to novel mutations is often difficult and interpretation of genetic results in the context of clinical, histological and, increasingly, muscle MRI findings is essential.

Diagnostic interview for genetic studies (DIGS): inter-rater and test-retest reliability of the French version.

The National Institute of Mental Health developed the semi-structured Diagnostic Interview for Genetic Studies (DIGS) for the assessment of major mood and psychotic disorders and their spectrum conditions. The DIGS was translated into French in a collaborative effort of investigators from sites in France and Switzerland. Inter-rater and test-retest reliability of the French version have been established in a clinical sample in Lausanne. Excellent inter-rater reliability was found for schizophrenia, bipolar disorder, major depression, and unipolar schizoaffective disorder while fair inter-rater reliability was demonstrated for bipolar schizoaffective disorder. Using a six-week test-retest interval, reliability for all diagnoses was found to be fair to good with the exception of bipolar schizoaffective disorder. The lower test-retest reliability was the result of a relatively long test-retest interval that favored incomplete symptom recall. In order to increase reliability for lifetime diagnoses in persons not currently affected, best-estimate procedures using additional sources of diagnostic information such as medical records and reports from relatives should supplement DIGS information in family-genetic studies. Within such a procedure, the DIGS appears to be a useful part of data collection for genetic studies on major mood disorders and schizophrenia in French-speaking populations.

Genetic Variability and Microdistribution of Triatoma infestans Genotypes and Trypanosoma cruzi Clones in Arequipa Region (Peru)

The genetic variability of Triatoma infestans and Trypanosoma cruzi populations was studied by isoenzyme analysis in two distinct areas of Arequipa province (Peru); one, Santa Rita de Siguas, being an endemic area for Chagas' disease, the second, Arequipa, recently infected. Analysis of T. infestans genetic variability indicates, (i) temporal stability of genotypes found in Santa Rita de Siguas, (ii) high genetic differences between Arequipa and Santa Rita de Siguas populations suggesting minor contact between them, (iii) multiple origin of the T. infestans population in Arequipa, and (iv) poor dispersal capacity of T. infestans: the panmictic unit could be reduce to a house. Parasite isoenzyme analysis was performed in 29 Peruvian stocks of T. cruzi, mainly isolated from bugs taken in a single locality, Santa Rita de Siguas. The results show, (i) a high genetic polymorphism, (ii) nine different multilocus genotypes were detected and clustered in two different clades, (iii) most of the parasite isolates pertained to one of the clade and were genetically similar to those analyzed 12 years before. This sample allowed the study of the mating system of T. cruzi in strict sympatic conditions and gave more strength to the hypothesis of the clonal structure of T. cruzi populations

Genetic differentiation in two European tree frog (Hyla arborea) metapopulations in contrasted landscapes of Western Switzerland

The survival of threatened species as the European tree frog (Hyla arborea) is strongly dependent on the genetic variability within populations, as well as gene flow between them. In Switzerland, only two sectors in its western part still harbour metapopulations. The first is characterised by a very heterogeneous and urbanized landscape, while the second is characterised by a uninterrupted array of suitable habitats. In this study, six microsatellite loci were used to establish levels of genetic differentiation among the populations from the two different locations. The results show that the metapopulations have: (i) weak levels of genetic differentiation (FST within metapopulation ≈ 0.04), (ii) no difference in levels of genetic structuring between them, (iii) significant (p = 0.019) differences in terms of genetic diversity (Hs) and observed heterozygozity (Ho), the metapopulation located in a disturbed landscape showing lower values. Our results suggest that even if the dispersal of H. arborea among contiguous ponds seems to be efficient in areas of heterogeneous landscape, a loss of genetic diversity can occur.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

DNA degradation in avian faecal samples and feasibility of non-invasive genetic studies applied to threatened capercaillie populations

We evaluated the feasibility of using faeces as a non-invasively collected DNA source for the genetic study of an endangered bird population (capercaillie; Tetrao urogallus). We used a multitube approach, and for our panel of 11 microsatellites genotyping reliability was estimated at 98% with five repetitions. Experiments showed that free DNases in faecal material were the major cause of DNA degradation. Our results demonstrate that using avian faeces as a source of DNA, reliable microsatellite genotyping can be obtained with a reasonable number of PCR replicates.

Estimation of Genetic Divergence and Gene Flow between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Argentina

Allele frequencies at seven polymorphic loci controlling the synthesis of enzymes were analyzed in six populations of Culex pipiens L. and Cx. quinquefasciatus Say. Sampling sites were situated along a north-south line of about 2,000 km in Argentina. The predominant alleles at Mdh, Idh, Gpdh and Gpi loci presented similar frequencies in all the samples. Frequencies at the Pgm locus were similar for populations pairs sharing the same geographic area. The loci Cat and Hk-1 presented significant geographic variation. The latter showed a marked latitudinal cline, with a frequency for allele b ranging from 0.99 in the northernmost point to 0.04 in the southernmost one, a pattern that may be explained by natural selection (FST = 0.46; p < 0.0001) on heat sensitive alleles. The average value of FST (0.088) and Nm (61.12) indicated a high gene flow between adjacent populations. A high correlation was found between genetic and geographic distance (r = 0.83; p < 0.001). The highest genetic identity (IN = 0.988) corresponded to the geographically closest samples from the central area. In one of these localities Cx. quinquefasciatus was predominant and hybrid individuals were detected, while in the other, almost all the specimens were identified as Cx. pipiens. To verify the fertility between Cx. pipiens and Cx. quinquefasciatus from the northern- and southernmost populations, experimental crosses were performed. Viable egg rafts were obtained from both reciprocal crosses. Hatching ranged from 76.5 to 100%. The hybrid progenies were fertile through two subsequent generations

Genetic Variation among Natural and Laboratory Colony Populations of Lutzomyia longipalpis (Lutz & Neiva, 1912)(Diptera:Psychodidae) from Colombia

Genetic diversity among three field populations of Lutzomyia longipalpis in Colombia was studied using isozyme analysis. Study sites were as much as 598 km apart and included populations separated by the eastern Cordillera of the Andes. Genetic variability among populations, estimated by heterozygosity, was within values typical for insects in general (8.1%). Heterozygosity for field populations were compared with a laboratory colony from Colombia (Melgar colony) and were only slightly lower. These results suggest that establishment and long term maintenance of the Melgar colony has had little effect on the level of isozyme variability it carries. Genetic divergences between populations was evaluated using estimates of genetic distance. Genetic divergence among the three field populations was low (D=0.021), suggesting they represent local populations within a single species. Genetic distance between field populations and the Melgar colony was also low (D=0.016), suggesting that this colony population does not depart significantly from natural populations. Finally, comparisons were made between Colombian populations and colonies from Brazil and Costa Rica. Genetic distance values were high between Colombian and both Brazil and Costa Rica colony populations (D=0.199 and 0.098 respectively) providing additional support for our earlier report that populations from the three countries represent distinct species

Evolution, maintenance and ecological consequences of genetic caste determination in " Pogonomyrmex " harvester ants

Summary Division of labor between reproducers (queens) and helpers (workers) is the main characteristic of social insect societies and at the root of their ecological success. Kin selection models predict that phenotypic differences between queens and workers should result from environmental rather than from genetic differences. However, genetic effects on queen and worker differentiation were found in two populations-of Pogonomyrmex harvester ants. Each of the two populations is composed of two genetically distinct lineages. Queens (which can be of either lineage) generally mate with males of their own and of the alternate lineage and produce two types of female offspring, those fertilized by males of the queens' lineage which develop into queens and those fertilized by males of the alternate lineage which develop into workers. All four lineages were further suggested to be themselves of hybrid origin between-the species P: barbatus and P. rugosus, in which queens and workers do not differ genetically. In a first set of experiments, we tested if female caste determination (the differentiation into queens and workers) in the lineages was genetically hardwired and if it was associated with costs in terms of the ability to optimally allocate resources to the production of queens and workers. To this end we first mated queens of-two lineages to a single male. Queens mated to a male of the alternate lineage successfully raised worker offspring whereas queens mated to a male of their own lineage almost always failed to produce workers. This reveals that pure-lineage individuals have lost the ability to develop into workers. Second, we analyzed offspring produced by naturally mated queens. During the stage of colony founding when only workers are produced, naturally mated queens laid a high proportion of pure-lineage eggs but the large majority of these eggs failed to develop. As a consequence, the number of offspring produced by incipient colonies decreased linearly with the proportion of pure-lineage eggs laid by queens. Moreover, queens of the lineage most commonly represented in a given population produced more pure-lineage eggs, in line with the view that they mate randomly with the two types of males and indiscriminately use their sperm. Altogether these results predict frequency-dependent founding success for pairs of lineages because queens of the more common lineage will produce more pure-lineage eggs and their colonies be less successful during the stage of colony founding. To describe the distribution of populations characterized with genetic caste determination relative to the populations with environmental caste determination we genotyped queens and workers collected during a large survey of -additional populations. Genetic caste determination associated with pairs of interbreeding lineages was frequent and widespread in the studied range and we identified four additional lineages displaying genetic caste determination. Overall, there were thus eight highly differentiated lineages with genetic caste determination. These lineages always co-occurred in the same complementary lineage pairs. Three of the four lineage pairs appeared to have a common origin, while their relationship with the forth could not be resolved. The genetic survey also revealed that, in addition to being genetically isolated from one another, all eight lineages were genetically distinct from P. rugosus and P. barbatus, even when colonies of interbreeding lineages co-occurred with colonies of either putative parent at the same site. This raised the question of the mechanisms involved in the reproductive isolation between the lineages and the parental species and between the two lineages of a lineage pair. At a site where one lineage pair co-occurred with P. rugosus, we identified two pre-zygotic mechanisms (differences in timing for mating flights between P. rugosus and the lineage pair and assortative mating) and one post-zygotic mechanism (high levels of hybrid unviablility) which in combination may largely account for the reproductive isolation between the lineages and their parental species. The mechanisms accounting for the reproductive isolation between the two lineages of a lineage pair varied across lineage pairs. In one lineage pair, inter-lineage individuals exclusively occurred in the sterile worker caste, raising the possibility that inter-lineage eggs have completely lost the ability to develop into queens in this lineage pair and that there is thus no opportunity for gene flow. In each of the three remaining lineage pairs, inter-lineage queens were produced by a minority of colonies. In these lineage pairs, colonies headed by inter-lineage queens failed to grow sufficiently to produce reproductive individuals which may account for the reproductive isolation between co-occurring lineages in three lineage pairs. In conclusion, the results of this thesis show that genetic caste determination is costly but widespread in Pogonomyrmex harvester ants. Reproductive isolation among the lineages and between the lineages and the parental species as well as frequency-dependent founding success for co-occurring lineages may contribute to the persistence of this extraordinary system. Résumé La division du travail entre individus reproducteurs (les reines) et individus non-reproducteurs (ouvrières) représente la caractéristique principale des sociétés d'insectes et est à la base de leur succès écologique. Des modèles de sélection de parentèle prédisent que les différences phénotypiques entre reines et ouvrières devraient provenir d'effets environnementaux plutôt que de différences génétiques. Malgré ce fait, des effets génétiques sur la différentiation entre reines et ouvrières ont été montrés dans deux populations de fourmis moissonneuses du genre Pogonomyrmex. Chacune des deux populations est composée de deux lignées génétiquement distinctes. Les reines de chaque lignée s'accouplent en général avec des mâles de leur propre lignée ainsi qu'avec des mâles de l'autre lignée et produisent deux types d'oeufs, ceux qui sont fécondés par les mâles de leur propre lignée qui se développent en nouvelles reines et ceux qui sont fécondés par les mâles de l'autre lignée qui se développent en ouvrières. Il a été suggéré que les lignées sont elles-mêmes des hybrides entre les deux espèces P. barbatus et P. rugosus. Dans ces deux espèces, les reines et ouvrières ne sont pas génétiquement distinctes. Dans une première série d'expériences, nous avons testé si la détermination de la caste femelle (le développement en reine ou en ouvrière) est génétiquement rigide et si elle est associée à des coûts en terme de capacité à allouer de façon optimale les ressources pour la production de reines et d'ouvrières. Pour cela nous avons accouplé des reines de deux lignées avec un seul mâle. Les reines accouplées avec un mâle de l'autre lignée ont élevé de nouvelles ouvrières avec succès alors que les reines accouplées avec un mâle de leur propre lignée ont presque toujours échoué à produire des ouvrières. Ceci montre que les individus de lignée pure ont perdu la capacité de se développer en ouvrière. Deuxièmement, nous avons analysé la descendance de reines qui se sont accouplées naturellement. Durant le stade de fondation de la colonie, où seules des ouvrières sont élevées, les reines accouplées naturellement ont pondu une grande proportion d'oeufs de lignée pure mais la majorité de ces derniers ne se sont pas développés. En conséquence, le nombre de descendants produits par des colonies fondatrices diminuait linéairement avec la proportion des oeufs de lignée pure pondus par la reine en accord avec l'hypothèse que les reines s'accouplent au hasard avec les deux types de mâles et utilisent leur sperme aléatoirement. Dans l'ensemble; ces résultats prédisent un succès de fondation fréquence-dépendant pour les deux lignées, car les reines de la lignée la plus fréquente produiront .plus d'oeufs de lignée pure et leurs colonies auront moins de succès lors de la fondation de colonies par rapport aux colonies de la lignée la moins fréquente. Pour décrire la distribution des-populations caractérisées par une détermination génétique des castes par rapport aux populations caractérisées par une détermination environnementale des castes, nous avons génotypé des reines et des ouvrières qui ont été collectées lors d'une analyse de populations supplémentaires. La détermination génétique des castes associée à des croisements entre lignées est fréquente et largement répartie dans l'aire étudiée. Nous avons identifié quatre lignées supplémentaires, ayant une détermination génétique des castes, pour un total de huit lignées. Ces huit lignées forment quatre paires de lignées et on ne trouve jamais deux lignées de paires différentes, dans une population. Trois des quatre paires de lignées s'avèrent avoir une origine commune alors que leur relation avec la quatrième paire de lignées n'a pas pu être résolue. L'analyse génétique de populations supplémentaires a également révélé qu'en plus d'être génétiquement isolées les unes des autres, les huit lignées sont génétiquement distinctes de P. rugosus et P. barbatus même si les colonies d'une paire de lignées se trouvent en sympatrie avec l'une ou l'autre des espèces parentales. Ceci relève la question des mécanismes impliqués dans l'isolation reproductive entre les lignées et les espèces parentales ainsi qu'entre les deux lignées d'une paire. En étudiant un site où une paire de lignées se trouve en sympatrie avec P. rugosus, nous avons identifié deux mécanismes pré-zygotiques (des différences dans le timing du vol nuptial entre P. rugosus et les lignées et des accouplements assortis) ainsi qu'un mécanisme post-zygotique (un niveau élevé de non-viabilité des hybrides). En combinaison, ces mécanismes peuvent largement expliquer l'isolement reproductif entre les lignées et leurs espèces parentales. Les mécanismes contribuant à l'isolement reproductif entre les deux lignées d'une paire variaient entre paires de lignées. Dans une paire, les individus de génotype inter-lignée se trouvent uniquement dans la caste stérile des ouvrières, suggérant qu'il n'y a pas d'opportunité pour avoir du flux de gènes entre les deux lignées ce cette paire. Dans chacune des trois autres paires de lignées des nouvelles reines de génotype inter-lignée sont produites par une minorité de colonies. Par contre, les colonies avec une reine mère de génotype inter-lignée ne se développent pas suffisamment pour produire des individus reproducteurs. Ceci peut donc expliquer pourquoi il n'y a pas de flux de gènes entre les deux lignées de trois paires. En conclusion, les résultats de cette thèse montrent que la détermination génétique de la caste est coûteuse mais très répandue chez les fourmis. moissonneuses du genre Pogonomyrmex. L'isolement reproductif des lignées entre elles et avec les espèces parentales, ainsi qu'un succès de fondation fréquence-dépendant contribuent à la persistance de ce système extraordinaire.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.

Preliminary Evaluation of the Genetic Relatedness of Three Species of the Subgenus Dendromyia Theobald and Other Species of the Genus Wyeomyia Theobald (Diptera: Culicidae)

An eletrophoretic analysis of three species of the subgenus Dendromyia (Wyeomyia luteoventralis, Wy. ypsipola and Wy. testei) and three species belonging to different groups in the genus Wyeomyia (Wy. negrensis, Wy. mystes and Wy.confusa) was performed. Eight enzyme loci were analyzed. High values of genetic identity were detected among the species of the subgenus Dendromyia: Wy. luteoventralis, Wy. ypsipola and Wy. testei (mean value 0.63). On the other hand low values of genetic identity were observed among Wy. negrensis, Wy. mystes and Wy. confusa (mean value 0.23), suggesting that they belong, at least, to distinct subgenera within the Genus Wyeomyia. The UPGMA phenogram revealed the grouping of the Dendromyia species, while the others clustered at lower identity levels.

Genetic and Antigenic Analysis of Adenovirus Type 3 Strains Showing Intermediate Behavior in Standard Seroneutralization Test

During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites

Integrated Genetic Epidemiology of Infectious Diseases: The Chagas Model

Genetic typing of pathogenic agents and of vectors has known impressive developments in the last 10 years, thanks to the progresses of molecular biology, and to the contribution of the concepts of evolutionary genetics. Moreover, we know more and more on the genetic susceptibility of man to infectious diseases. I propose here to settle a new, synthetic field of research, which I call `integrated genetic epidemiology of infectious diseases' (IGEID). I aim at evaluating, by an evolutionary genetic approach, the respective impact, on the transmission and pathogenicity of infectious diseases, of the host's, the pathogen's and the vector's genetic diversity, and their possible interactions (co-evolution phenomena). Chagas' disease constitutes a fine model to develop the IGEID methodology, by both field and experimental studies.