M-protein and other intrinsic virulence factors of Streptococcus pyogenes are encoded on an ancient pathogenicity island.

BACKGROUND: The increasing number of completely sequenced bacterial genomes allows comparing their architecture and genetic makeup. Such new information highlights the crucial role of lateral genetic exchanges in bacterial evolution and speciation. RESULTS: Here we analyzed the twelve sequenced genomes of Streptococcus pyogenes by a naïve approach that examines the preferential nucleotide usage along the chromosome, namely the usage of G versus C (GC-skew) and T versus A (TA-skew). The cumulative GC-skew plot presented an inverted V-shape composed of two symmetrical linear segments, where the minimum and maximum corresponded to the origin and terminus of DNA replication. In contrast, the cumulative TA-skew presented a V-shape, which segments were interrupted by several steep slopes regions (SSRs), indicative of a different nucleotide composition bias. Each S. pyogenes genome contained up to nine individual SSRs, encompassing all described strain-specific prophages. In addition, each genome contained a similar unique non-phage SSR, the core of which consisted of 31 highly homologous genes. This core includes the M-protein, other mga-related factors and other virulence genes, totaling ten intrinsic virulence genes. In addition to a high content in virulence-related genes and to a peculiar nucleotide bias, this SSR, which is 47 kb-long in a M1GAS strain, harbors direct repeats and a tRNA gene, suggesting a mobile element. Moreover, its complete absence in a M-protein negative group A Streptococcus natural isolate demonstrates that it could be spontaneously lost, but in vitro deletion experiments indicates that its excision occurred at very low rate. The stability of this SSR, combined to its presence in all sequenced S. pyogenes sequenced genome, suggests that it results from an ancient acquisition. CONCLUSION: Thus, this non-phagic SSR is compatible with a pathogenicity island, acquired before S. pyogenes speciation. Its potential excision might bear relevance for vaccine development, because vaccines targeting M-protein might select for M-protein-negative variants that still carry other virulence determinants.

Evolución de la agregación y separación de sexos: ¿Qué hemos aprendido de las poblaciones ibéricas de Mercurialis annua?

Most plant species are hermaphrodites, with both male and female functions performed by the same individuals. However, separate sexes (dioecy) have evolved on numerous independent occasions, probably either in response to selection for inbreeding avoidance, or because it pays individuals to specialize in one gender or the other. Although the evolution of dioecy from hermaphroditism tends to be thought of as a one-way path, dioecy has broken down to yield hermaphroditic populations on several occasions. One such case is found in the mainly dioecious genus Mercurialis (Euphorbiaceae). In the species complex M. annua, diploids are dioecious, but polyploid populations are variously monoecious or androdioecious (where males co-exist with functional hermaphrodites). This species complex offers rich material for addressing questions concerning the evolution and ecology of combined versus separate sexes, the evolution of secondary sexual dimorphism, which likely contributes to the stability of dioecy in the genus, and the evolution and genetics of sex determination and sex chromosomes. The species also offers itself as a valuable teaching tool for addressing topics ranging from sex-ratio selection to inter-sexual competition.

G-banded karyotype of the alpine shrew, Sorex alpinus (Mammalia, Soricidae), from the Sumava Mts

The G-banded karyotype of two individuals of the alpine shrew Sorer alpinus collected from the Sumava Mountains (Czech Republic) is presented. The diploid number of chromosomes was 56 and both the morphology and the G-banding pattern of chromosomes appeared to be very similar Gto those reported from the Alps and the Jura Mountains. This is the first report on the G-banded chromosomes of this species outside Switzerland and eastern France.

Topological origins of chromosomal territories.

Using freely jointed polymer model we compare equilibrium properties of crowded polymer chains whose segments are either permeable or not permeable for other segments to pass through. In particular, we addressed the question whether non-permeability of long chain molecules, in the absence of excluded volume effect, is sufficient to compartmentalize highly crowded polymer chains, similarly to what happens during formation of chromosomal territories in interphase nuclei. Our results indicate that even polymers without excluded volume compartmentalize and show strongly reduced intermingling when they are mutually non-permeable. Judging from the known fact that chromatin fibres originating from different chromosomes show very limited intermingling in interphase nuclei, we propose that regular chromatin fibres during chromosome decondensation can hardly serve as a substrate of cellular type II DNA topoisomerases.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

Distal trisomy 14 (q24 --> qter) and aorto-pulmonary window: a case report and review of the literature.

A newborn female with partial trisomy for the distal part of the long arm of the chromosome 14 (14q24 --> qter) resulting from a paternal balanced translocation (3;14) is described. We compare her phenotype with eight other individuals with trisomy 14q24 --> qter.

Computational and statistical analysis of copy number variants in normal and cancer genomes

AbstractAlthough the genomes from any two human individuals are more than 99.99% identical at the sequence level, some structural variation can be observed. Differences between genomes include single nucleotide polymorphism (SNP), inversion and copy number changes (gain or loss of DNA). The latter can range from submicroscopic events (CNVs, at least 1kb in size) to complete chromosomal aneuploidies. Small copy number variations have often no (lethal) consequences to the cell, but a few were associated to disease susceptibility and phenotypic variations. Larger re-arrangements (i.e. complete chromosome gain) are frequently associated with more severe consequences on health such as genomic disorders and cancer. High-throughput technologies like DNA microarrays enable the detection of CNVs in a genome-wide fashion. Since the initial catalogue of CNVs in the human genome in 2006, there has been tremendous interest in CNVs both in the context of population and medical genetics. Understanding CNV patterns within and between human populations is essential to elucidate their possible contribution to disease. But genome analysis is a challenging task; the technology evolves rapidly creating needs for novel, efficient and robust analytical tools which need to be compared with existing ones. Also, while the link between CNV and disease has been established, the relative CNV contribution is not fully understood and the predisposition to disease from CNVs of the general population has not been yet investigated.During my PhD thesis, I worked on several aspects related to CNVs. As l will report in chapter 3, ! was interested in computational methods to detect CNVs from the general population. I had access to the CoLaus dataset, a population-based study with more than 6,000 participants from the Lausanne area. All these individuals were analysed on SNP arrays and extensive clinical information were available. My work explored existing CNV detection methods and I developed a variety of metrics to compare their performance. Since these methods were not producing entirely satisfactory results, I implemented my own method which outperformed two existing methods. I also devised strategies to combine CNVs from different individuals into CNV regions.I was also interested in the clinical impact of CNVs in common disease (chapter 4). Through an international collaboration led by the Centre Hospitalier Universitaire Vaudois (CHUV) and the Imperial College London I was involved as a main data analyst in the investigation of a rare deletion at chromosome 16p11 detected in obese patients. Specifically, we compared 8,456 obese patients and 11,856 individuals from the general population and we found that the deletion was accounting for 0.7% of the morbid obesity cases and was absent in healthy non- obese controls. This highlights the importance of rare variants with strong impact and provides new insights in the design of clinical studies to identify the missing heritability in common disease.Furthermore, I was interested in the detection of somatic copy number alterations (SCNA) and their consequences in cancer (chapter 5). This project was a collaboration initiated by the Ludwig Institute for Cancer Research and involved other groups from the Swiss Institute of Bioinformatics, the CHUV and Universities of Lausanne and Geneva. The focus of my work was to identify genes with altered expression levels within somatic copy number alterations (SCNA) in seven metastatic melanoma ceil lines, using CGH and SNP arrays, RNA-seq, and karyotyping. Very few SCNA genes were shared by even two melanoma samples making it difficult to draw any conclusions at the individual gene level. To overcome this limitation, I used a network-guided analysis to determine whether any pathways, defined by amplified or deleted genes, were common among the samples. Six of the melanoma samples were potentially altered in four pathways and five samples harboured copy-number and expression changes in components of six pathways. In total, this approach identified 28 pathways. Validation with two external, large melanoma datasets confirmed all but three of the detected pathways and demonstrated the utility of network-guided approaches for both large and small datasets analysis.RésuméBien que le génome de deux individus soit similaire à plus de 99.99%, des différences de structure peuvent être observées. Ces différences incluent les polymorphismes simples de nucléotides, les inversions et les changements en nombre de copies (gain ou perte d'ADN). Ces derniers varient de petits événements dits sous-microscopiques (moins de 1kb en taille), appelés CNVs (copy number variants) jusqu'à des événements plus large pouvant affecter des chromosomes entiers. Les petites variations sont généralement sans conséquence pour la cellule, toutefois certaines ont été impliquées dans la prédisposition à certaines maladies, et à des variations phénotypiques dans la population générale. Les réarrangements plus grands (par exemple, une copie additionnelle d'un chromosome appelée communément trisomie) ont des répercutions plus grave pour la santé, comme par exemple dans certains syndromes génomiques et dans le cancer. Les technologies à haut-débit telle les puces à ADN permettent la détection de CNVs à l'échelle du génome humain. La cartographie en 2006 des CNV du génome humain, a suscité un fort intérêt en génétique des populations et en génétique médicale. La détection de différences au sein et entre plusieurs populations est un élément clef pour élucider la contribution possible des CNVs dans les maladies. Toutefois l'analyse du génome reste une tâche difficile, la technologie évolue très rapidement créant de nouveaux besoins pour le développement d'outils, l'amélioration des précédents, et la comparaison des différentes méthodes. De plus, si le lien entre CNV et maladie a été établit, leur contribution précise n'est pas encore comprise. De même que les études sur la prédisposition aux maladies par des CNVs détectés dans la population générale n'ont pas encore été réalisées.Pendant mon doctorat, je me suis concentré sur trois axes principaux ayant attrait aux CNV. Dans le chapitre 3, je détaille mes travaux sur les méthodes d'analyses des puces à ADN. J'ai eu accès aux données du projet CoLaus, une étude de la population de Lausanne. Dans cette étude, le génome de plus de 6000 individus a été analysé avec des puces SNP et de nombreuses informations cliniques ont été récoltées. Pendant mes travaux, j'ai utilisé et comparé plusieurs méthodes de détection des CNVs. Les résultats n'étant pas complètement satisfaisant, j'ai implémenté ma propre méthode qui donne de meilleures performances que deux des trois autres méthodes utilisées. Je me suis aussi intéressé aux stratégies pour combiner les CNVs de différents individus en régions.Je me suis aussi intéressé à l'impact clinique des CNVs dans le cas des maladies génétiques communes (chapitre 4). Ce projet fut possible grâce à une étroite collaboration avec le Centre Hospitalier Universitaire Vaudois (CHUV) et l'Impérial College à Londres. Dans ce projet, j'ai été l'un des analystes principaux et j'ai travaillé sur l'impact clinique d'une délétion rare du chromosome 16p11 présente chez des patients atteints d'obésité. Dans cette collaboration multidisciplinaire, nous avons comparés 8'456 patients atteint d'obésité et 11 '856 individus de la population générale. Nous avons trouvés que la délétion était impliquée dans 0.7% des cas d'obésité morbide et était absente chez les contrôles sains (non-atteint d'obésité). Notre étude illustre l'importance des CNVs rares qui peuvent avoir un impact clinique très important. De plus, ceci permet d'envisager une alternative aux études d'associations pour améliorer notre compréhension de l'étiologie des maladies génétiques communes.Egalement, j'ai travaillé sur la détection d'altérations somatiques en nombres de copies (SCNA) et de leurs conséquences pour le cancer (chapitre 5). Ce projet fut une collaboration initiée par l'Institut Ludwig de Recherche contre le Cancer et impliquant l'Institut Suisse de Bioinformatique, le CHUV et les Universités de Lausanne et Genève. Je me suis concentré sur l'identification de gènes affectés par des SCNAs et avec une sur- ou sous-expression dans des lignées cellulaires dérivées de mélanomes métastatiques. Les données utilisées ont été générées par des puces ADN (CGH et SNP) et du séquençage à haut débit du transcriptome. Mes recherches ont montrées que peu de gènes sont récurrents entre les mélanomes, ce qui rend difficile l'interprétation des résultats. Pour contourner ces limitations, j'ai utilisé une analyse de réseaux pour définir si des réseaux de signalisations enrichis en gènes amplifiés ou perdus, étaient communs aux différents échantillons. En fait, parmi les 28 réseaux détectés, quatre réseaux sont potentiellement dérégulés chez six mélanomes, et six réseaux supplémentaires sont affectés chez cinq mélanomes. La validation de ces résultats avec deux larges jeux de données publiques, a confirmée tous ces réseaux sauf trois. Ceci démontre l'utilité de cette approche pour l'analyse de petits et de larges jeux de données.Résumé grand publicL'avènement de la biologie moléculaire, en particulier ces dix dernières années, a révolutionné la recherche en génétique médicale. Grâce à la disponibilité du génome humain de référence dès 2001, de nouvelles technologies telles que les puces à ADN sont apparues et ont permis d'étudier le génome dans son ensemble avec une résolution dite sous-microscopique jusque-là impossible par les techniques traditionnelles de cytogénétique. Un des exemples les plus importants est l'étude des variations structurales du génome, en particulier l'étude du nombre de copies des gènes. Il était établi dès 1959 avec l'identification de la trisomie 21 par le professeur Jérôme Lejeune que le gain d'un chromosome supplémentaire était à l'origine de syndrome génétique avec des répercussions graves pour la santé du patient. Ces observations ont également été réalisées en oncologie sur les cellules cancéreuses qui accumulent fréquemment des aberrations en nombre de copies (telles que la perte ou le gain d'un ou plusieurs chromosomes). Dès 2004, plusieurs groupes de recherches ont répertorié des changements en nombre de copies dans des individus provenant de la population générale (c'est-à-dire sans symptômes cliniques visibles). En 2006, le Dr. Richard Redon a établi la première carte de variation en nombre de copies dans la population générale. Ces découvertes ont démontrées que les variations dans le génome était fréquentes et que la plupart d'entre elles étaient bénignes, c'est-à-dire sans conséquence clinique pour la santé de l'individu. Ceci a suscité un très grand intérêt pour comprendre les variations naturelles entre individus mais aussi pour mieux appréhender la prédisposition génétique à certaines maladies.Lors de ma thèse, j'ai développé de nouveaux outils informatiques pour l'analyse de puces à ADN dans le but de cartographier ces variations à l'échelle génomique. J'ai utilisé ces outils pour établir les variations dans la population suisse et je me suis consacré par la suite à l'étude de facteurs pouvant expliquer la prédisposition aux maladies telles que l'obésité. Cette étude en collaboration avec le Centre Hospitalier Universitaire Vaudois a permis l'identification d'une délétion sur le chromosome 16 expliquant 0.7% des cas d'obésité morbide. Cette étude a plusieurs répercussions. Tout d'abord elle permet d'effectuer le diagnostique chez les enfants à naître afin de déterminer leur prédisposition à l'obésité. Ensuite ce locus implique une vingtaine de gènes. Ceci permet de formuler de nouvelles hypothèses de travail et d'orienter la recherche afin d'améliorer notre compréhension de la maladie et l'espoir de découvrir un nouveau traitement Enfin notre étude fournit une alternative aux études d'association génétique qui n'ont eu jusqu'à présent qu'un succès mitigé.Dans la dernière partie de ma thèse, je me suis intéressé à l'analyse des aberrations en nombre de copies dans le cancer. Mon choix s'est porté sur l'étude de mélanomes, impliqués dans le cancer de la peau. Le mélanome est une tumeur très agressive, elle est responsable de 80% des décès des cancers de la peau et est souvent résistante aux traitements utilisés en oncologie (chimiothérapie, radiothérapie). Dans le cadre d'une collaboration entre l'Institut Ludwig de Recherche contre le Cancer, l'Institut Suisse de Bioinformatique, le CHUV et les universités de Lausanne et Genève, nous avons séquencés l'exome (les gènes) et le transcriptome (l'expression des gènes) de sept mélanomes métastatiques, effectués des analyses du nombre de copies par des puces à ADN et des caryotypes. Mes travaux ont permis le développement de nouvelles méthodes d'analyses adaptées au cancer, d'établir la liste des réseaux de signalisation cellulaire affectés de façon récurrente chez le mélanome et d'identifier deux cibles thérapeutiques potentielles jusqu'alors ignorées dans les cancers de la peau.

Further delineation of the facial 13q14 deletion syndrome in 13 retinoblastoma patients.

Thirteen years ago, Motegi and colleagues (J Med Genet 1987;24:696-697) summarized the specific facial phenotype of six Japanese retinoblastoma patients with interstitial 13q14 deletions. Among a series of 228 propositi with retinoblastoma referred to the Lausanne Retinoblastoma Clinic for treatment and genetic counseling between 1986 and 1997, 13 (5.7%) were diagnosed with a cytogenetic de-novo 13q14 deletion. We confirm the presence of the reported facial phenotype in our population of Caucasian patients and describe additional clinical traits, thus extending the facial phenotype associated with the 13q14 deletion. Del(13q14) comprises, among others, cranial anomalies, frontal bossing, deeply grooved and long philtrum, depressed and broad nasal bridge, bulbous tip of the nose, thick lower lip, thin upper lip, broad cheeks, and large ears and lobules. Recognition of this particular facial appearance was instrumental in the genetic diagnosis of 13q deletions and in the presymptomatic diagnosis of retinoblastoma in a significant number of our cases. Identification of this phenotype in a retinoblastoma patient allows for efficient diagnosis of recurrence in his progeny and/or sibship, while its ignorance will compromise genetic counseling due to the possible difficulties in detecting large deletions by standard molecular mutation analysis. Recognition of this syndrome in newborns without known familial risk for retinoblastoma is even more important as it is a clear warning sign that indicates immediate ophthalmic examination.

ALK inhibitors in the treatment of advanced NSCLC.

Pharmacologic agents that target protein products of oncogenes in tumors are playing an increasing clinical role in the treatment of cancer. Currently, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represent the standard of care for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. Subsequently other genetic abnormalities with "driver" characteristics - implying transforming and tumor maintenance capabilities have been extensively reported in several small distinct subsets of NSCLC. Among these rare genetic changes, anaplastic lymphoma kinase (ALK) gene rearrangements, most often consisting in a chromosome 2 inversion leading to a fusion with the echinoderm microtubule-associated protein like 4 (EML4) gene, results in the abnormal expression and activation of this tyrosine kinase in the cytoplasm of cancer cells. This rearrangement occurs in 2-5% of NSCLC, predominantly in young (50 years or younger), never- or former-smokers with adenocarcinoma. This aberration most commonly occurs a independently of EGFR and KRAS gene mutations. A fluorescent in situ hybridization assay was approved by the US Food and Drug Administration (FDA) as the standard method for the detection of ALK gene rearrangement in clinical practice and is considered the gold standard. Crizotinib, a first-in-class dual ALK and c-MET inhibitor, has been shown to be particularly effective against ALK positive NSCLC, showing dramatic and prolonged responses with low toxicity, predominantly restricted to the gastro-intestinal and visual systems, and generally self-limiting or easily managed. However, resistance to crizotinib inevitably emerges. The molecular mechanisms of resistance are currently under investigation, as are therapeutic approaches including crizotinib-based combination therapy and novel agents such as Hsp90 inhibitors. This review aims to present the current knowledge on this fusion gene, the clinic-pathological profile of ALK rearranged NSCLC, and to review the existing literature on ALK inhibitors, focusing on their role in the treatment of NSCLC.

Genes, environment and sex : perspectives from ectothermic vertebrates

RESUMELes modèles classiques sur l'évolution des chromosomes sexuels supposent que des gènes sexe- antagonistes s'accumulent sur les chromosomes sexuels, entraînant ainsi l'apparition d'une région non- recombinante, qui se répand progressivement en favorisant l'accumulation de mutations délétères. En accord avec cette théorie, les chromosomes sexuels que l'on observe aujourd'hui chez les mammifères et les oiseaux sont considérablement différenciés. En revanche, chez la plupart des vertébrés ectothermes, les chromosomes sexuels sont indifférenciés et il existe une impressionnante diversité de mécanismes de détermination du sexe. Au cours de cette thèse, j'ai étudié l'évolution des chromosomes sexuels chez les vertébrés ectothermes, en outre pour mieux comprendre ce contraste avec les vertébrés endothermes. L'hypothèse « high-turnover » postule que les chromosomes sexuels sont remplacés régulièrement à partir d'autosomes afin d'éviter leur dégénérescence. L'hypothèse « fountain-of-youth » propose que la recombinaison entre le chromosome X et le chromosome Y au sein de femelles XY empêche la dégénérescence. Les résultats de ma thèse, basés sur des études théoriques et empiriques, suggèrent que les deux processus peuvent être entraînés par l'environnement et ainsi jouent un rôle important dans l'évolution des chromosomes sexuels chez les vertébrés ectothermes.SUMMARYClassical models of sex-chromosome evolution assume that sexually antagonistic genes accumulate on sex chromosomes leading to a non-recombining region, which progressively expands and favors the accumulation of deleterious mutations. Concordant with this theory, sex chromosomes in extant mammals and birds are considerably differentiated. In most ectothermic vertebrates, such as frogs, however, sex chromosomes are undifferentiated and a striking diversity of sex determination systems is observed. This thesis was aimed to investigate this apparent contrast of sex chromosome evolution between endothermic and ectothermic vertebrates. The "high-turnover" hypothesis holds that sex chromosomes arose regularly from autosomes preventing decay. The "fountain-of-youth" hypothesis posits that sex chromosomes undergo episodic X-Y recombination in sex-reversed XY females, thereby purging ("rejuvenating") the Y chromosome. We suggest that both processes likely played an important role in sex chromosome evolution of ectothermic vertebrates. The literature largely views sex determination as a dichotomous process: individual sex is assumed to be determined either by genetic (genotypic sex determination, GSD) or by environmental factors (environmental sex determination, ESD), most often temperature (temperature sex determination, TSD). We endorsed an alternative view, which sees GSD and TSD as the ends of a continuum. The conservatism of molecular processes among different systems of sex determination strongly supports the continuum view. We proposed to define sex as a threshold trait underlain by a liability factor, and reaction norms allowing modeling interactions between genotypic and temperature effects. We showed that temperature changes (due to e.g., climatic changes or range expansions) are expected to provoke turnovers in sex-determination mechanisms maintaining homomorphic sex chromosomes. The balanced lethal system of crested newts might be the result of such a sex determination turnover, originating from two variants of ancient Y-chromosomes. Observations from a group of tree frogs, on the other hand, supported the 'fountain of youth' hypothesis. We then showed that low rates of sex- reversals in species with GSD might actually be adaptive considering joint effects of deleterious mutation purging and sexually antagonistic selection. Ongoing climatic changes are expected to threaten species with TSD by biasing population sex ratios. In contrast, species with GSD are implicitly assumed immune against such changes, because genetic systems are thought to necessarily produce even sex ratios. We showed that this assumption may be wrong and that sex-ratio biases by climatic changes may represent a previously unrecognized extinction threat for some GSD species.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Sex-chromosome turnovers induced by deleterious mutation load.

In sharp contrast with mammals and birds, many cold-blooded vertebrates present homomorphic sex chromosomes. Empirical evidence supports a role for frequent turnovers, which replace nonrecombining sex chromosomes before they have time to decay. Three main mechanisms have been proposed for such turnovers, relying either on neutral processes, sex-ratio selection, or intrinsic benefits of the new sex-determining genes (due, e.g., to linkage with sexually antagonistic mutations). Here, we suggest an additional mechanism, arising from the load of deleterious mutations that accumulate on nonrecombining sex chromosomes. In the absence of dosage compensation, this load should progressively lower survival rate in the heterogametic sex. Turnovers should occur when this cost outweighs the benefits gained from any sexually antagonistic genes carried by the nonrecombining sex chromosome. We use individual-based simulations of a Muller's ratchet process to test this prediction, and investigate how the relevant parameters (effective population size, strength and dominance of deleterious mutations, size of nonrecombining segment, and strength of sexually antagonistic selection) are expected to affect the rate of turnovers.

Subtelomeric 6p deletion: clinical and array-CGH characterization in two patients

We report on two patients with de novo subtelomeric terminal deletion of chromosome 6p. Patient 1 is an 8-month-old female born with normal growth parameters, typical facial features of 6pter deletion, bilateral corectopia, and protruding tongue. She has severe developmental delay, profound bilateral neurosensory deafness, poor visual contact, and hypsarrhythmia since the age of 6 months. Patient 2 is a 5-year-old male born with normal growth parameters and unilateral hip dysplasia; he has a characteristic facial phenotype, bilateral embryotoxon, and moderate mental retardation. Further characterization of the deletion, using high-resolution array comparative genomic hybridization (array-CGH; Agilent Human Genome kit 244 K), revealed that Patient 1 has a 8.1 Mb 6pter-6p24.3 deletion associated with a contiguous 5.8 Mb 6p24.3-6p24.1 duplication and Patient 2 a 5.7 Mb 6pter-6p25.1 deletion partially overlapping with that of Patient 1. Complementary FISH and array analysis showed that the inv del dup(6) in Patient 1 originated de novo. Our results demonstrate that simple rearrangements are often more complex than defined by standard techniques. We also discuss genotype-phenotype correlations including previously reported cases of deletion 6p.