Copy number variation modifies expression profiles over developmental time

Abstract : A preliminary understanding of the phenotypic effect of copy number variation (CNV) of DNA segments is emerging. These rearrangements were shown to influence, in a somewhat dose-dependent manner, the expression of genes mapping within them. They were also shown to modify the expression of genes located on their Hanks, sometimes at great distance. Here, we demonstrate by monitoring these effects at multiple life stages, that these controls over expression are effective throughout mouse development. Similarly, we observe that the more specific spatial expression patterns of CNV genes are maintained through life. However, 'we find that some brain- expressed genes mapping within CNVS appear to be under compensatory loops only at specific time-points, indicating that the effect of CNVS on these genes is modulated during development. Notably, we also observe that CNV genes are significantly enriched within transcripts that show variable time-course expression between strains. Thus, modifying the copy number of a gene may potentially alter not only its expression level, but its timing of expression as well. Résume : Nous commençons à comprendre les effets phénotypiques liés aux séquences d'ADN qui changent de nombre de copies d'un individu a l'autre. Des travaux précédents ont montré que ces variante de nombre de copies (CNVS) avaient une influence sur l'expression non seulement des gènes se trouvant dans le réarrangement, mais aussi sur ceux se trouvant à une certaine distance. Le présent travail étudie ces effets à différents stades du développement de la souris allant d'un embryon de deux semaines à la souris adulte. Nous avons observé que certains gènes exprimés dans le cerveau semblent soumis à un contrôle plus strict a certaines étapes du développement suggèrent que l'effet des CNVs est modulé différemment au cours de la vie. Notre travail sur trois souches différentes de souris a permis de montrer que les gènes ayant un profil d'expression différent dans le temps entre souches sont enrichis en gènes se trouvant dans des CNVs. Ceci nous amène à penser que les CNVs ont, non seulement une influence sur le niveau d'expression des gènes, mais aussi sur les moments durant lesquels ils seront exprimés. Résumé pour un large public : De nombreuses maladies sont dues soit a un gain (on parle alors de duplication) soit à une perte de matériel génétique (il s'agit dune délétion). Bien que les recherches visant à identifier les mécanismes moléculaires liés à ces réarrangements de notre génome progressent continuellement, la plupart des causes des maladies génétiques restent à élucider. Certaines parties de notre génome sont présentes en un nombre de copies qui diffère d'un individu à l'autre sans pour autant provoquer une ou des maladies. Ces segments d'ADN qui varient en nombre sont appelés Copy Number Variant (CNVs). Ils couvrent environ 12% de notre matériel génétique. Des études menées sur différents modèles animaux ont montré que les CNVs avaient une influence aussi bien sur les gènes qui sont a l'intérieur des CNVs que sur ceux qui sont dans leur voisinage. Ce travail étudie l'effet des CNVs à travers différents stades du développement de la souris. Nous avons démontré que les segments d'ADN qui varient en nombre de copies ont des effets variables selon le stade auxquels ils sont mesurés. Ainsi, les CNVs ont non seulement un impact sur l'expression des gènes présents dans ces régions et dans leur voisinage, mais influencent également leurs profils d'expression au cours du temps.

SCRIB and PUF60 Are Primary Drivers of the Multisystemic Phenotypes of the 8q24.3 Copy-Number Variant.

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions.

Copy number variants, diseases and gene expression.

Copy number variation (CNV) has recently gained considerable interest as a source of genetic variation likely to play a role in phenotypic diversity and evolution. Much effort has been put into the identification and mapping of regions that vary in copy number among seemingly normal individuals in humans and a number of model organisms, using bioinformatics or hybridization-based methods. These have allowed uncovering associations between copy number changes and complex diseases in whole-genome association studies, as well as identify new genomic disorders. At the genome-wide scale, however, the functional impact of CNV remains poorly studied. Here we review the current catalogs of CNVs, their association with diseases and how they link genotype and phenotype. We describe initial evidence which revealed that genes in CNV regions are expressed at lower and more variable levels than genes mapping elsewhere, and also that CNV not only affects the expression of genes varying in copy number, but also have a global influence on the transcriptome. Further studies are warranted for complete cataloguing and fine mapping of CNVs, as well as to elucidate the different mechanisms by which they influence gene expression.

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.

Genetic factors of obesity and eating disorders: Copy number variations (CNV) involving SH2B1

Context : It is now clearly shown that genetic factors in association with environment play a key role in obesity and eating disorders. This project studies the clinical symptoms and molecular abnormalities in patients carrying a strong hereditary predisposition to obesity and eating behavior disorders. We have previously published the association between the 16:29.5-30.1 deletion and a very penetrant form of morbid obesity and macrocephaly. We have also demonstrated the association between the reciprocal 16:29.5-30.1 duplication and underweight and small head circumference. These 2 studies demonstrate that gene dosage of one or several genes in this region regulates BMI as well as brain growth. At present, there are no data pointing towards particular candidate genes. We are currently investigating a second non-overlapping recurrent CNV encompassing SH2B1, upstream of the aforementioned rearrangement. SNPs in this gene have been associated with BMI in GWAS studies and mice models confirmed this association. Bokuchova et al have reported an association between deletions encompassing this gene and severe early onset obesity, as well as insulin resistance. We are currently collecting and analyzing data to fully characterize the phenotype and the transcriptional patterns associated with this rearrangement. Aims : 1. Identify carriers of any CNVs in the greater 16p11.2 region (between 16:28MB and 32MB) in the EGG consortium. 2. Perform association studies between SNPs in the greater 16p11.2 region (16:28-32MB) and anthropometric measures with adjusted "locus-wide significance", to identify or prioritize candidate genes potentially driving the association observed in patients with the CNVs (and thus worthy of further validation and sequencing). 3. Explore associations between GSV genome-wide and brain volume. 4. Explore relationship between brain volumes (whole brain and regional for those who underwent brain MRI), head circumference and BMI. 5. Extrapolate this procedure to other regions covered by the Metabochip. Methods : - Examine and collect clinical informations, as well as molecular informations in these patients. - Analysis of MRI data in children and adults with BMI > 2SD. Compare changes to MRI data obtained in patients with monogenic forms of obesity (data from Lausanne study) and to underweight (BMI<-2SD) individuals from EGG. - Test whether opposite extremes of the phenotypic distribution may be highly informative Expected results : This is a highly focused study, pertaining to approximately 1 0/00 of the human genome. Yet it is clear that if successful, the lessons learned from this study could be extrapolated to other segments of the genome and would need validation and replication by additional studies. Altogether they will contribute to further explore the missing heritability and point to etiologic genes and pathways underlying these important health burdens.

Copy number variation modifies expression time courses.

A preliminary understanding into the phenotypic effect of DNA segment copy number variation (CNV) is emerging. These rearrangements were demonstrated to influence, in a somewhat dose-dependent manner, the expression of genes that map within them. They were also shown to modify the expression of genes located on their flanks and sometimes those at a great distance from their boundary. Here we demonstrate, by monitoring these effects at multiple life stages, that these controls over expression are effective throughout mouse development. Similarly, we observe that the more specific spatial expression patterns of CNV genes are maintained through life. However, we find that some brain-expressed genes mapping within CNVs appear to be under compensatory loops only at specific time points, indicating that the effect of CNVs on these genes is modulated during development. Notably, we also observe that CNV genes are significantly enriched within transcripts that show variable time courses of expression between strains. Thus, modifying the copy number of a gene may potentially alter not only its expression level, but also the timing of its expression.

Copy number variation in the porcine genome inferred from a 60 k SNP BeadChip

Background: Recent studies in pigs have detected copy number variants (CNVs) using the Comparative Genomic Hybridization technique in arrays designed to cover specific porcine chromosomes. The goal of this study was to identify CNV regions (CNVRs) in swine species based on whole genome SNP genotyping chips. Results: We used predictions from three different programs (cnvPartition, PennCNV and GADA) to analyze data from the Porcine SNP60 BeadChip. A total of 49 CNVRs were identified in 55 animals from an Iberian x Landrace cross (IBMAP) according to three criteria: detected in at least two animals, contained three or more consecutive SNPs and recalled by at least two programs. Mendelian inheritance of CNVRs was confirmed in animals belonging to several generations of the IBMAP cross. Subsequently, a segregation analysis of these CNVRs was performed in 372 additional animals from the IBMAP cross and its distribution was studied in 133 unrelated pig samples from different geographical origins. Five out of seven analyzed CNVRs were validated by real time quantitative PCR, some of which coincide with well known examples of CNVs conserved across mammalian species. Conclusions: Our results illustrate the usefulness of Porcine SNP60 BeadChip to detect CNVRs and show that structural variants can not be neglected when studying the genetic variability in this species.

Copy number variations and cognitive phenotypes in unselected populations

IMPORTANCE: The association of copy number variations (CNVs), differing numbers of copies of genetic sequence at locations in the genome, with phenotypes such as intellectual disability has been almost exclusively evaluated using clinically ascertained cohorts. The contribution of these genetic variants to cognitive phenotypes in the general population remains unclear. OBJECTIVE: To investigate the clinical features conferred by CNVs associated with known syndromes in adult carriers without clinical preselection and to assess the genome-wide consequences of rare CNVs (frequency ≤0.05%; size ≥250 kilobase pairs [kb]) on carriers' educational attainment and intellectual disability prevalence in the general population. DESIGN, SETTING, AND PARTICIPANTS: The population biobank of Estonia contains 52,000 participants enrolled from 2002 through 2010. General practitioners examined participants and filled out a questionnaire of health- and lifestyle-related questions, as well as reported diagnoses. Copy number variant analysis was conducted on a random sample of 7877 individuals and genotype-phenotype associations with education and disease traits were evaluated. Our results were replicated on a high-functioning group of 993 Estonians and 3 geographically distinct populations in the United Kingdom, the United States, and Italy. MAIN OUTCOMES AND MEASURES: Phenotypes of genomic disorders in the general population, prevalence of autosomal CNVs, and association of these variants with educational attainment (from less than primary school through scientific degree) and prevalence of intellectual disability. RESULTS: Of the 7877 in the Estonian cohort, we identified 56 carriers of CNVs associated with known syndromes. Their phenotypes, including cognitive and psychiatric problems, epilepsy, neuropathies, obesity, and congenital malformations are similar to those described for carriers of identical rearrangements ascertained in clinical cohorts. A genome-wide evaluation of rare autosomal CNVs (frequency, ≤0.05%; ≥250 kb) identified 831 carriers (10.5%) of the screened general population. Eleven of 216 (5.1%) carriers of a deletion of at least 250 kb (odds ratio [OR], 3.16; 95% CI, 1.51-5.98; P = 1.5e-03) and 6 of 102 (5.9%) carriers of a duplication of at least 1 Mb (OR, 3.67; 95% CI, 1.29-8.54; P = .008) had an intellectual disability compared with 114 of 6819 (1.7%) in the Estonian cohort. The mean education attainment was 3.81 (P = 1.06e-04) among 248 (≥250 kb) deletion carriers and 3.69 (P = 5.024e-05) among 115 duplication carriers (≥1 Mb). Of the deletion carriers, 33.5% did not graduate from high school (OR, 1.48; 95% CI, 1.12-1.95; P = .005) and 39.1% of duplication carriers did not graduate high school (OR, 1.89; 95% CI, 1.27-2.8; P = 1.6e-03). Evidence for an association between rare CNVs and lower educational attainment was supported by analyses of cohorts of adults from Italy and the United States and adolescents from the United Kingdom. CONCLUSIONS AND RELEVANCE: Known pathogenic CNVs in unselected, but assumed to be healthy, adult populations may be associated with unrecognized clinical sequelae. Additionally, individually rare but collectively common intermediate-size CNVs may be negatively associated with educational attainment. Replication of these findings in additional population groups is warranted given the potential implications of this observation for genomics research, clinical care, and public health.

Phenotypic Association Analyses With Copy Number Variation in Recurrent Depressive Disorder.

BACKGROUND: Defining the molecular genomic basis of the likelihood of developing depressive disorder is a considerable challenge. We previously associated rare, exonic deletion copy number variants (CNV) with recurrent depressive disorder (RDD). Sex chromosome abnormalities also have been observed to co-occur with RDD. METHODS: In this reanalysis of our RDD dataset (N = 3106 cases; 459 screened control samples and 2699 population control samples), we further investigated the role of larger CNVs and chromosomal abnormalities in RDD and performed association analyses with clinical data derived from this dataset. RESULTS: We found an enrichment of Turner's syndrome among cases of depression compared with the frequency observed in a large population sample (N = 34,910) of live-born infants collected in Denmark (two-sided p = .023, odds ratio = 7.76 [95% confidence interval = 1.79-33.6]), a case of diploid/triploid mosaicism, and several cases of uniparental isodisomy. In contrast to our previous analysis, large deletion CNVs were no more frequent in cases than control samples, although deletion CNVs in cases contained more genes than control samples (two-sided p = .0002). CONCLUSIONS: After statistical correction for multiple comparisons, our data do not support a substantial role for CNVs in RDD, although (as has been observed in similar samples) occasional cases may harbor large variants with etiological significance. Genetic pleiotropy and sample heterogeneity suggest that very large sample sizes are required to study conclusively the role of genetic variation in mood disorders.

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer.

PURPOSE: Prospective-retrospective assessment of theTOP1gene copy number andTOP1mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan.TOP1copy number status was analyzed by fluorescencein situhybridization (FISH) using aTOP1/CEN20 dual-probe combination.TOP1mRNA data were available from previous analyses. RESULTS: TOP1FISH and follow-up data were obtained from 534 patients.TOP1gain was identified in 27% using a single-probe enumeration strategy (≥4TOP1signals per cell) and in 31% when defined by aTOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent onTOP1FISH status.TOP1mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized byTOP1mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment byTOP1mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasingTOP1mRNA values appeared to be associated with increasing benefit of irinotecan. CONCLUSIONS: In contrast to theTOP1copy number, a trend was demonstrated for a predictive property ofTOP1mRNA expression. On the basis ofTOP1mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.Clin Cancer Res; 22(7); 1621-31. ©2015 AACR.

Copy number variations of colorectal cancer by whole exome sequencing data

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death worldwide. About 85% of the cases of CRC are known to have chromosomal instability, an allelic imbalance at several chromosomal loci, and chromosome amplification and translocation. The aim of this study is to determine the recurrent copy number variant (CNV) regions present in stage II of CRC through whole exome sequencing, a rapidly developing targeted next-generation sequencing (NGS) technology that provides an accurate alternative approach for accessing genomic variations. 42 normal-tumor paired samples were sequenced by Illumina Genome Analyzer. Data was analyzed with Varscan2 and segmentation was performed with R package R-GADA. Summary of the segments across all samples was performed and the result was overlapped with DEG data of the same samples from a previous study in the group1. Major and more recurrent segments of CNV were: gain of chromosome 7pq(13%), 13q(31%) and 20q(75%) and loss of 8p(25%), 17p(23%), and 18pq(27%). This results are coincident with the known literature of CNV in CRC or other cancers, but our methodology should be validated by array comparative genomic hybridisation (aCGH) profiling, which is currently the gold standard for genetic diagnosis of CNV.

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different Solanum species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1-4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613-884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species.

Copy number variants on the X chromosome in women with primary ovarian insufficiency

Objective: To investigate whether submicroscopic copy number variants (CNVs) on the X chromosome can be identified in women with primary ovarian insufficiency (POI), defined as spontaneous secondary amenorrhea before 40 years of age accompanied by follicle-stimulating hormone levels above 40 IU/L on at least two occasions. Design: Analysis of intensity data of single nucleotide polymorphism (SNP) probes generated by genomewide Illumina 370k CNV BeadChips, followed by the validation of identified loci using a custom designed ultra-high-density comparative genomic hybridization array containing 48,325 probes evenly distributed over the X chromosome. Setting: Multicenter genetic cohort study in the Netherlands. Patient(s): 108 Dutch Caucasian women with POI, 97 of whom passed quality control, who had a normal karyogram and absent fragile X premutation, and 235 healthy Dutch Caucasian women as controls. Intervention(s): None. Main Outcome Measure(s): Amount and locus of X chromosomal microdeletions or duplications. Result(s): Intensity differences between SNP probes identify microdeletions and duplications. The initial analysis identified an overrepresentation of deletions in POI patients. Moreover, CNVs in two genes on the Xq21.3 locus (i.e., PCDH11X and TGIF2LX) were statistically significantly associated with the POI phenotype. Mean size of identified CNVs was 262 kb. However, in the validation study the identified putative Xq21.3 deletions samples did not show deviations in intensities in consecutive probes. Conclusion(s): X chromosomal submicroscopic CNVs do not play a major role in Caucasian POI patients. We provide guidelines on how submicroscopic cytogenetic POI research should be conducted. (Fertil Steril (R) 2011;95:1584-8. (C) 2011 by American Society for Reproductive Medicine.)

Copy number of P elements, KP/full-sized P element ratio and their relationships with environmental factors in Brazilian Drosophila melanogaster populations

The P transposable element copy numbers and the KP/full-sized P element ratios were determined in eight Brazilian strains of Drosophila melanogaster. Strains from tropical regions showed lower overall P element copy numbers than did strains from temperate regions. Variable numbers of full-sized and defective elements were detected, but the full-sized P and KP elements were the predominant classes of elements in all strains. The full-sized P and KP element ratios were calculated and compared with latitude. The northernmost and southernmost Brazilian strains showed fewer full-sized elements than KP elements per genome, and the strains from less extreme latitudes had many more full-sized P than KP elements. However, no clinal variation was observed. Strains from different localities, previously classified as having P cytotype, displayed a higher or a lower proportion of KP elements than of full-sized P elements, as well as an equal number of the two element types, showing that the same phenotype may be produced by different underlying genomic components of the P-M system.

Characterization of hobo element copy number and integrity in Brasilian populations of Drosophila melanogaster

We have determined the copy number and the presence of full-size hobo transposable elements in eight Brasilian strains of Drosophila melanogaster. Genomic DNA was digested with AvaII and XhoI restriction enzymes, respectively, and probed with a 963 bp sequence of the hobo element. Variable numbers of full-sized and defective elements were detected in all strains. The range of the copy number was 22.13 +/- 4.52. Blots showed the presence of a 2.6 kb fragment, corresponding to the complete element, in all strains exception of one and the 1.0 kb sequence, correponding to the Th1 and Th2 repressor elements. There was neither association among copy numbers of hobo elements and latitude nor the mean annual temperatures in the original geographical region of each strain.