Genomic microarrays in chromosomal analysis of leukemia

Chromosomal alterations in leukemia have been shown to have prognostic and predictive significance and are also important minimal residual disease (MRD) markers in the follow-up of leukemia patients. Although specific oncogenes and tumor suppressors have been discovered in some of the chromosomal alterations, the role and target genes of many alterations in leukemia remain unknown. In addition, a number of leukemia patients have a normal karyotype by standard cytogenetics, but have variability in clinical course and are often molecularly heterogeneous. Cytogenetic methods traditionally used in leukemia analysis and diagnostics; G-banding, various fluorescence in situ hybridization (FISH) techniques, and chromosomal comparative genomic hybridization (cCGH), have enormously increased knowledge about the leukemia genome, but have limitations in resolution or in genomic coverage. In the last decade, the development of microarray comparative genomic hybridization (array-CGH, aCGH) for DNA copy number analysis and the SNP microarray (SNP-array) method for simultaneous copy number and loss of heterozygosity (LOH) analysis has enabled investigation of chromosomal and gene alterations genome-wide with high resolution and high throughput. In these studies, genetic alterations were analyzed in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). The aim was to screen and characterize genomic alterations that could play role in leukemia pathogenesis by using aCGH and SNP-arrays. One of the most important goals was to screen cryptic alterations in karyotypically normal leukemia patients. In addition, chromosomal changes were evaluated to narrow the target regions, to find new markers, and to obtain tumor suppressor and oncogene candidates. The work presented here shows the capability of aCGH to detect submicroscopic copy number alterations in leukemia, with information about breakpoints and genes involved in the alterations, and that genome-wide microarray analyses with aCGH and SNP-array are advantageous methods in the research and diagnosis of leukemia. The most important findings were the cryptic changes detected with aCGH in karyotypically normal AML and CLL, characterization of amplified genes in 11q marker chromosomes, detection of deletion-based mechanisms of MLL-ARHGEF12 fusion gene formation, and detection of LOH without copy number alteration in karyotypically normal AML. These alterations harbor candidate oncogenes and tumor suppressors for further studies.

Genetic background of bipolar disorder and related cognitive impairments

Bipolar disorder (BP) is a complex psychiatric disorder characterized by episodes of mania and depression. BP affects approximately 1% of the world’s population and shows no difference in lifetime prevalence between males and females. BP arises from complex interactions among genetic, developmental and environmental factors, and it is likely that several predisposing genes are involved in BP. The genetic background of BP is still poorly understood, although intensive and long-lasting research has identified several chromosomal regions and genes involved in susceptibility to BP. This thesis work aims to identify the genetic variants that influence bipolar disorder in the Finnish population by candidate gene and genome-wide linkage analyses in families with many BP cases. In addition to diagnosis-based phenotypes, neuropsychological traits that can be seen as potential endophenotypes or intermediate traits for BP were analyzed. In the first part of the thesis, we examined the role of the allelic variants of the TSNAX/DISC1 gene cluster to psychotic and bipolar spectrum disorders and found association of distinct allelic haplotypes with these two groups of disorders. The haplotype at the 5’ end of the Disrupted-in-Schizophrenia-1 gene (DISC1) was over-transmitted to males with psychotic disorder (p = 0.008; for an extended haplotype p = 0.0007 with both genders), whereas haplotypes at the 3’ end of DISC1 associated with bipolar spectrum disorder (p = 0.0002; for an extended haplotype p = 0.0001). The variants of these haplotypes also showed association with different cognitive traits. The haplotypes at the 5’ end associated with perseverations and auditory attention, while the variants at the 3’ end associated with several cognitive traits including verbal fluency and psychomotor processing speed. Second, in our complete set of BP families with 723 individuals we studied six functional candidate genes from three distinct signalling systems: serotonin-related genes (SLC6A4 and TPH2), BDNF -related genes (BDNF, CREB1 and NTRK2) and one gene related to the inflammation and cytokine system (P2RX7). We replicated association of the functional variant Val66Met of BDNF with BP and better performance in retention. The variants at the 5’ end of SLC6A4 also showed some evidence of association among males (p = 0.004), but the widely studied functional variants did not yield any significant results. A protective four-variant haplotype on P2RX7 showed evidence of association with BP and executive functions: semantic and phonemic fluency (p = 0.006 and p = 0.0003, respectively). Third, we analyzed 23 bipolar families originating from the North-Eastern region of Finland. A genome-wide scan was performed using the 6K single nucleotide polymorphism (SNP) array. We identified susceptibility loci at chromosomes 7q31 with a LOD score of 3.20 and at 9p13.1 with a LOD score of 4.02. We followed up both linkage findings in the complete set of 179 Finnish bipolar families. The finding on chromosome 9p13 was supported (maximum LOD score of 3.02), but the susceptibility gene itself remains unclarified. In the fourth part of the thesis, we wanted to test the role of the allelic variants that have associated with bipolar disorder in recent genome-wide association studies (GWAS). We could confirm findings for the DFNB31, SORCS2, SCL39A3, and DGKH genes. The best signal in this study comes from DFNB31, which remained significant after multiple testing corrections. Two variants of SORCS2 were allelic replications and presented the same signal as the haplotype analysis. However, no association was detected with the PALB2 gene, which was the most significantly associated region in the previous GWAS. Our results indicate that BP is heterogeneous and its genetic background may accordingly vary in different populations. In order to fully understand the allelic heterogeneity that underlies common diseases such as BP, complete genome sequencing for many individuals with and without the disease is required. Identification of the specific risk variants will help us better understand the pathophysiology underlying BP and will lead to the development of treatments with specific biochemical targets. In addition, it will further facilitate the identification of environmental factors that alter risk, which will potentially provide improved occupational, social and psychological advice for individuals with high risk of BP.

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.

Genome-wide association study of Lp-PLA(2) activity and mass in the Framingham Heart Study.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an emerging risk factor and therapeutic target for cardiovascular disease. The activity and mass of this enzyme are heritable traits, but major genetic determinants have not been explored in a systematic, genome-wide fashion. We carried out a genome-wide association study of Lp-PLA(2) activity and mass in 6,668 Caucasian subjects from the population-based Framingham Heart Study. Clinical data and genotypes from the Affymetrix 550K SNP array were obtained from the open-access Framingham SHARe project. Each polymorphism that passed quality control was tested for associations with Lp-PLA(2) activity and mass using linear mixed models implemented in the R statistical package, accounting for familial correlations, and controlling for age, sex, smoking, lipid-lowering-medication use, and cohort. For Lp-PLA(2) activity, polymorphisms at four independent loci reached genome-wide significance, including the APOE/APOC1 region on chromosome 19 (p = 6 x 10(-24)); CELSR2/PSRC1 on chromosome 1 (p = 3 x 10(-15)); SCARB1 on chromosome 12 (p = 1x10(-8)) and ZNF259/BUD13 in the APOA5/APOA1 gene region on chromosome 11 (p = 4 x 10(-8)). All of these remained significant after accounting for associations with LDL cholesterol, HDL cholesterol, or triglycerides. For Lp-PLA(2) mass, 12 SNPs achieved genome-wide significance, all clustering in a region on chromosome 6p12.3 near the PLA2G7 gene. Our analyses demonstrate that genetic polymorphisms may contribute to inter-individual variation in Lp-PLA(2) activity and mass.

Étude de facteurs génétiques prédictifs dans le neuroblastome, en particulier les anomalies du chromosmoe 14q

Le neuroblastome (NB) représente 8% de tous les cancers pédiatriques et est caractérisé par sa grande hétérogénéité clinique. Afin d’évaluer son pronostic, plusieurs facteurs génétiques sont utilisés : amplification de MYCN, délétion 1p, gain 11q et gain 17q. Les buts de notre travail étaient d’abord de vérifier si l’hybridation in situ en fluorescence (FISH) permet une analyse complète de ces anomalies et ensuite, en utilisant une analyse globale du génome telle le polymorphisme nucléotidique simple (SNP), de vérifier la concordance avec les résultats de la FISH et le pronostic potentiel des anomalies du 14q, en particulier du gène AKT. Nous avons donc établi un panel de sondes pour la FISH qui a été appliqué sur 16 tumeurs non-fixées. Après isolation de l’ADN de 36 tumeurs, nous avons effectué une analyse génotypique par SNP utilisant les puces « Affymetrix Genome-Wide Human SNP Array 6.0 » contenant 945,826 sondes non polymorphiques et 906,000 sondes polymorphiques. Nos résultats ont démontré que la FISH permet l’évaluation complète des anomalies génétiques importantes du NB et que les anomalies déséquilibrées sont détectées très précisément par SNP. Les anomalies du 14q tendent à être associées avec des facteurs cliniques comme le grade et l’évolution, contrairement aux anomalies d’AKT. L’analyse du 14q a révélé trois gènes d’intérêt, MAX, BCL11B et GPHN, qui devraient être analysés sur un plus grand échantillon. Ainsi, l’étude par FISH semble adaptée pour détecter les anomalies génétiques classiques du NB, alors que celles retrouvées en 14q représentent de potentielles cibles thérapeutiques pour cette tumeur.

Analyse génotypique des cellules initiatrices de tumeurs exprimant CD133 dans le neuroblastome

Le neuroblastome (NB) est la tumeur solide extracranienne la plus fréquente et mortelle chez les jeunes enfants. Il se caractérise par une résistance à la chimiothérapie possiblement en partie dû à la présence de cellules initiatrices de tumeurs (TICs). Des études ont mis en évidence le rôle de CD133 comme un marqueur des TICs dans divers types de cancers. Les buts de notre travail étaient d’abord de démontrer les vertus de TICs des cellules exprimant CD133 et ensuite, en utilisant une analyse globale du génome avec des polymorphismes nucléotidiques simples (SNPs), d’effectuer une analyse différentielle entre les TICs et les autres cellules du NB afin d’en identifier les anomalies génétiques spécifiques. Des lignées cellulaires de NB ont été triées par cytométrie de flux afin d’obtenir deux populations: une enrichie en CD133 (CD133high), l’autre faible en CD133 (CD133low). Afin de déterminer si ces populations cellulaires présentent des propriétés de TICs, des essais sur les neurosphères, les colonies en agar mou et les injections orthotopiques de 500 cellules sélectionnées dans 11 souris ont été réalisées. Après une isolation de l’ADN des populations sélectionnées, nous avons effectué une analyse génotypique par SNP utilisant les puces « Affymetrix Genome-Wide Human SNP Array 6.0 ». Pour vérifier l’expression des gènes identifiés, des Western Blots ont été réalisés. Nos résultats ont démontré que la population CD133 avait des propriétés de TICs in vitro et in vivo. L’analyse génotypique différentielle a permis d’identifier deux régions communes (16p13.3 and 19p13.3) dans la population CD133high ayant des gains et deux autres régions (16q12.1 and 21q21.3) dans la population CD133low possédant des pertes d’hétérozygoties (LOH). Aucune perte n’a été observée. Parmi les gènes étudiés, l’expression protéique d’éphrine-A2 était corrélée à celle de CD133 dans 6 tumeurs et 2 lignées cellulaires de NB. De plus, l’augmentation de la concentration d’anticorps anti-éphrine-A2 dans le milieu diminue la taille des neurosphères. Ainsi, la population CD133high, qui a des vertus de TICs, possède des caractéristiques génotypiques différentes par rapport à celle CD133low. La présence d’éphrine-A2 dans les cellules exprimant CD133 souligne son importance dans le développement des TICs. Ces résultats suggèrent la présence de potentielle cible pour de nouvelles thérapeutiques ciblant les TICs mise en évidence par l’étude génomique.

An eight-parent multiparent advanced generation inter-cross population for winter-sown wheat: creation, properties, and validation

MAGIC populations represent one of a new generation of crop genetic mapping resources combining high genetic recombination and diversity. We describe the creation and validation of an eight-parent MAGIC population consisting of 1091 F7 lines of winter-sown wheat (Triticum aestivum L.). Analyses based on genotypes from a 90,000-single nucleotide polymorphism (SNP) array find the population to be well-suited as a platform for fine-mapping quantitative trait loci (QTL) and gene isolation. Patterns of linkage disequilibrium (LD) show the population to be highly recombined; genetic marker diversity among the founders was 74% of that captured in a larger set of 64 wheat varieties, and 54% of SNPs segregating among the 64 lines also segregated among the eight founder lines. In contrast, a commonly used reference bi-parental population had only 54% of the diversity of the 64 varieties with 27% of SNPs segregating. We demonstrate the potential of this MAGIC resource by identifying a highly diagnostic marker for the morphological character "awn presence/absence" and independently validate it in an association-mapping panel. These analyses show this large, diverse, and highly recombined MAGIC population to be a powerful resource for the genetic dissection of target traits in wheat, and it is well-placed to efficiently exploit ongoing advances in phenomics and genomics. Genetic marker and trait data, together with instructions for access to seed, are available at http://www.niab.com/MAGIC/.

Genome-wide association mapping in a wild avian population identifies a link between genetic and phenotypic variation in a life-history trait

Understanding the genetic basis of traits involved in adaptation is a major challenge in evolutionary biology but remains poorly understood. Here, we use genome-wide association mapping using a custom 50 k single nucleotide polymorphism (SNP) array in a natural population of collared flycatchers to examine the genetic basis of clutch size, an important life-history trait in many animal species. We found evidence for an association on chromosome 18 where one SNP significant at the genome-wide level explained 3.9% of the phenotypic variance. We also detected two suggestive quantitative trait loci (QTLs) on chromosomes 9 and 26. Fitness differences among genotypes were generally weak and not significant, although there was some indication of a sex-by-genotype interaction for lifetime reproductive success at the suggestive QTL on chromosome 26. This implies that sexual antagonism may play a role in maintaining genetic variation at this QTL. Our findings provide candidate regions for a classic avian life-history trait that will be useful for future studies examining the molecular and cellular function of, as well as evolutionary mechanisms operating at, these loci.

Prospection of genomic regions divergently selected in racing line of Quarter Horses in relation to cutting line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prospection of genomic regions divergently selected in cutting line of quarter horses in relation to racing line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The search for Multiple Myeloma Stem cells: molecular characterization and self-renewal mechanisms involved in the disease persistence

The existence of Multiple Myeloma Stem cells (MMSCs)is supposed to be one of the major causes of MM drug-resistance. However, very little is known about the molecular characteristics of MMSCs, even if some studies suggested that these cells resembles the memory B cells. In order to molecularly characterize MMSCs, we isolated the 138+138- population. For each cell fraction we performed a VDJ rearrangement analysis. The complete set of aberrations were performed by SNP Array 6.0 and HG-U133 Plus 2.0 microarray analyses (Affymetrix). The VDJ rearrangement analyses confirmed the clonal relationship between the 138+ clone and the immature clone. Both BM and PBL 138+ clones showed exactly the same genomic macroalterations. In the BM and PBL 138-19+27+ cell fractions several micro-alterations (range: 1-350 Kb) unique of the memory B cells clone were highlighted. Any micro-alterations detected were located out of any genomic variants region and are presumably associated to the MM pathogenesis, as confirmed by the presence of KRAS, WWOX and XIAP genes among the amplified regions. To get insight into the biology of the clonotypic B cell population, we compared the gene expression profile of 8 MM B cells samples 5 donor B cells vs, thus showing a differential expression of 11480 probes (p-value: <0,05). Among the self-renewal mechanisms, we observed the down-regulation of Hedgehog pathway and the iperactivation of Notch and Wnt signaling. Moreover, these immature cells showed a particular phenotype correlated to resistance to proteasome inhibitors (IRE1α-XBP1: -18.0; -19.96. P<0,05). Data suggested that the MM 138+ clone might resume the end of the complex process of myelomagenesis, whereas the memory B cells have some intriguing micro-alterations and a specific transcriptional program, supporting the idea that these post germinal center cells might be involved in the transforming event that originate and sustain the neoplastic clone.

DNA-Reparatur-assoziierte genetische und epigenetische Faktoren für die Zweittumorentstehung nach Tumoren im Kindes- und Jugendalter

Die Ursachen der Zweittumorentwicklung bei Personen, die eine Krebserkrankung in der Kindheit überlebten, sind weitgehend unklar. Strahlenexposition oder Chemotherapie führen in normalen somatischen Zellen zu DNA-Schäden, welche bei fehlerhafter Reparatur eine Karzinogenese auslösen können. Es ist denkbar, dass genetische Unterschiede z. B. in den Signalwegen der Zellzykluskontrolle und der DNA-Reparatur nach therapieinduzierten DNA-Schäden eine entscheidende Rolle bei der Zweittumorentwicklung spielen. Im Rahmen dieser Arbeit wurden 20 Personen, die eine Krebserkrankung in der Kindheit überlebten und einen unabhängigen Zweittumor entwickelten, mit 20 gematchten Kontrollpersonen ohne Zweittumorentwicklung verglichen. Die primären Fibroblasten der Patienten wurden auf somatische, genetische und/oder epigenetische Unterschiede in DNA-Reparaturnetzwerken untersucht. Die biologisch relevantesten Ergebnisse lieferten Proteinuntersuchungen mittels Antikörper-Microarrays. Hierbei wurde eine konstitutiv erniedrigte Menge an RAD9A und einigen anderen DNA-Reparatur-Proteinen (BRCA1, DDIT3, MSH6, p53, RAD51) in den Zweittumorpatienten im Vergleich zu den Eintumorpatienten festgestellt. Nach einer DNA-Schädigung durch 1 Gray Bestrahlung erhöhte sich die RAD9A-Proteinmenge, wobei die Zweittumorpatienten eine geringere Induktion als die Eintumorpatienten zeigten. Bei der Quantifizierung der mRNA-Expression mittels RTq-PCR wurde ein niedrigerer RAD9A-mRNA-Level sowohl in den unbehandelten und als auch in den 1 Gray bestrahlten Zellen der Zweittumorpatienten festgestellt. SNP-Array und Methylierungsanalysen konnten keine Auffälligkeiten im RAD9A-Lokus nachweisen. Diese Ergebnisse unterstützen die Hypothese, dass Modulationen von RAD9A und anderen Zellzyklusarrest- und DNA-Reparaturproteinen zum Risiko einer Zweittumorentwicklung in Kinderkrebspatienten beitragen. Bei einem diskordanten monozygoten Zwillingspaar wurde in ca. 20% der Zellen des Zweittumorzwillings eine Hypermethylierung des Tumorsuppressorgens BRCA1 festgestellt, die mit einer konstitutiv erniedrigten BRCA1-Proteinexpression einhergeht und einen möglichen Krebsrisikofaktor darstellt. Die partielle Deletion des Gens RSPO3, die wahrscheinlich als somatisches Zellmosaik beim Zweittumorzwilling vorliegt, korreliert mit einer niedrigeren RSPO3-mRNA-Expression und ist vermutlich auch mit einer erhöhten Krebsprädisposition assoziiert.

LUPA: a European initiative taking advantage of the canine genome architecture for unravelling complex disorders in both human and dogs

The domestic dog offers a unique opportunity to explore the genetic basis of disease, morphology and behaviour. Humans share many diseases with our canine companions, making dogs an ideal model organism for comparative disease genetics. Using newly developed resources, genome-wide association studies in dog breeds are proving to be exceptionally powerful. Towards this aim, veterinarians and geneticists from 12 European countries are collaborating to collect and analyse the DNA from large cohorts of dogs suffering from a range of carefully defined diseases of relevance to human health. This project, named LUPA, has already delivered considerable results. The consortium has collaborated to develop a new high density single nucleotide polymorphism (SNP) array. Mutations for four monogenic diseases have been identified and the information has been utilised to find mutations in human patients. Several complex diseases have been mapped and fine mapping is underway. These findings should ultimately lead to a better understanding of the molecular mechanisms underlying complex diseases in both humans and their best friend.

Microphthalmia in Texel sheep is associated with a missense mutation in the paired-like homeodomain 3 (PITX3) gene

Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia.

A chromosomal region on ECA13 is associated with maxillary prognathism in horses.

Hereditary variations in head morphology and head malformations are known in many species. The most common variation encountered in horses is maxillary prognathism. Prognathism and brachygnathism are syndromes of the upper and lower jaw, respectively. The resulting malocclusion can negatively affect teeth wear, and is considered a non-desirable trait in breeding programs. We performed a case-control analysis for maxillary prognathism in horses using 96 cases and 763 controls. All horses had been previously genotyped with a commercially available 50 k SNP array. We analyzed the data with a mixed-model considering the genomic relationships in order to account for population stratification. Two SNPs within a region on the distal end of chromosome ECA 13 reached the Bonferroni corrected genome-wide significance level. There is no known prognathism candidate gene located within this region. Therefore, our findings in the horse offer the possibility of identifying a novel gene involved in the complex genetics of prognathism that might also be relevant for humans and other livestock species.