Characterization of simple and complex genomic structural variation : a study of human populations and leukaemia

Over the last ten years, improvements in molecular techniques and the arrival of the next-generation sequencing technologies have revealed a large amount of structural variation (SV) in the human genome. Consequently, there has been a significant increase in interest from the scientific community to understand the role of the SV in diseases, such as cancer, or in determining phenotypic traits in the general population. The objective of this thesis has been to study in depth the characterization and the functional importance of the SV, through the analysis of different methods for its detection and its biological impact in two different contexts. First, we have analysed the presence of copy-number variants in several human populations using a microarray approach and, by validating one of the detected regions, we have confirmed the reliability of this method for the detection of this type of SV. Second, through the chronic lymphocytic leukaemia (CLL) genome project, we have identified structural variants in patients with CLL by whole-genome sequencing. To obtain a comprehensive analysis of the SV in cancer genomes, we have developed a computational tool with the capacity to characterize and define all forms of the SV using next-generation sequencing data. With this tool we have detected, on one hand, some novel variants in CLL and, on the other hand, a high level of genomic complexity in one of the patients studied. From this last case, we have carried out the evaluation of the phenotypic impact of the complex variants in the progression of the CLL, which has allowed us to determine the importance of analysing cancer as a dynamic process undergoing evolutionary changes over timeEn els últims deu anys, les millores en tècniques moleculars i l’aparició d’una nova generació de tècniques de seqüenciació han revelat que existeix una gran quantitat de variació estructural (SV, en anglès) en el genoma humà. En conseqüència, hi ha hagut un augment significatiu en l’interès de la comunitat científica per entendre el paper que la SV juga en malalties com el càncer, o en la determinació de trets fenotípics en la població general. L’objectiu d’aquesta tesi ha estat aprofundir en la caracterització i la importància funcional de la SV, analitzant diferents mètodes per a la seva detecció i el seu impacte biològic en dos contextos específics. En primer lloc, hem analitzat la presència de variants en nombre de còpia en diferents poblacions humanes mitjançant una tècnica de microarray i, a través de la validació d’una de les regions trobades, hem pogut confirmar la fiabilitat d’aquesta tècnica per detectar aquest tipus de SV. En segon lloc, a través del projecte del genoma de la leucèmia limfàtica crònica (LLC), hem caracteritzat variants estructurals en pacients amb LLC mitjançant la seqüenciació completa del seu genoma. Per tal d’obtenir un anàlisi el màxim d’exhaustiu de la SV en genomes de càncer, hem desenvolupat una eina computacional capaç de caracteritzar i definir totes les formes possibles de SV utilitzant dades de seqüenciació. Amb aquesta eina hem pogut detectar, per una banda, algunes variants noves en LLC i, per l’altra, un alt nivell de complexitat genòmica en un dels pacients. A partir d’aquest últim cas hem dut a terme l’avaluació de l’impacte fenotípic d’un patró de SV complex en la progressió de la LLC, la qual cosa ens ha permès determinar la importància d’analitzar el càncer com a un procés dinàmic sotmès a canvis evolutius al llarg del temps

A role for CD154, the CD40 ligand, in granulomatous inflammation

Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation

Worldwide population distribution of the common LCE3C-LCE3B deletion associated with psoriasis and other autoimmune disorders

Background: There is increasing evidence of the importance of copy number variants (CNV) in genetic diversity among individuals and populations, as well as in some common genetic diseases. We previously characterized a common 32-kb insertion/deletion variant of the PSORS4 locus at chromosome 1q21 that harbours the LCE3C and LCE3B genes. This variant allele (LCE3C_LCE3B-del) is common in patients with psoriasis and other autoimmune disorders from certain ethnic groups./nResults: Using array-CGH (Agilent 244 K) in samples from the HapMap and Human Genome Diversity Panel (HGDP) collections, we identified 54 regions showing population differences in comparison to Africans. We provided here a comprehensive population-genetic analysis of one of these regions, which involves the 32-kb deletion of the PSORS4 locus. By a PCR-based genotyping assay we characterised the profiles of the LCE3C_LCE3B-del and the linkage disequilibrium (LD) pattern between the variant allele and the tag SNP rs4112788. Our results show that most populations tend to have a higher frequency of the deleted allele than Sub-Saharan Africans. Furthermore, we found strong LD between rs4112788G and LCE3C_LCE3B-del in most non-African populations (r2 >0.8), in contrast to the low concordance between loci (r2 <0.3) in the African populations. Conclusions: These results are another example of population variability in terms of biomedical interesting CNV. The frequency distribution of the LCE3C_LCE3B-del allele and the LD pattern across populations suggest that the differences between ethnic groups might not be due to natural selection, but the consequence of genetic drift caused by the strong bottleneck that occurred during “out of Africa” expansion.The study was supported by grants from European Commission (AnEuploidy -LSHG-CT-2006-037627- and ENGAGE -ENGAGE_201413-), the “Plan Nacional” Programme of the Spanish Ministry of Economy and Competivity (NOVADIS SAF2008-00357), and the Generalitat de Catalunya (2009 SGR 0008). Mario Cáceres was supported by the Ramón y Cajal Program (Spanish Ministry of Science and Education

PeSV-Fisher: identification of somatic and non-somatic structural variants using nextgeneration sequencing data

Next-generation sequencing technologies expedited research to develop efficient computational tools for the identification of structural variants (SVs) and their use to study human diseases. As deeper data is obtained, the existence of higher complexity SVs in some genomes becomes more evident, but the detection and definition of most of these complex rearrangements is still in its infancy. The full characterization of SVs is a key aspect for discovering their biological implications. Here we present a pipeline (PeSV-Fisher) for the detection of deletions, gains, intra- and inter-chromosomal translocations, and inversions, at very reasonable computational costs. We further provide comprehensive information on co-localization of SVs in the genome, a crucial aspect for studying their biological consequences. The algorithm uses a combination of methods based on paired-reads and read-depth strategies. PeSV-Fisher has been designed with the aim to facilitate identification of somatic variation, and, as such, it is capable of analysing two or more samples simultaneously, producing a list of non-shared variants between samples. We tested PeSV-Fisher on available sequencing data, and compared its behaviour to that of frequently deployed tools (BreakDancer and VariationHunter). We have also tested this algorithm on our own sequencing data, obtained from a tumour and a normal blood sample of a patient with chronic lymphocytic leukaemia, on which we have also validated the results by targeted re-sequencing of different kinds of predictions. This allowed us to determine confidence parameters that influence the reliability of breakpoint predictions.This work was supported by AGAUR (Generalitat de Catalunya, 2009 SGR 1502) (X.E.); CIBERESP (Instituto de Salud Carlos III) (G.E.); ESGI (European Commission, 262055_ESGI) (R.R., X.E.), ENGAGE (European Commission, ENGAGE_201413), TECHGENE (European Commission, TECHGENE_223143), and GEUVADIS (European Commission, 261123_GEUVADIS) (X.E.); NOVADIS (Ministerio de Ciencia y Technologia, SAF2008-00357) (X.E.); Galicia Government Xunta de Galicia (Spain) through the project number 10PXIB918057 (J.M.C.T.); MAEC-AEC1 Predoctoral Fellowship (Ministerio de Asuntos Exteriores y Cooperación, Spain) (A.M.F.); and Ramón y Cajal position and grant BFU2007-60930 (Ministerio de Educación y Ciencia) (M.C.)

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer