Molecular genomics of a genetic code alteration

The genetic code is not universal. Alterations to its standard form have been discovered in both prokaryotes and eukaryotes and demolished the dogma of an immutable code. For instance, several Candida species translate the standard leucine CUG codon as serine. In the case of the human pathogen Candida albicans, a serine tRNA (tRNACAGSer) incorporates in vivo 97% of serine and 3% of leucine in proteins at CUG sites. Such ambiguity is flexible and the level of leucine incorporation increases significantly in response to environmental stress. To elucidate the function of such ambiguity and clarify whether the identity of the CUG codon could be reverted from serine back to leucine, we have developed a forced evolution strategy to increase leucine incorporation at CUGs and a fluorescent reporter system to monitor such incorporation in vivo. Leucine misincorporation increased from 3% up to nearly 100%, reverting CUG identity from serine back to leucine. Growth assays showed that increasing leucine incorporation produced impressive arrays of phenotypes of high adaptive potential. In particular, strains with high levels of leucine misincorporation exhibited novel phenotypes and high level of tolerance to antifungals. Whole genome re-sequencing revealed that increasing levels of leucine incorporation were associated with accumulation of single nucleotide polymorphisms (SNPs) and loss of heterozygozity (LOH) in the higher misincorporating strains. SNPs accumulated preferentially in genes involved in cell adhesion, filamentous growth and biofilm formation, indicating that C. albicans uses its natural CUG ambiguity to increase genetic diversity in pathogenesis and drug resistance related processes. The overall data provided evidence for unantecipated flexibility of the C. albicans genetic code and highlighted new roles of codon ambiguity on the evolution of genetic and phenotypic diversity.

Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n 5 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

A recent study characterizing bacteriophage populations within human caecal effluent demonstrated the presence of numerous Podoviridae, Siphoviridae and Myoviridae within this material (Hoyles et al., 2014, Res Microbiol 165, 803–812). Further to this work, anaerobic bacteria were isolated on fastidious anaerobe agar from the caecal effluent of a healthy 31-year-old woman. Ten colonies were selected at random, streaked to purity and screened against the remaining caecal effluent (filter-sterilized, 0.45 μm pore size) in an attempt to isolate lytic bacteriophages. Bacteriophages within the effluent [2×105 ± 2.65×103 (n=3) pfu/ml] were active against five of the isolates, all identified by 16S rRNA gene sequence analysis as Klebsiella pneumoniae. One of the five isolates, L4-FAA5, was characterized further and found to be K. pneumoniae subsp. pneumoniae capsule type K2 rmpA+, and was used to propagate a bacteriophage (which we named KLPN1) to purity. Bacteriophage KLPN1 was a member of the Siphoviridae with a rosette-like tail tip and exhibited depolymerase activity, demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of 21 clinical strains representing unknown K. pneumoniae subsp. pneumoniae capsule types and types K1, K2, K5, K20, K54 and K57, KLPN1 infected only K2 strains, but did not exhibit depolymerase activity against these. Whole-genome sequence analysis of KLPN1 showed the bacteriophage to have a genome of 49,037 bp (50.53 GC mol%) comprising 73 predicted ORFs, of which 22 encoded genes associated with structure, host recognition, packaging, DNA replication and cell lysis. The host recognition-associated gene was a potential depolymerase. This is the first report of the isolation of a bacterium–bacteriophage combination from the human caecum, and only the third member of the Siphoviridae known to infect K. pneumoniae subsp. pneumoniae.

Ankylosing spondylitis: genomic and functional characterization of candidate genes and their repercussion in clinical practice

RESUMO: Introdução: A espondilite anquilosante (EA) é uma doença inflamatória crónica caracterizada pela inflamação das articulações sacroilíacas e da coluna. A anquilose progressiva motiva uma deterioração gradual da função física e da qualidade de vida. O diagnóstico e o tratamento precoces podem contribuir para um melhor prognóstico. Neste contexto, a identificação de biomarcadores, assume-se como sendo muito útil para a prática clínica e representa hoje um grande desafio para a comunidade científica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar possíveis associações entre genes, MHC e não-MHC, com a suscetibilidade e as características fenotípicas da EA; 3 - identificar genes candidatos associados a EA através da tecnologia de microarray. Material e Métodos: Foram recrutados doentes com EA, de acordo com os critérios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demográficos, clínicos e radiológicos e colhidas amostras de sangue periférico. Selecionaram-se de forma aleatória, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os haplótipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes alélicas dos genes IL23R, ERAP1 e ANKH foram estudadas através de ensaios de discriminação alélica TaqMan. A análise de associação foi realizada utilizando testes da Cochrane-Armitage e de regressão linear, tal como implementado pelo PLINK, para variáveis qualitativas e quantitativas, respetivamente. O estudo de expressão génica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram incluídos 369 doentes (62,3% do sexo masculino, com idade média de 45,4 ± 13,2 anos, duração média da doença de 11,4 ± 10,5 anos). No momento da avaliação, 49,9% tinham doença axial, 2,4% periférica, 40,9% mista e 7,1% entesopática. A uveíte anterior aguda (33,6%) foi a manifestação extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os haplótipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir proteção em termos de atividade, repercussão funcional e radiológica da doença. Três variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associação com a doença, confirmando a associação destes genes com a EA na população Portuguesa. O mesmo não se verificou com as variantes estudadas do ANKH. Não se verificou associação entre as variantes génicas não-MHC e as manifestações clínicas da EA. Foi identificado um perfil de expressão génica para a EA, tendo sido validados catorze genes - alguns têm um papel bem documentado em termos de inflamação, outros no metabolismo da cartilagem e do osso. Conclusões: Foi estabelecido um perfil demográfico e clínico dos doentes com EA em Portugal. A identificação de variantes génicas e de um perfil de expressão contribuem para uma melhor compreensão da sua fisiopatologia e podem ser úteis para estabelecer modelos com relevância em termos de diagnóstico, prognóstico e orientação terapêutica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4±13.2 years; mean disease duration 11.4±10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.

Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Pervasive positive selection on duplicated and nonduplicated vertebrate protein coding genes.

A stringent branch-site codon model was used to detect positive selection in vertebrate evolution. We show that the test is robust to the large evolutionary distances involved. Positive selection was detected in 77% of 884 genes studied. Most positive selection concerns a few sites on a single branch of the phylogenetic tree: Between 0.9% and 4.7% of sites are affected by positive selection depending on the branches. No functional category was overrepresented among genes under positive selection. Surprisingly, whole genome duplication had no effect on the prevalence of positive selection, whether the fish-specific genome duplication or the two rounds at the origin of vertebrates. Thus positive selection has not been limited to a few gene classes, or to specific evolutionary events such as duplication, but has been pervasive during vertebrate evolution.

Genetics of calcium homeostasis in humans: continuum between monogenic diseases and continuous phenotypes

Extracellular calcium participates in several key physiological functions, such as control of blood coagulation, bone calcification or muscle contraction. Calcium homeostasis in humans is regulated in part by genetic factors, as illustrated by rare monogenic diseases characterized by hypo or hypercalcaemia. Both serum calcium and urinary calcium excretion are heritable continuous traits in humans. Serum calcium levels are tightly regulated by two main hormonal systems, i.e. parathyroid hormone and vitamin D, which are themselves also influenced by genetic factors. Recent technological advances in molecular biology allow for the screening of the human genome at an unprecedented level of detail and using hypothesis-free approaches, such as genome-wide association studies (GWAS). GWAS identified novel loci for calcium-related phenotypes (i.e. serum calcium and 25-OH vitamin D) that shed new light on the biology of calcium in humans. The substantial overlap (i.e. CYP24A1, CASR, GATA3; CYP2R1) between genes involved in rare monogenic diseases and genes located within loci identified in GWAS suggests a genetic and phenotypic continuum between monogenic diseases of calcium homeostasis and slight disturbances of calcium homeostasis in the general population. Future studies using whole-exome and whole-genome sequencing will further advance our understanding of the genetic architecture of calcium homeostasis in humans. These findings will likely provide new insight into the complex mechanisms involved in calcium homeostasis and hopefully lead to novel preventive and therapeutic approaches. Keyword: calcium, monogenic, genome-wide association studies, genetics.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

The Stealthy Superbug: the Role of Asymptomatic Enteric Carriage in Maintaining a Long-Term Hospital Outbreak of ST228 Methicillin-Resistant Staphylococcus aureus.

UNLABELLED: Whole-genome sequencing (WGS) of 228 isolates was used to elucidate the origin and dynamics of a long-term outbreak of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 228 (ST228) SCCmec I that involved 1,600 patients in a tertiary care hospital between 2008 and 2012. Combining of the sequence data with detailed metadata on patient admission and movement confirmed that the outbreak was due to the transmission of a single clonal variant of ST228, rather than repeated introductions of this clone into the hospital. We note that this clone is significantly more frequently recovered from groin and rectal swabs than other clones (P < 0.0001) and is also significantly more transmissible between roommates (P < 0.01). Unrecognized MRSA carriers, together with movements of patients within the hospital, also seem to have played a major role. These atypical colonization and transmission dynamics can help explain how the outbreak was maintained over the long term. This "stealthy" asymptomatic colonization of the gut, combined with heightened transmissibility (potentially reflecting a role for environmental reservoirs), means the dynamics of this outbreak share some properties with enteric pathogens such as vancomycin-resistant enterococci or Clostridium difficile. IMPORTANCE: Using whole-genome sequencing, we showed that a large and prolonged outbreak of methicillin-resistant Staphylococcus aureus was due to the clonal spread of a specific strain with genetic elements adapted to the hospital environment. Unrecognized MRSA carriers, the movement of patients within the hospital, and the low detection with clinical specimens were also factors that played a role in this occurrence. The atypical colonization of the gut means the dynamics of this outbreak may share some properties with enteric pathogens.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (similar to 22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression "signatures" are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.

Performances de la puce exon et son application dans l’analyse de l’épissage alternatif associé à la métastase du cancer de sein

Nous montrons l’utilisation de la puce exon d’Affymetrix pour l’analyse simultanée de l’expression des gènes et de la variation d’isoformes. Nous avons utilisé les échantillons d’ARN du cerveau et des tissus de référence qui ont été antérieurement utilisés dans l’étude du consortium MicroArray Quality Control (MAQC). Nous démontrons une forte concordance de la quantification de l’expression des gènes entre trois plateformes d’expression populaires à savoir la puce exon d’Affymetrix, la puce Illumina et la puce U133A d’Affymetrix. Plus intéressant nous montrons que la majorité des discordances entre les trois plateformes résulterait des positions différentes des sondes à travers les plateformes et que les variations d’isoforme exactes ne peuvent être identifiées que par la puce exon. Nous avons détecté avec succès, entre les tissus de référence et ceux du cerveau, une centaine de cas d’évènements d’épissage alternatif. La puce exon est requise dans l’analyse de l’épissage alternatif associé aux pathologies telles que les cancers et les troubles neurologiques. Comme application de cette technologie, nous avons analysé les variations d’épissage dans la métastase du cancer de sein développé dans le model de la souris. Nous avons utilisé une gamme bien définie de trois lignées de tumeur mammaire ayant différents potentiels métastatiques. Par des analyses statistiques, nous avons répertorié 2623 transcripts présentant des variations d’expression et d’isoformes entre les types de tumeur. Une analyse du réseau de gènes montre qu’environ la moitié d’entre eux est impliquée dans plusieurs activités cellulaires, ainsi que dans nombreux cancers et désordres génétiques.

Étude clinique et génétique d’une nouvelle forme d’ataxie spinocérébelleuse pure associée à l’Érythrokératodermie

Nous présentons ici la description clinique et génétique d’un syndrome neurocutané unique. Le laboratoire du Dr Cossette a entrepris la caractérisation clinique et génétique d'une famille canadienne-française qui a été identifiée par les Drs Giroux et Barbeau en 1972 et qui comprend plus de 100 personnes sur six générations. Les membres atteints de cette famille présentent des lésions typiques d'érythrokératodermie (EK) (OMIM 133190, EKV1 et EKV2), associées à une ataxie spinocérébelleuse pure. Dans cette famille, l'ataxie est caractérisée par des troubles de la coordination et de la démarche causés par une dégénérescence du cervelet et de la moelle épinière. Cette ataxie est transmise selon un mode autosomique dominant. Une étude antérieure de cette variante d'EK avec ataxie avait suggéré une liaison sur le chromosome 1p34-p35, soit la même région que les formes EKV de type 1 et 2, causées respectivement par des mutations dans les gènes connexin-31 (GJB3; OMIM 603324) et connexin-30.3 (GJB4; OMIM 605425). Cependant, aucune mutation n'a été retrouvée dans ces gènes pour la famille canadienne-française. Nous avons récemment recontacté la famille et effectué des examens détaillés, incluant une imagerie par résonance magnétique (IRM) et un électromyogramme (EMG). Les manifestations neurologiques des individus atteints sont compatibles avec une nouvelle forme d’ataxie cérébelleuse pure à transmission autosomique dominante (ADCA de type III dans la classification de Harding) que nous avons appelée SCA34. Une cartographie complète du génome nous a permis de localiser le gène SCA34 sur le chromosome 6p12.3-q16.2. Également, en collaboration avec les Drs Alexis Brice (Hôpital Pitié-La Salpêtrière, Paris) et Alfredo Brusco (Hôpital San Giovanni Battista di Torino, Italie), nous avons confirmé que trois autres familles européennes avec SCA inexpliquée étaient également liées au locus SCA34. Notre laboratoire a récemment entrepris la recherche des mutations responsables de SCA34. Les résultats de ce criblage de gènes candidats sont présentés dans le chapitre 3 de cette thèse.

Algorithmes pour la reconstruction de génomes ancestraux

L’inférence de génomes ancestraux est une étape essentielle pour l’étude de l’évolution des génomes. Connaissant les génomes d’espèces éteintes, on peut proposer des mécanismes biologiques expliquant les divergences entre les génomes des espèces modernes. Diverses méthodes visant à résoudre ce problème existent, se classant parmis deux grandes catégories : les méthodes de distance et les méthodes de synténie. L’état de l’art des distances génomiques ne permettant qu’un certain répertoire de réarrangements pour le moment, les méthodes de synténie sont donc plus appropriées en pratique. Nous proposons une méthode de synténie pour la reconstruction de génomes ancestraux basée sur une définition relaxée d’adjacences de gènes, permettant un contenu en gène inégal dans les génomes modernes causé par des pertes de gènes de même que des duplications de génomes entiers (DGE). Des simulations sont effectuées, démontrant une capacité de former une solution assemblée en un nombre réduit de régions ancestrales contigües par rapport à d’autres méthodes tout en gardant une bonne fiabilité. Des applications sur des données de levures et de plantes céréalières montrent des résultats en accord avec d’autres publications, notamment la présence de fusion imbriquée de chromosomes pendant l’évolution des céréales.

Mécanismes moléculaires de la réplication préférentielle du VIH-1 dans les cellules à polarisation Th1Th17 versus Th1 : rôle de PPARG dans la régulation négative de la réplication virale

Les cellules T CD4+ humaines sont hétérogènes du point de vue de la permissivité à l’infection par le virus de l’immunodéficience humaine de type 1 (VIH-1). Notre laboratoire a préalablement démontré que les cellules Th1 à phénotype CXCR3+CCR6- sont relativement résistantes à l’infection par le VIH-1 alors que les cellules Th1Th17 à phénotype CXCR3+CCR6+ y sont hautement permissives. La réplication du VIH dépend de plusieurs facteurs cellulaires de restriction ou de permissivité agissant à différentes étapes du cycle viral. Toutefois, malgré plusieurs avancées, la compréhension des voies de signalisation cellulaire impliquées dans la régulation de la réplication du VIH est encore limitée. L’objectif majeur de ce projet de maîtrise est de caractériser les mécanismes moléculaires de la permissivité et de la résistance au VIH respectivement dans les cellules Th1Th17 et Th1. Ce mémoire est divisé en quatre parties qui visent: (i) l’identification des voies canoniques et des fonctions biologiques différemment régulées dans les cellules Th1Th17 versus Th1 par l’analyse de leur transcriptome au niveau du génome entier; (ii) la validation de l’expression différentielle des gènes d’intérêt identifiés par biopuces au niveau des transcrits et des protéines; (iii) la caractérisation du rôle fonctionnel de certains de ces facteurs (i.e., PPARG, AhR) sur la réplication du VIH dans les cellules Th1Th17 versus Th1; et (iv) l’identification du niveau auquel ces facteurs interfèrent avec le cycle de réplication du VIH. Nos résultats d’analyse du transcriptome du génome entier par Gene Set Enrichment Analysis et Ingenuity Pathway Analysis indiquent que les cellules à profil Th1Th17 sont plus susceptibles à l’activation cellulaire et à l’apoptose, favorisent plus l’inflammation et expriment moins fortement les gènes liés à la dégradation protéosomale comparé aux cellules à profil Th1. Ces différences dans la régulation de diverses voies et fonctions biologiques permettent en partie d’expliquer la susceptibilité à l’infection par le VIH dans ces cellules. Nous avons ensuite confirmé l’expression différentielle de certains gènes d’intérêt dans les cellules Th1Th17 (CXCR6, PPARG, ARNTL, CTSH, PTPN13, MAP3K4) versus Th1 (SERPINB6, PTK2) au niveau de l’ARNm et des protéines. Finalement, nous avons démontré le rôle des facteurs de transcription PPARG et AhR dans la régulation de la réplication du VIH. L’activation de la voie PPARG par la rosiglitazone induit la diminution importante de la réplication du VIH dans les cellules T CD4+, alors que l’activation de la voie AhR par les ligands exogènes TCDD et FICZ augmente de façon significative la réplication virale. Nous proposons que la voie PPARG agit comme un régulateur négatif de la réplication du VIH dans ces cellules, en interférant avec la polarisation Th17 et probablement en inhibant l’activité transcriptionnelle du facteur NF-kB. Les rôles des formes nucléaires versus cytoplasmiques du récepteur Ahr semblent être diamétralement opposés, dans la mesure où l’interférence ARN contre AhR s’associe également à l’augmentation de la réplication virale. Il est ainsi possible que la forme cytoplasmique d’AhR, connue par son activité E3 ligase, participe à la dégradation protéosomale des particules virales. Le mécanisme par lequel le AhR nucléaire versus cytoplasmique interfère avec la réplication virale est en cours d’étude au laboratoire. Cette étude représente la première caractérisation de l’expression différentielle de gènes au niveau du génome entier de sous-populations T CD4+ permissives versus résistantes à l’infection par le VIH. Nos résultats identifient de nouvelles cibles moléculaires pour de nouvelles stratégies thérapeutiques visant à limiter la réplication du VIH dans les lymphocytes T CD4+ primaires.