Évaluation du caryotype moléculaire en tant qu’outil diagnostique chez les enfants avec déficience intellectuelle et/ou malformations congénitales

Le caryotype moléculaire permet d’identifier un CNV chez 10-14% des individus atteints de déficience intellectuelle et/ou de malformations congénitales. C’est pourquoi il s’agit maintenant de l’analyse de première intention chez ces patients. Toutefois, le rendement diagnostique n’est pas aussi bien défini en contexte prénatal et l’identification de CNVs de signification clinique incertaine y est particulièrement problématique à cause du risque d’interruption de grossesse. Nous avons donc testé 49 fœtus avec malformations majeures et un caryotype conventionnel normal avec une micropuce CGH pangénomique, et obtenu un diagnostic dans 8,2% des cas. Par ailleurs, des micropuces à très haute résolution combinant le caryotype moléculaire et le génotypage de SNPs ont récemment été introduites sur le marché. En plus d’identifier les CNVs, ces plateformes détectent les LOHs, qui peuvent indiquer la présence d’une mutation homozygote ou de disomie uniparentale. Ces anomalies pouvant être associées à la déficience intellectuelle ou à des malformations, leur détection est particulièrement intéressante pour les patients dont le phénotype reste inexpliqué. Cependant, le rendement diagnostique de ces plateformes n’est pas confirmé, et l’utilité clinique réelle des LOHs n’est toujours pas établie. Nous avons donc testé 21 enfants atteints de déficience intellectuelle pour qui les méthodes standards d’analyse génétique n’avaient pas résulté en un diagnostic, et avons pu faire passer le rendement diagnostique de 14,3% à 28,6% grâce à l’information fournie par les LOHs. Cette étude démontre l’utilité clinique d’une micropuce CGH pangénomique chez des fœtus avec malformations, de même que celle d’une micropuce SNP chez des enfants avec déficience intellectuelle.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized L-histidine, L-glutamine, L-proline, L-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Comparative genomic indexing reveals the phylogenomics of Escherichia coli pathogens

The Escherichia coli O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing E. coli O26 strains represent the same clonal lineage and that W-expressing E. coli O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the E. coli species and identified regions of variation from the E. coli K-12 chromosomal backbone.

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.)

Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype (similar to 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

Chromosome imbalances in syndromic hearing loss

The cause of hearing impairment has not been elucidated in a large proportion of patients. We screened by 1-Mb array-based comparative genomic hybridization (aCGH) 29 individuals with syndromic hearing impairment whose clinical features were not typical of known disorders. Rare chromosomal copy number changes were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage-sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2-q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but its role as a predisposition gene remains a possibility. Our results show that syndromic deafness is frequently associated with chromosome microimbalances (14-27%), and the use of aCGH for defining disease etiology is recommended.

Complex rearrangements in patients with duplications of MECP2 can occur by fork stalling and template switching

Duplication at the Xq28 band including the MECP2 gene is one of the most common genomic rearrangements identified in neurodevelopmentally delayed males. Such duplications are non-recurrent and can be generated by a non-homologous end joining (NHEJ) mechanism. We investigated the potential mechanisms for MECP2 duplication and examined whether genomic architectural features may play a role in their origin using a custom designed 4-Mb tiling-path oligonucleotide array CGH assay. Each of the 30 patients analyzed showed a unique duplication varying in size from similar to 250 kb to similar to 2.6 Mb. Interestingly, in 77% of these non-recurrent duplications, the distal breakpoints grouped within a 215 kb genomic interval, located 47 kb telomeric to the MECP2 gene. The genomic architecture of this region contains both direct and inverted low-copy repeat (LCR) sequences; this same region undergoes polymorphic structural variation in the general population. Array CGH revealed complex rearrangements in eight patients; in six patients the duplication contained an embedded triplicated segment, and in the other two, stretches of non-duplicated sequences occurred within the duplicated region. Breakpoint junction sequencing was achieved in four duplications and identified an inversion in one patient, demonstrating further complexity. We propose that the presence of LCRs in the vicinity of the MECP2 gene may generate an unstable DNA structure that can induce DNA strand lesions, such as a collapsed fork, and facilitate a Fork Stalling and Template Switching event producing the complex rearrangements involving MECP2.

Tumor microenvironmental genomic alterations in juvenile nasopharyngeal angiofibroma

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

Genomic content and new insights on the origin of the B chromosome of the cichlid fish Astatotilapia latifasciata

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

Comparative genomic analysis of plant-associated bacteria

This review deals with a comparative analysis of seven genome sequences from plant-associated bacteria. These are the genomes of Agrobacterium tumefaciens, Mesorhizobium loti, Sinorhizobium meliloti, Xanthomonas campestris pv campestris, Xanthomonas axonopodis pv citri, Xylella fastidiosa, and Ralstonia solanacearum. Genome structure and the metabolism pathways available highlight the compromise between the genome size and lifestyle. Despite the recognized importance of the type III secretion system in controlling host compatibility, its presence is not universal in all necrogenic pathogens. Hemolysins, hemagglutinins, and some adhesins, previously reported only for mammalian pathogens, are present in most organisms discussed. Different numbers and combinations of cell wall degrading enzymes and genes to overcome the oxidative burst generally induced by the plant host are characterized in these genomes. A total of 19 genes not involved in housekeeping functions were found common to all these bacteria.

Optimal clone identifier for genomic shotgun libraries: OC Identifier tool

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray. ©FUNPEC-RP.

Alterações no número de cópias genômicas em mastocitomas cutâneos caninos

Pós-graduação em Medicina Veterinária - FCAV

Alterações genômicas na endometriose

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Alterações no número de cópias genômicas em carcinomas de mama

Pós-graduação em Ciências Biológicas (Genética) - IBB

ROCK1 as a novel prognostic marker in vulvar cancer

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