Protein-binding microarrays: probing disease markers at the interface of proteomics and genomics.

DNA-binding proteins mediate a variety of crucial molecular functions, such as transcriptional regulation and chromosome maintenance, replication and repair, which in turn control cell division and differentiation. The roles of these proteins in disease are currently being investigated using microarray-based approaches. However, these assays can be difficult to adapt to routine diagnosis of complex diseases such as cancer. Here, we review promising alternative approaches involving protein-binding microarrays (PBMs) that probe the interaction of proteins from crude cell or tissue extracts with large collections of synthetic or natural DNA sequences. Recent studies have demonstrated the use of these novel PBM approaches to provide rapid and unbiased characterization of DNA-binding proteins as molecular markers of disease, for example cancer progression or infectious diseases.

The expansion of amino-acid repeats is not associated to adaptive evolution in mammalian genes.

BACKGROUND: The expansion of amino acid repeats is determined by a high mutation rate and can be increased or limited by selection. It has been suggested that recent expansions could be associated with the potential of adaptation to new environments. In this work, we quantify the strength of this association, as well as the contribution of potential confounding factors. RESULTS: Mammalian positively selected genes have accumulated more recent amino acid repeats than other mammalian genes. However, we found little support for an accelerated evolutionary rate as the main driver for the expansion of amino acid repeats. The most significant predictors of amino acid repeats are gene function and GC content. There is no correlation with expression level. CONCLUSIONS: Our analyses show that amino acid repeat expansions are causally independent from protein adaptive evolution in mammalian genomes. Relaxed purifying selection or positive selection do not associate with more or more recent amino acid repeats. Their occurrence is slightly favoured by the sequence context but mainly determined by the molecular function of the gene.

Towards the identification of a genetic basis for Landau-Kleffner syndrome.

OBJECTIVE: To establish the genetic basis of Landau-Kleffner syndrome (LKS) in a cohort of two discordant monozygotic (MZ) twin pairs and 11 isolated cases. METHODS: We used a multifaceted approach to identify genetic risk factors for LKS. Array comparative genomic hybridization (CGH) was performed using the Agilent 180K array. Whole genome methylation profiling was undertaken in the two discordant twin pairs, three isolated LKS cases, and 12 control samples using the Illumina 27K array. Exome sequencing was undertaken in 13 patients with LKS including two sets of discordant MZ twins. Data were analyzed with respect to novel and rare variants, overlapping genes, variants in reported epilepsy genes, and pathway enrichment. RESULTS: A variant (cG1553A) was found in a single patient in the GRIN2A gene, causing an arginine to histidine change at site 518, a predicted glutamate binding site. Following copy number variation (CNV), methylation, and exome sequencing analysis, no single candidate gene was identified to cause LKS in the remaining cohort. However, a number of interesting additional candidate variants were identified including variants in RELN, BSN, EPHB2, and NID2. SIGNIFICANCE: A single mutation was identified in the GRIN2A gene. This study has identified a number of additional candidate genes including RELN, BSN, EPHB2, and NID2. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

MCL1 is deregulated in subgroups of diffuse large B-cell lymphoma.

Myeloid cell leukemia-1 (MCL1) is an anti-apoptotic member of the BCL2 family that is deregulated in various solid and hematological malignancies. However, its role in the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL) is unclear. We analyzed gene expression profiling data from 350 DLBCL patient samples and detected that activated B-cell-like (ABC) DLBCLs express MCL1 at significantly higher levels compared with germinal center B-cell-like DLBCL patient samples (P=2.7 × 10(-10)). Immunohistochemistry confirmed high MCL1 protein expression predominantly in ABC DLBCL in an independent patient cohort (n=249; P=0.001). To elucidate molecular mechanisms leading to aberrant MCL1 expression, we analyzed array comparative genomic hybridization data of 203 DLBCL samples and identified recurrent chromosomal gains/amplifications of the MCL1 locus that occurred in 26% of ABC DLBCLs. In addition, aberrant STAT3 signaling contributed to high MCL1 expression in this subtype. Knockdown of MCL1 as well as treatment with the BH3-mimetic obatoclax induced apoptotic cell death in MCL1-positive DLBCL cell lines. In summary, MCL1 is deregulated in a significant fraction of ABC DLBCLs and contributes to therapy resistance. These data suggest that specific inhibition of MCL1 might be utilized therapeutically in a subset of DLBCLs.

Subtelomeric 6p deletion: clinical and array-CGH characterization in two patients

We report on two patients with de novo subtelomeric terminal deletion of chromosome 6p. Patient 1 is an 8-month-old female born with normal growth parameters, typical facial features of 6pter deletion, bilateral corectopia, and protruding tongue. She has severe developmental delay, profound bilateral neurosensory deafness, poor visual contact, and hypsarrhythmia since the age of 6 months. Patient 2 is a 5-year-old male born with normal growth parameters and unilateral hip dysplasia; he has a characteristic facial phenotype, bilateral embryotoxon, and moderate mental retardation. Further characterization of the deletion, using high-resolution array comparative genomic hybridization (array-CGH; Agilent Human Genome kit 244 K), revealed that Patient 1 has a 8.1 Mb 6pter-6p24.3 deletion associated with a contiguous 5.8 Mb 6p24.3-6p24.1 duplication and Patient 2 a 5.7 Mb 6pter-6p25.1 deletion partially overlapping with that of Patient 1. Complementary FISH and array analysis showed that the inv del dup(6) in Patient 1 originated de novo. Our results demonstrate that simple rearrangements are often more complex than defined by standard techniques. We also discuss genotype-phenotype correlations including previously reported cases of deletion 6p.

Beta-catenin Status in P?aediatric Medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics.

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/beta-catenin pathway occurs in 10-15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for beta-catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of beta-catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all beta-catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/beta-catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear beta-catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/beta-catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow-up of 75.7 months (range 27.5-121.2 months) from diagnosis. All three patients with focal nuclear staining of beta-catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1-mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de-escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved.

Fibrosarcoma-like lipomatous neoplasm: a reappraisal of so-called spindle cell liposarcoma defining a unique lipomatous tumor unrelated to other liposarcomas

The term "spindle cell liposarcoma" has been applied to liposarcomas (LPSs) composed predominantly or exclusively of spindled cells. These tumors have been considered variants of well-differentiated LPS (WDL), myxoid LPS, and spindle cell lipoma, suggesting that this is a heterogenous group of lesions. Using strict morphologic criteria and molecular and immunohistochemical analyses, we have identified a homogenous group of spindle cell lipomatous tumors, histologically and genetically distinct from other forms of LPS, which we have called "fibrosarcoma-like lipomatous neoplasm." Cases classified as "spindle cell LPS" or "low-grade LPS with spindle cell features" were reviewed. Final selection criteria included: (1) an exclusive low-grade spindle cell component resembling fibrosarcoma; (2) a mixture of bland fibroblastic cells resembling the preadipocyte and early-adipocyte stage of embryonic fat; and (3) molecular-genetic analysis that excluded other forms of lipomatous tumors. Of the initial 25 cases identified, comparative genomic hybridization (CGH) was uninformative in 2 cases; 5 were reclassified as WDL on the basis of molecular data (MDM2 amplification) and 6 as spindle cell lipoma (CGH profiles with a few gains and losses including a constant loss of chromosome 13 and frequent losses of chromosomes 16 and 6). The 12 remaining cases showed flat CGH profiles; of these cases, 11 were negative for DDIT3 gene rearrangements, and 1 result was uninterpretable. Patients ranged in age from 15 to 82 years (mean 50 y); male patients were affected slightly more often (7:5). Tumors arose in the deep (6) and superficial (3) soft tissue of the groin (4), buttock (3), thigh (2), flank (1), shoulder (1), and paratesticular tissue (1) and ranged in size from 2 to 20 cm (mean 7.5 cm). Clinical follow-up in 11 patients (9 mo to 20 y; mean 68 mo) showed no recurrences or metastases. As defined above, "fibrosarcoma-like lipomatous neoplasm" is a unique lipomatous tumor that should be distinguished from WDL/(low-grade) dedifferentiated LPS and myxoid LPS on combined histologic/molecular features because of its better prognosis.

Genetic aberrations in pediatric follicular lymphoma

Background: Pediatric follicular lymphoma (FL) is a rare disease that differs from its adult counterpart both genetically and clinically. Excluding pediatric FL with IRF4-translocation, the genetic events associated with pediatric FL have not yet been defined. Objectives: The aim of this study was to perform a complete genetic characterization of IRF4-translocation negative pediatric follicular lymphomas to elucidate the genetic profile of these rare pediatric cases and determine common genetic alterations that could be associated to this phenotype. Design/Methods: We applied array-comparative genomic hybridization and molecular inversion probe assay adapted to formalin-fixed paraffin-embedded tissues from 18 patients aged £18 years diagnosed with FL. With the exception of one case with only focal involvement by lymphoma, the tumor cell content exceeded 50% in the evaluable samples. Eleven of 18 patients were treated according to NHL-BFM group multicenter trials whereas the remaining according to different protocols. All lacked t(14;18) translocation. Mutational analysis of TNFRSF14 gene was performed in 17 cases. Results: Only six pediatric cases displayed chromosomal imbalances, with gain/amplification of 6pter-p24.3 (including IRF4) and deletion/ copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being the most frequent alterations. Sequencing of the candidate gene TNFRSF14 at 1p36.32 showed nine mutations in seven cases. Conclusion: Combination of molecular and genetic features differentiated a recurrent pattern of genomic imbalances as well as of TNFRSF14 mutations in pediatric FL which together with other genetic alterations distinguishes two subsets of pediatric follicular lymphomas. The first group shows genomic aberrations and is associated with more aggressive histopathologic and clinical features. The second group lacks genetic alterations detectable with the present approaches and is associated with a more limited disease. Despite the absence of genomic aberrations, these cases resembled FL by their histopathological features.

p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.

A common cognitive, psychiatric, and dysmorphic phenotype in carriers of NRXN1 deletion

Deletions in the 2p16.3 region that includes the neurexin (NRXN1) gene are associated with intellectual disability and various psychiatric disorders, in particular, autism and schizophrenia. We present three unrelated patients, two adults and one child, in whom we identified an intragenic 2p16.3 deletion within the NRXN1 gene using an oligonucleotide comparative genomic hybridization array. The three patients presented dual diagnosis that consisted of mild intellectual disability and autism and bipolar disorder. Also, they all shared a dysmorphic phenotype characterized by a long face, deep set eyes, and prominent premaxilla. Genetic analysis of family members showed two inherited deletions. A comprehensive neuropsychological examination of the 2p16.3 deletion carriers revealed the same phenotype, characterized by anxiety disorder, borderline intelligence, and dysexecutive syndrome. The cognitive pattern of dysexecutive syndrome with poor working memory and reduced attention switching, mental flexibility, and verbal fluency was the same than those of the adult probands. We suggest that in addition to intellectual disability and psychiatric disease, NRXN1 deletion is a risk factor for a characteristic cognitive and dysmorphic profile. The new cognitive phenotype found in the 2p16.3 deletion carriers suggests that 2p16.3 deletions might have a wide variable expressivity instead of incomplete penetrance

Cytoplasmic accumulation of NCoR in malignant melanoma: Consequences of altered gene repression and prognostic significance

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy available in advanced stages. Nuclear corepressor (NCoR) is an essential regulator of gene transcription, and its function has been found deregulated in different types of cancer. In colorectal cancer cells, loss of nuclear NCoR is induced by Inhibitor of kappa B kinase (IKK) through the phosphorylation of specific serine residues. We here investigate whether NCoR function impacts in MM, which might have important diagnostic and prognostic significance. By IHC, we here determined the subcellular distribution of NCoR in a cohort of 63 primary invasive MM samples, and analyzed its possible correlation with specific clinical parameters. We therefore used a microarray-based strategy to determine global gene expression differences in samples with similar tumor stage, which differ in the presence of cytoplasmic or nuclear NCoR. We found that loss of nuclear NCoR results in upregulation of a specific cancer-related genetic signature, and is significantly associated with MM progression. Inhibition of IKK activity in melanoma cells reverts NCoR nuclear distribution and specific NCoR-regulated gene transcription. Analysis of public database demonstrated that inactivating NCoR mutations are highly prevalent in MM, showing features of driver oncogene.

Alternative procedures for parent choice in a breeding program for the common bean (Phaseolus vulgaris L.)

Six common bean cultivars were crossed in diallel and the segregant populations were assessed in the F2 and F3 generations to compare methodologies for parental selection in a breeding program based on hybridization. The cultivars involved in the diallel were A 114, A 77, ESAL 686, Milionário, Carioca, and Flor de Mayo. The segregant F2 and F3 generations were assessed on the experimental campus of the Universidade Federal de Larvas, in July 1994. It was found that the cultivars differed in their general combining ability (GCA). Flor de Mayo, which belongs to the Durango race, had the largest positive GCA estimate for grain field, and the cultivars from the Mesoamerican race, Milionário and A 114, the smallest GCA estimates. For flowering, the cultivar that most contributed to reduced plant cycle was ESAL 686. There was agreement among the results obtained from the diallel and the estimates of the parameter m + a of the populations. However, it was evident that the estimate of genetic variance of the populations should be considered as a condition to identify the hybrid population that will produce a line with high performance.

Amplification of 9q34 in childhood adrenocortical tumors: a specific feature unrelated to ethnic origin or living conditions

Adrenocortical tumors (ACT) in children under 15 years of age exhibit some clinical and biological features distinct from ACT in adults. Cell proliferation, hypertrophy and cell death in adrenal cortex during the last months of gestation and the immediate postnatal period seem to be critical for the origin of ACT in children. Studies with large numbers of patients with childhood ACT have indicated a median age at diagnosis of about 4 years. In our institution, the median age was 3 years and 5 months, while the median age for first signs and symptoms was 2 years and 5 months (N = 72). Using the comparative genomic hybridization technique, we have reported a high frequency of 9q34 amplification in adenomas and carcinomas. This finding has been confirmed more recently by investigators in England. The lower socioeconomic status, the distinctive ethnic groups and all the regional differences in Southern Brazil in relation to patients in England indicate that these differences are not important to determine 9q34 amplification. Candidate amplified genes mapped to this locus are currently being investigated and Southern blot results obtained so far have discarded amplification of the abl oncogene. Amplification of 9q34 has not been found to be related to tumor size, staging, or malignant histopathological features, nor does it seem to be responsible for the higher incidence of ACT observed in Southern Brazil, but could be related to an ACT from embryonic origin.

Voluntary exercise prior to traumatic brain injury alters miRNA expression in the injured mouse cerebral cortex

MicroRNAs (miRNAs) may be important mediators of the profound molecular and cellular changes that occur after traumatic brain injury (TBI). However, the changes and possible roles of miRNAs induced by voluntary exercise prior to TBI are still not known. In this report, the microarray method was used to demonstrate alterations in miRNA expression levels in the cerebral cortex of TBI mice that were pretrained on a running wheel (RW). Voluntary RW exercise prior to TBI: i) significantly decreased the mortality rate and improved the recovery of the righting reflex in TBI mice, and ii) differentially changed the levels of several miRNAs, upregulating some and downregulating others. Furthermore, we revealed global upregulation of miR-21, miR-92a, and miR-874 and downregulation of miR-138, let-7c, and miR-124 expression among the sham-non-runner, TBI-non-runner, and TBI-runner groups. Quantitative reverse transcription polymerase chain reaction data (RT-qPCR) indicated good consistency with the microarray results. Our microarray-based analysis of miRNA expression in mice cerebral cortex after TBI revealed that some miRNAs such as miR-21, miR-92a, miR-874, miR-138, let-7c, and miR-124 could be involved in the prevention and protection afforded by voluntary exercise in a TBI model.

Étude sur le rôle des déséquilibres génomiques dans le Syndrome d’Impatiences Musculaires de l’Éveil

Le Syndrome d’Impatiences Musculaires de l’Éveil (SIME) est une maladie neurologique caractérisée par un besoin urgent de bouger les jambes. C’est également l’une des causes les plus fréquentes d’insomnie. C’est une maladie très répandue, avec une prévalence de presque 15 % dans la population générale. Les maladies multifactorielles comme le SIME sont souvent le résultat de l’évolution d’une composante génétique et d’une composante environnementale. Dans le cadre du SIME, les études d’association génomique ont permis l’identification de 4 variants à effet modéré ou faible. Cependant, ces quatre variants n’expliquent qu’une faible partie de la composante génétique de la maladie, ce qui confirme que plusieurs nouveaux variants sont encore à identifier. Le rôle des déséquilibres génomiques (Copy Number Variations ou CNVs) dans le mécanisme génétique du SIME est à ce jour inconnu. Cependant, les CNVs se sont récemment positionnés comme une source d’intérêt majeur de variation génétique potentiellement responsable des phénotypes. En collaboration avec une équipe de Munich, nous avons réalisé deux études CNVs à échelle génomique (biopuces à SNP et hybridation génomique comparée (CGH)) sur des patients SIME d’ascendance germanique. À l’aide d’une étude cas-contrôle, nous avons pu identifier des régions avec une occurrence de CNVs différentes pour les patients SIME, comparés à différents groupes contrôles. L’une de ces régions est particulièrement intéressante, car elle est concordante à la fois avec des précédentes études familiales ainsi qu’avec les récentes études d’associations génomiques.