De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼ 1%-2% of CdLS-like phenotypes.

APPRIS: annotation of principal and alternative splice isoforms.

Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform.

Genome sequence of OXA-48 carbapenemase-producing Klebsiella pneumoniae KpO3210.

Klebsiella pneumoniae KpO3210 is a OXA-48 carbapenemase-producing isolate obtained from a blood culture in the context of an outbreak in Hospital Universitario La Paz (Madrid, Spain) in 2010. It belongs to the major clone detected during the outbreak and is resistant to all beta-lactams and to several other antibiotics.

Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway.

The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

CDKN1C (p57(Kip2)) analysis in Beckwith-Wiedemann syndrome (BWS) patients: Genotype-phenotype correlations, novel mutations, and polymorphisms.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by macroglossia, macrosomia, and abdominal wall defects. It is a multigenic disorder caused in most patients by alterations in growth regulatory genes. A small number of individuals with BWS (5-10%) have mutations in CDKN1C, a cyclin-dependent kinase inhibitor of G1 cyclin complexes that functions as a negative regulator of cellular growth and proliferation. Here, we report on eight patients with BWS and CDKN1C mutations and review previous reported cases. We analyzed 72 patients (50 BWS, 17 with isolated hemihyperplasia (IH), three with omphalocele, and two with macroglossia) for CDKN1C defects with the aim to search for new mutations and to define genotype-phenotype correlations. Our findings suggest that BWS patients with CDKN1C mutations have a different pattern of clinical malformations than those with other molecular defects. Polydactyly, genital abnormalities, extra nipple, and cleft palate are more frequently observed in BWS with mutations in CDKN1C. The clinical observation of these malformations may help to decide which genetic characterization should be undertaken (i.e., CDKN1C screening), thus optimizing the laboratory evaluation for BWS.

Determination and validation of principal gene products.

MOTIVATION: Alternative splicing has the potential to generate a wide range of protein isoforms. For many computational applications and for experimental research, it is important to be able to concentrate on the isoform that retains the core biological function. For many genes this is far from clear. RESULTS: We have combined five methods into a pipeline that allows us to detect the principal variant for a gene. Most of the methods were based on conservation between species, at the level of both gene and protein. The five methods used were the conservation of exonic structure, the detection of non-neutral evolution, the conservation of functional residues, the existence of a known protein structure and the abundance of vertebrate orthologues. The pipeline was able to determine a principal isoform for 83% of a set of well-annotated genes with multiple variants.

Population structure of OXA-48-producing Klebsiella pneumoniae ST405 isolates during a hospital outbreak characterised by genomic typing.

OBJECTIVES: The aim of this study was to investigate the structure of a broad and sustained hospital outbreak of OXA-48-producing Klebsiella pneumoniae (KpO48) belonging to sequence type 405 (ST405). METHODS: Whole-genome sequencing and comparison of ten ST405 KpO48 isolates obtained from clinical samples in our hospital was performed. Using stringent criteria, 36 single nucleotide polymorphisms (SNPs) were detected (range 0-21 in pairwise comparisons), and allele-specific PCR was used to call the SNPs among a larger set of isolates. RESULTS: Several haplotypes were identified within the population. The haplotypes did not show a spatial structure, but a temporal evolution of sequential haplotype replacements was observed. CONCLUSIONS: The dispersed spatial distribution suggests a reservoir formed by a large pool of colonised patients, and the temporal replacement pattern suggests that the sustained outbreak was composed of several small outbreaks that appeared and rapidly dispersed to several units.

Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize.

BACKGROUND: While most cells in multicellular organisms carry the same genetic information, in each cell type only a subset of genes is being transcribed. Such differentiation in gene expression depends, for a large part, on the activation and repression of regulatory sequences, including transcriptional enhancers. Transcriptional enhancers can be located tens of kilobases from their target genes, but display characteristic chromatin and DNA features, allowing their identification by genome-wide profiling. Here we show that integration of chromatin characteristics can be applied to predict distal enhancer candidates in Zea mays, thereby providing a basis for a better understanding of gene regulation in this important crop plant. RESULT: To predict transcriptional enhancers in the crop plant maize (Zea mays L. ssp. mays), we integrated available genome-wide DNA methylation data with newly generated maps for chromatin accessibility and histone 3 lysine 9 acetylation (H3K9ac) enrichment in young seedling and husk tissue. Approximately 1500 intergenic regions, displaying low DNA methylation, high chromatin accessibility and H3K9ac enrichment, were classified as enhancer candidates. Based on their chromatin profiles, candidate sequences can be classified into four subcategories. Tissue-specificity of enhancer candidates is defined based on the tissues in which they are identified and putative target genes are assigned based on tissue-specific expression patterns of flanking genes. CONCLUSIONS: Our method identifies three previously identified distal enhancers in maize, validating the new set of enhancer candidates and enlarging the toolbox for the functional characterization of gene regulation in the highly repetitive maize genome.

Developmental ORIgins of Healthy and Unhealthy AgeiNg: the role of maternal obesity--introduction to DORIAN.

Europe has the highest proportion of elderly people in the world. Cardiovascular disease, type 2 diabetes, sarcopenia and cognitive decline frequently coexist in the same aged individual, sharing common early risk factors and being mutually reinforcing. Among conditions which may contribute to establish early risk factors, this review focuses on maternal obesity, since the epidemic of obesity involves an ever growing number of women of reproductive age and children, calling for appropriate studies to understand the consequences of maternal obesity on the offspring's health and for developing effective measures and policies to improve people's health before their conception and birth. Though the current knowledge suggests that the long-term impact of maternal obesity on the offspring's health may be substantial, the outcomes of maternal obesity over the lifespan have not been quantified, and the molecular changes induced by maternal obesity remain poorly characterized. We hypothesize that maternal insulin resistance and reduced placental glucocorticoid catabolism, leading to oxidative stress, may damage the DNA, either in its structure (telomere shortening) or in its function (via epigenetic changes), resulting in altered gene expression/repair, disease during life, and pathological ageing. This review illustrates the background to the EU-FP7-HEALTH-DORIAN project.

Technical phosphoproteomic and bioinformatic tools useful in cancer research.

Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools.

The implications of alternative splicing in the ENCODE protein complement.

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.

Determining a unique defining DNA sequence for yeast species using hashing techniques.

MOTIVATION: Yeasts are often still identified with physiological growth tests, which are both time consuming and unsuitable for detection of a mixture of organisms. Hence, there is a need for molecular methods to identify yeast species. RESULTS: A hashing technique has been developed to search for unique DNA sequences in 702 26S rRNA genes. A unique DNA sequence has been found for almost every yeast species described to date. The locations of the unique defining sequences are in accordance with the variability map of large subunit ribosomal RNA and provide detail of the evolution of the D1/D2 region. This approach will be applicable to the rapid identification of unique sequences in other DNA sequence sets. AVAILABILITY: Freely available upon request from the authors. SUPPLEMENTARY INFORMATION: Results are available at http://www.sys.uea.ac.uk/~jjw/project/paper