Automating sequence-based detection and genotyping of SNPs from diploid samples

The detection of sequence variation, for which DNA sequencing has emerged as the most sensitive and automated approach, forms the basis of all genetic analysis. Here we describe and illustrate an algorithm that accurately detects and genotypes SNPs from fluorescence-based sequence data. Because the algorithm focuses particularly on detecting SNPs through the identification of heterozygous individuals, it is especially well suited to the detection of SNPs in diploid samples obtained after DNA amplification. It is substantially more accurate than existing approaches and, notably, provides a useful quantitative measure of its confidence in each potential SNP detected and in each genotype called. Calls assigned the highest confidence are sufficiently reliable to remove the need for manual review in several contexts. For example, for sequence data from 47-90 individuals sequenced on both the forward and reverse strands, the highest-confidence calls from our algorithm detected 93% of all SNPs and 100% of high-frequency SNPs, with no false positive SNPs identified and 99.9% genotyping accuracy. This algorithm is implemented in a software package, PolyPhred version 5.0, which is freely available for academic use.     

Characterization of apoptosis induced by marine natural products in non small cell lung cancer A549 cells

The effects of different marine derived agents were studied in A549 cell growth. These drugs induced cell cycle arrest at the G2-M phase associated with the up-regulation of GADD45alpha-gamma and down-regulation of c-Myc. In treated cells, GADD45alpha-gamma and c-Myc were up- and down-regulated, respectively. A cascade of events leading to apoptotic mitochondrial 'intrinsic' pathway was observed in treated cells: (1) dephosphorylation of BAD serine136; (2) BAD dissociation from 14-3-3 followed by its association with BCL-XL; (3) cytochrome c release; (4) caspase-3 activation, and (5) cleavage of vimentin. Caspase(s) inhibitor prevented the formation of cleavage products and, in turn, apoptosis was inhibited through a p53-independent mechanism. Moreover, these compounds did not activate NF-kappaB. Our findings may offer new insights into the mechanisms of action of these agents in A549 cells. The better understanding of their effects might be important to fully exploit the potential of these new drugs.     

A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31-2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31-2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.     

Common deletion polymorphisms in the human genome

The locations and properties of common deletion variants in the human genome are largely unknown. We describe a systematic method for using dense SNP genotype data to discover deletions and its application to data from the International HapMap Consortium to characterize and catalogue segregating deletion variants across the human genome. We identified 541 deletion variants (94% novel) ranging from 1 kb to 745 kb in size; 278 of these variants were observed in multiple, unrelated individuals, 120 in the homozygous state. The coding exons of ten expressed genes were found to be commonly deleted, including multiple genes with roles in sex steroid metabolism, olfaction and drug response. These common deletion polymorphisms typically represent ancestral mutations that are in linkage disequilibrium with nearby SNPs, meaning that their association to disease can often be evaluated in the course of SNP-based whole-genome association studies.     

Biological weapons

Anthrax has been a major cause of death in grazing animals and an occasional cause of death in humans for thousands of years. Since the late 1800s there has been an exceptional international history of anthrax vaccine development. Due to animal vaccinations, the rate of infection has dropped dramatically. Anthrax vaccines have progressed from uncharacterized whole-cell vaccines in 1881, to pXO2-negative spores in the 1930s, to culture filtrates absorbed to aluminum hydroxide in 1970, and likely to recombinant protective antigen in the near future. Each of these refinements has increased safety without significant loss of efficacy. The threat of genetically engineered, antibiotic and vaccine resistant strains of Bacillus anthracis is fueling hypothesis-driven research and global techniques--including genomics, proteomics and transposon site hybridization--to facilitate the discovery of novel vaccine targets. This review highlights historical achievements and new developments in anthrax vaccine research.     

Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29

Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.     

Epigenetic remodeling in colorectal cancer results in coordinate gene suppression across an entire chromosome band

We report a new mechanism in carcinogenesis involving coordinate long-range epigenetic gene silencing. Epigenetic silencing in cancer has always been envisaged as a local event silencing discrete genes. However, in this study of silencing in colorectal cancer, we found common repression of the entire 4-Mb band of chromosome 2q.14.2, associated with global methylation of histone H3 Lys9. DNA hypermethylation within the repressed genomic neighborhood was localized to three separate enriched CpG island 'suburbs', with the largest hypermethylated suburb spanning 1 Mb. These data change our understanding of epigenetic gene silencing in cancer cells: namely, epigenetic silencing can span large regions of the chromosome, and both DNA-methylated and neighboring unmethylated genes can be coordinately suppressed by global changes in histone modification. We propose that loss of gene expression can occur through long-range epigenetic silencing, with similar implications as loss of heterozygosity in cancer.     

Mutations in embryonic myosin heavy chain (MYH3) cause Freeman-Sheldon syndrome and Sheldon-Hall syndrome

The genetic basis of most conditions characterized by congenital contractures is largely unknown. Here we show that mutations in the embryonic myosin heavy chain (MYH3) gene cause Freeman-Sheldon syndrome (FSS), one of the most severe multiple congenital contracture (that is, arthrogryposis) syndromes, and nearly one-third of all cases of Sheldon-Hall syndrome (SHS), the most common distal arthrogryposis. FSS and SHS mutations affect different myosin residues, demonstrating that MYH3 genotype is predictive of phenotype. A structure-function analysis shows that nearly all of the MYH3 mutations are predicted to interfere with myosin's catalytic activity. These results add to the growing body of evidence showing that congenital contractures are a shared outcome of prenatal defects in myofiber force production. Elucidation of the genetic basis of these syndromes redefines congenital contractures as unique defects of the sarcomere and provides insights about what has heretofore been a poorly understood group of disorders.