Structural genomics: beyond the human genome project.

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.     

Mutagenic insertion and chromosome engineering resource (MICER)

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.     

Dwarf8 polymorphisms associate with variation in flowering time.

Historically, association tests have been used extensively in medical genetics, but have had virtually no application in plant genetics. One obstacle to their application is the structured populations often found in crop plants, which may lead to nonfunctional, spurious associations. In this study, statistical methods to account for population structure were extended for use with quantitative variation and applied to our evaluation of maize flowering time. Mutagenesis and quantitative trait locus (QTL) studies suggested that the maize gene Dwarf8 might affect the quantitative variation of maize flowering time and plant height. The wheat orthologs of this gene contributed to the increased yields seen in the 'Green Revolution' varieties. We used association approaches to evaluate Dwarf8 sequence polymorphisms from 92 maize inbred lines. Population structure was estimated using a Bayesian analysis of 141 simple sequence repeat (SSR) loci. Our results indicate that a suite of polymorphisms associate with differences in flowering time, which include a deletion that may alter a key domain in the coding region. The distribution of nonsynonymous polymorphisms suggests that Dwarf8 has been a target of selection.     

Efficiency and power in genetic association studies

We investigated selection and analysis of tag SNPs for genome-wide association studies by specifically examining the relationship between investment in genotyping and statistical power. Do pairwise or multimarker methods maximize efficiency and power? To what extent is power compromised when tags are selected from an incomplete resource such as HapMap? We addressed these questions using genotype data from the HapMap ENCODE project, association studies simulated under a realistic disease model, and empirical correction for multiple hypothesis testing. We demonstrate a haplotype-based tagging method that uniformly outperforms single-marker tests and methods for prioritization that markedly increase tagging efficiency. Examining all observed haplotypes for association, rather than just those that are proxies for known SNPs, increases power to detect rare causal alleles, at the cost of reduced power to detect common causal alleles. Power is robust to the completeness of the reference panel from which tags are selected. These findings have implications for prioritizing tag SNPs and interpreting association studies.     

Tissue-specific and reversible RNA interference in transgenic mice

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.     

A robust statistical method for case-control association testing with copy number variation

Copy number variation (CNV) is pervasive in the human genome and can play a causal role in genetic diseases. The functional impact of CNV cannot be fully captured through linkage disequilibrium with SNPs. These observations motivate the development of statistical methods for performing direct CNV association studies. We show through simulation that current tests for CNV association are prone to false-positive associations in the presence of differential errors between cases and controls, especially if quantitative CNV measurements are noisy. We present a statistical framework for performing case-control CNV association studies that applies likelihood ratio testing of quantitative CNV measurements in cases and controls. We show that our methods are robust to differential errors and noisy data and can achieve maximal theoretical power. We illustrate the power of these methods for testing for association with binary and quantitative traits, and have made this software available as the R package CNVtools.     

Fine points in mapping autoimmunity

An efficient way to design genotyping arrays for fine mapping is to group phenotypes with common biology. The first application of the Immunochip to celiac disease provides an insightful view of what this strategy can achieve.     

Biological and biomedical implications of the co-evolution of pathogens and their hosts

Co-evolution between host and pathogen is, in principle, a powerful determinant of the biology and genetics of infection and disease. Yet co-evolution has proven difficult to demonstrate rigorously in practice, and co-evolutionary thinking is only just beginning to inform medical or veterinary research in any meaningful way, even though it can have a major influence on how genetic variation in biomedically important traits is interpreted. Improving our understanding of the biomedical significance of co-evolution will require changing the way in which we look for it, complementing the phenomenological approach traditionally favored by evolutionary biologists with the exploitation of the extensive data becoming available on the molecular biology and molecular genetics of host-pathogen interactions.     

Apports et limites de la biologie moléculaire en mycologie médicale

For medical mycology, molecular biology is a fantastic tool for epidemiological investigation. Molecular typing of environmental and patient isolates allows to define the way of transmission and the origin of nosocomial fungal outbreaks (candidiasis, aspergillosis). For diagnosis, detection of fungal DNA by PCR is actually no superior compared to blood culture (Candida) or serum antigen detection (Aspergillus). For identification of species, the interest is quite limited because of good results obtained with classical methods (biochimical tests, microscopy).     

Extensive allelic variation and ultrashort telomeres in senescent human cells

By imposing a limit on the proliferative lifespan of most somatic cells, telomere erosion represents an innate mechanism for tumor suppression and may contribute to age-related disease. A detailed understanding of the pathways that link shortened telomeres to replicative senescence has been severely hindered by the inability of current methods to analyze telomere dynamics in detail. Here we describe single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. STELA analysis of human XpYp telomeres in fibroblasts identifies several features of telomere biology. We observe bimodal distributions of telomeres in normal fibroblasts; these distributions result from inter-allelic differences of up to 6.5 kb, indicating that unexpectedly large-scale differences in zygotic telomere length are maintained throughout development. Most telomeres shorten in a gradual fashion consistent with simple losses through end replication, and the rates of erosion are independent of allele size. Superimposed on this are occasional, more substantial changes in length, which may be the consequence of additional mutational mechanisms. Notably, some alleles show almost complete loss of TTAGGG repeats at senescence.     

Diagnostic moléculaire de la résistance de Mycobacterium tuberculosis aux antituberculeux

The development of new technical tools for molecular biology has modified the methods used for the identification of resistance in Mycobacterium tuberculosis. Although the proportion method generally remains the reference method, more rapid and sophisticated techniques are now available, such as PCR-SSCP analysis, LiPA hybridization and DNA sequencing. At present, PCR-SSCP analysis is no longer used in routine. The INNO-LiPA assay, which is well adapted to routine, allows the rapid determination of rifampin resistance, but is not developed yet for detecting resistance to other antimycobacterial drugs. DNA sequencing is a powerful tool for the detection of resistance to rifampin, pyrazinamide and fluoroquinolones, but not to isoniazid. However, the technique requires specific facilities which are very costly. Finally, the development of high-density oligonucleotide arrays that can be used to rapidly examine large amounts of DNA sequences could allow to simultaneously investigate a large number of mechanisms of resistance. This new approach represents a very promising alternative for the rapid analysis of genomic regions involved in drug resistance.     

Nouveaux traitements de l'hépatite C

The treatment of chronic hepatitis C is now based on the combination of interferon alpha and ribavirin. The posology of interferon is 3 MIU t.i.w. subcutaneous during at least a 6 month period and the posology of ribavirin is 1 000 to 1 200 mg/day. This combination is overall more efficacient than interferon alone and also according to genotype and viral load. It is preferable to use this association during 6 months if the patients had a genotype non 1 and a low viral load. On the other hand, the duration of treatment should be 12 months if patient has a genotype 1 and high viral load. The combination of interferon and ribavirin should be also prescribed in relapser patients to first treatment by interferon alone. In non responder patients no treatment had shown an efficacy. Protocols are actually performed in order to test bitherapy using daily dose of interferon or a new interferon (pegylated interferon) and finally new molecules as amantadine which in combination with interferon and ribavirin could be more efficient than interferon alone or interferon and ribavirin. It is probably that in the future, it will be possible to realize new progress, because of a better knowledge of virus, especially in terms of molecular biology and kinetic of viral elimination.     

Exhaustive allelic transmission disequilibrium tests as a new approach to genome-wide association studies

Genome-wide disease-association mapping has been heralded as the study design of the next generation, but the lack of analytical methods to use genotype data fully is a large stumbling block. Here we describe an algorithm and statistical method that efficiently and exhaustively exploits haplotype information by subjecting alleles (a marker or contiguous sets of markers) from sliding windows of all sizes to transmission disequilibrium tests. By applying our method to simulated data and to Hirschsprung disease, we show that it can detect both common and rare disease variants of small effect. These results show that the theoretical benefits of genome-wide association studies are at last realizable.     

Population genomic analysis of outcrossing and recombination in yeast

The budding yeast Saccharomyces cerevisiae has been used by humans for millennia to make wine, beer and bread. More recently, it became a key model organism for studies of eukaryotic biology and for genomic analysis. However, relatively little is known about the natural lifestyle and population genetics of yeast. One major question is whether genetically diverse yeast strains mate and recombine in the wild. We developed a method to infer the evolutionary history of a species from genome sequences of multiple individuals and applied it to whole-genome sequence data from three strains of Saccharomyces cerevisiae and the sister species Saccharomyces paradoxus. We observed a pattern of sequence variation among yeast strains in which ancestral recombination events lead to a mosaic of segments with shared genealogy. Based on sequence divergence and the inferred median size of shared segments (approximately 2,000 bp), we estimated that although any two strains have undergone approximately 16 million cell divisions since their last common ancestor, only 314 outcrossing events have occurred during this time (roughly one every 50,000 divisions). Local correlations in polymorphism rates indicate that linkage disequilibrium in yeast should extend over kilobases. Our results provide the initial foundation for population studies of association between genotype and phenotype in S. cerevisiae.     

Les phlébotomes et leur rôle dans la transmission des leishmanioses

This paper deals with phlebotomine sandflies morphology, geographical distribution, biology and transmission of leishmaniases, methods of investigation and fight. It includes illustrations and a simplified key for generic and sub-generic diagnosis.     

Genetic clonal diversity predicts progression to esophageal adenocarcinoma

Neoplasms are thought to progress to cancer through genetic instability generating cellular diversity and clonal expansions driven by selection for mutations in cancer genes. Despite advances in the study of molecular biology of cancer genes, relatively little is known about evolutionary mechanisms that drive neoplastic progression. It is unknown, for example, which may be more predictive of future progression of a neoplasm: genetic homogenization of the neoplasm, possibly caused by a clonal expansion, or the accumulation of clonal diversity. Here, in a prospective study, we show that clonal diversity measures adapted from ecology and evolution can predict progression to adenocarcinoma in the premalignant condition known as Barrett's esophagus, even when controlling for established genetic risk factors, including lesions in TP53 (p53; ref. 6) and ploidy abnormalities. Progression to cancer through accumulation of clonal diversity, on which natural selection acts, may be a fundamental principle of neoplasia with important clinical implications.     

Chromosome-scale selective sweeps shape Caenorhabditis elegans genomic diversity

The nematode Caenorhabditis elegans is central to research in molecular, cell and developmental biology, but nearly all of this research has been conducted on a single strain of C. elegans. Little is known about the population genomic and evolutionary history of this species. We characterized C. elegans genetic variation using high-throughput selective sequencing of a worldwide collection of 200 wild strains and identified 41,188 SNPs. Notably, C. elegans genome variation is dominated by a set of commonly shared haplotypes on four of its six chromosomes, each spanning many megabases. Population genetic modeling showed that this pattern was generated by chromosome-scale selective sweeps that have reduced variation worldwide; at least one of these sweeps probably occurred in the last few hundred years. These sweeps, which we hypothesize to be a result of human activity, have drastically reshaped the global C. elegans population in the recent past.