Receptor binding and cell entry of Old World arenaviruses reveal novel aspects of virus-host interaction.

Ten years ago, the first cellular receptor for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the highly pathogenic Lassa virus (LASV) was identified as alpha-dystroglycan (alpha-DG), a versatile receptor for proteins of the extracellular matrix (ECM). Biochemical analysis of the interaction of alpha-DG with arenaviruses and ECM proteins revealed a strikingly similar mechanism of receptor recognition that critically depends on specific sugar modification on alpha-DG involving a novel class of putative glycosyltransferase, the LARGE proteins. Interestingly, recent genome-wide detection and characterization of positive selection in human populations revealed evidence for positive selection of a locus within the LARGE gene in populations from Western Africa, where LASV is endemic. While most enveloped viruses that enter the host cell in a pH-dependent manner use clathrin-mediated endocytosis, recent studies revealed that the Old World arenaviruses LCMV and LASV enter the host cell predominantly via a novel and unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the virus is rapidly delivered to endosomes via an unusual route of vesicular trafficking that is largely independent of the small GTPases Rab5 and Rab7. Since infection of cells with LCMV and LASV depends on DG, this unusual endocytotic pathway could be related to normal cellular trafficking of the DG complex. Alternatively, engagement of arenavirus particles may target DG for an endocytotic pathway not normally used in uninfected cells thereby inducing an entry route specifically tailored to the pathogen's needs.

Machine-learning methods for structure prediction of β-barrel membrane proteins

Different types of proteins exist with diverse functions that are essential for living organisms. An important class of proteins is represented by transmembrane proteins which are specifically designed to be inserted into biological membranes and devised to perform very important functions in the cell such as cell communication and active transport across the membrane. Transmembrane β-barrels (TMBBs) are a sub-class of membrane proteins largely under-represented in structure databases because of the extreme difficulty in experimental structure determination. For this reason, computational tools that are able to predict the structure of TMBBs are needed. In this thesis, two computational problems related to TMBBs were addressed: the detection of TMBBs in large datasets of proteins and the prediction of the topology of TMBB proteins. Firstly, a method for TMBB detection was presented based on a novel neural network framework for variable-length sequence classification. The proposed approach was validated on a non-redundant dataset of proteins. Furthermore, we carried-out genome-wide detection using the entire Escherichia coli proteome. In both experiments, the method significantly outperformed other existing state-of-the-art approaches, reaching very high PPV (92%) and MCC (0.82). Secondly, a method was also introduced for TMBB topology prediction. The proposed approach is based on grammatical modelling and probabilistic discriminative models for sequence data labeling. The method was evaluated using a newly generated dataset of 38 TMBB proteins obtained from high-resolution data in the PDB. Results have shown that the model is able to correctly predict topologies of 25 out of 38 protein chains in the dataset. When tested on previously released datasets, the performances of the proposed approach were measured as comparable or superior to the current state-of-the-art of TMBB topology prediction.

The Growing Importance of CNVs: New Insights for Detection and Clinical Interpretation.

Differences between genomes can be due to single nucleotide variants, translocations, inversions, and copy number variants (CNVs, gain or loss of DNA). The latter can range from sub-microscopic events to complete chromosomal aneuploidies. Small CNVs are often benign but those larger than 500 kb are strongly associated with morbid consequences such as developmental disorders and cancer. Detecting CNVs within and between populations is essential to better understand the plasticity of our genome and to elucidate its possible contribution to disease. Hence there is a need for better-tailored and more robust tools for the detection and genome-wide analyses of CNVs. While a link between a given CNV and a disease may have often been established, the relative CNV contribution to disease progression and impact on drug response is not necessarily understood. In this review we discuss the progress, challenges, and limitations that occur at different stages of CNV analysis from the detection (using DNA microarrays and next-generation sequencing) and identification of recurrent CNVs to the association with phenotypes. We emphasize the importance of germline CNVs and propose strategies to aid clinicians to better interpret structural variations and assess their clinical implications.

Evidence for polygenic adaptation to pathogens in the human genome.

Most approaches aiming at finding genes involved in adaptive events have focused on the detection of outlier loci, which resulted in the discovery of individually "significant" genes with strong effects. However, a collection of small effect mutations could have a large effect on a given biological pathway that includes many genes, and such a polygenic mode of adaptation has not been systematically investigated in humans. We propose here to evidence polygenic selection by detecting signals of adaptation at the pathway or gene set level instead of analyzing single independent genes. Using a gene-set enrichment test to identify genome-wide signals of adaptation among human populations, we find that most pathways globally enriched for signals of positive selection are either directly or indirectly involved in immune response. We also find evidence for long-distance genotypic linkage disequilibrium, suggesting functional epistatic interactions between members of the same pathway. Our results show that past interactions with pathogens have elicited widespread and coordinated genomic responses, and suggest that adaptation to pathogens can be considered as a primary example of polygenic selection.

Effects of long-term averaging of quantitative blood pressure traits on the detection of genetic associations.

Blood pressure (BP) is a heritable, quantitative trait with intraindividual variability and susceptibility to measurement error. Genetic studies of BP generally use single-visit measurements and thus cannot remove variability occurring over months or years. We leveraged the idea that averaging BP measured across time would improve phenotypic accuracy and thereby increase statistical power to detect genetic associations. We studied systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP) averaged over multiple years in 46,629 individuals of European ancestry. We identified 39 trait-variant associations across 19 independent loci (p < 5 × 10(-8)); five associations (in four loci) uniquely identified by our LTA analyses included those of SBP and MAP at 2p23 (rs1275988, near KCNK3), DBP at 2q11.2 (rs7599598, in FER1L5), and PP at 6p21 (rs10948071, near CRIP3) and 7p13 (rs2949837, near IGFBP3). Replication analyses conducted in cohorts with single-visit BP data showed positive replication of associations and a nominal association (p < 0.05). We estimated a 20% gain in statistical power with long-term average (LTA) as compared to single-visit BP association studies. Using LTA analysis, we identified genetic loci influencing BP. LTA might be one way of increasing the power of genetic associations for continuous traits in extant samples for other phenotypes that are measured serially over time.

PhylomeDB v4: zooming into the plurality of evolutionary histories of a genome

Phylogenetic trees representing the evolutionary relationships of homologous genes are the entry point for many evolutionary analyses. For instance, the use of a phylogenetic tree can aid in the inference of orthology and paralogy relationships, and in the detection of relevant evolutionary events such as gene family expansions and contractions, horizontal gene transfer, recombination or incomplete lineage sorting. Similarly, given the plurality of evolutionary histories among genes encoded in a given genome, there is a need for the combined analysis of genome-wide collections of phylogenetic trees (phylomes). Here, we introduce a new release of PhylomeDB (http://phylomedb.org), a public repository of phylomes. Currently, PhylomeDB hosts 120 public phylomes, comprising >1.5 million maximum likelihood trees and multiple sequence alignments. In the current release, phylogenetic trees are annotated with taxonomic, protein-domain arrangement, functional and evolutionary information. PhylomeDB is also a major source for phylogeny-based predictions of orthology and paralogy, covering >10 million proteins across 1059 sequenced species. Here we describe newly implemented PhylomeDB features, and discuss a benchmark of the orthology predictions provided by the database, the impact of proteome updates and the use of the phylome approach in the analysis of newly sequenced genomes and transcriptomes.

Regulation of self-renewal and detection of karyotypic changes of pluripotent human embryonic stem cells

Human embryonic stem cells are pluripotent cells capable of renewing themselves and differentiating to specialized cell types. Because of their unique regenerative potential, pluripotent cells offer new opportunities for disease modeling, development of regenerative therapies, and treating diseases. Before pluripotent cells can be used in any therapeutic applications, there are numerous challenges to overcome. For instance, the key regulators of pluripotency need to be clarified. In addition, long term culture of pluripotent cells is associated with the accumulation of karyotypic abnormalities, which is a concern regarding the safe use of the cells for therapeutic purposes. The goal of the work presented in this thesis was to identify new factors involved in the maintenance of pluripotency, and to further characterize molecular mechanisms of selected candidate genes. Furthermore, we aimed to set up a new method for analyzing genomic integrity of pluripotent cells. The experimental design applied in this study involved a wide range of molecular biology, genome-wide, and computational techniques to study the pluripotency of stem cells and the functions of the target genes. In collaboration with instrument and reagent company Perkin Elmer, KaryoliteTM BoBsTM was implemented for detecting karyotypic changes of pluripotent cells. Novel genes were identified that are highly and specifically expressed in hES cells. Of these genes, L1TD1 and POLR3G were chosen for further investigation. The results revealed that both of these factors are vital for the maintenance of pluripotency and self-renewal of the hESCs. KaryoliteTM BoBsTM was validated as a novel method to detect karyotypic abnormalities in pluripotent stem cells. The results presented in this thesis offer significant new information on the regulatory networks associated with pluripotency. The results will facilitate in understanding developmental and cancer biology, as well as creating stem cell based applications. KaryoliteTM BoBsTM provides rapid, high-throughput, and cost-efficient tool for screening of human pluripotent cell cultures.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

BACKGROUND: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. METHODS: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. RESULTS: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. CONCLUSIONS: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Whole-genome genotyping of grape using a panel of microsatellite

The use of microsatellite markers in large-scale genetic studies is limited by its low throughput and high cost and labor requirements. Here, we provide a panel of 45 multiplex PCRs for fast and cost-efficient genome-wide fluorescence-based microsatellite analysis in grapevine. The developed multiplex PCRs panel (with up to 15-plex) enables the scoring of 270 loci covering all the grapevine genome (9 to 20 loci/chromosome) using only 45 PCRs and sequencer runs. The 45 multiplex PCRs were validated using a diverse grapevine collection of 207 accessions, selected to represent most of the cultivated Vitis vinifera genetic diversity. Particular attention was paid to quality control throughout the whole process (assay replication, null allele detection, ease of scoring). Genetic diversity summary statistics and features of electrophoretic profiles for each studied marker are provided, as are the genotypes of 25 common cultivars that could be used as references in other studies.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

An assessment of population structure in eight breeds of cattle using a whole genome SNP panel

Background: Analyses of population structure and breed diversity have provided insight into the origin and evolution of cattle. Previously, these studies have used a low density of microsatellite markers, however, with the large number of single nucleotide polymorphism markers that are now available, it is possible to perform genome wide population genetic analyses in cattle. In this study, we used a high-density panel of SNP markers to examine population structure and diversity among eight cattle breeds sampled from Bos indicus and Bos taurus. Results: Two thousand six hundred and forty one single nucleotide polymorphisms ( SNPs) spanning all of the bovine autosomal genome were genotyped in Angus, Brahman, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black, Limousin and Nelore cattle. Population structure was examined using the linkage model in the program STRUCTURE and Fst estimates were used to construct a neighbor-joining tree to represent the phylogenetic relationship among these breeds. Conclusion: The whole-genome SNP panel identified several levels of population substructure in the set of examined cattle breeds. The greatest level of genetic differentiation was detected between the Bos taurus and Bos indicus breeds. When the Bos indicus breeds were excluded from the analysis, genetic differences among beef versus dairy and European versus Asian breeds were detected among the Bos taurus breeds. Exploration of the number of SNP loci required to differentiate between breeds showed that for 100 SNP loci, individuals could only be correctly clustered into breeds 50% of the time, thus a large number of SNP markers are required to replace the 30 microsatellite markers that are currently commonly used in genetic diversity studies.

Familial varieties of primary aldosteronism

1. Improved approaches to screening and diagnosis have revealed primary aldosteronism (PAL) to be much more common than previously thought, with most patients normokalaemic. The spectrum of this disorder has been further broadened by the study of familial varieties. 2. Familial hyperaldosteronism type I (FH-I) is a glucocorticoid-remediable form of PAL caused by the inheritance of an adrenocorticotrophic hormone (ACTH)- regulated, hybrid CYP11B1/CYP11B2 gene. Diagnosis has been greatly facilitated by the advent of genetic testing. The severity of hypertension varies widely in FH-I, even among members of the same family, and has demonstrated relationships with gender, degree of biochemical disturbance and hybrid gene crossover point position. Hormone day curve studies show that the hybrid gene dominates over wild-type CYP11B2 in terms of aldosterone regulation. This may be due, in part, to a defect in wild-type CYP11B2-induced aldosterone production. Control of hypertension in FH-I requires only partial suppression of ACTH and much smaller glucocorticoid doses than previously recommended. 3. Familial hyperaldosteronism type II (FH-II) is not glucocorticoid remediable and is not associated with the hybrid gene mutation. Familial hyperaldosteronism type II is clinically, biochemically and morphologically indistinguishable from apparently non-familial PAL. Linkage studies in one informative family did not show segregation of FH-II with the CYP11B2, AT1 or MEN1 genes, but a genome-wide search has revealed linkage with a locus in chromosome 7. As has already occurred in FH-I, elucidation of causative mutations is likely to facilitate earlier detection of PAL.

Familial hyperaldosteronism

Primary aldosteronism (PAL) may be as much as ten times more common than has been traditionally thought, with most patients normokalemic. The study of familial varieties has facilitated a fuller appreciation of the nature and diversity of its clinical, biochemical, morphological and molecular aspects. In familial hyperaldosteronism type I (FH-I), glucocorticoid-remediable PAL is caused by inheritance of an ACTH-regulated, hybrid CYP11B1/CYP11B2 gene. Genetic testing has greatly facilitated diagnosis. Hypertension severity varies widely, demonstrating relationships with gender, affected parent's gender, urinary kallikrein level, degree of biochemical disturbance and hybrid gene crossover point position. Analyses of aldosterone/PRA/cortisol 'day-curves' have revealed that (1) the hybrid gene dominates over wild type CYP11B2 in terms of aldosterone regulation and (2) correction of hypertension in FH-I requires only partial suppression of ACTH, and much smaller glucocorticoid doses than those previously recommended. Familial hyperaldosteronism type II is not glucocorticoid-remediable, and is clinically, biochemically and morphologically indistinguishable from apparently sporadic PAL. In one informative family available for linkage analysis, FH-II does not segregate with either the CYP11B2, AT1 or MEN1 genes, but a genome-wide search has revealed linkage with a locus in chromosome 7. As has already occurred in FH-I, elucidation of causative mutations is likely to facilitate earlier detection of PAL and other curable or specifically treatable forms of hypertension. (C) 2001 Elsevier Science Ltd. All rights reserved.

The impact of genotyping error on haplotype reconstruction and frequency estimation

The choice of genotyping families vs unrelated individuals is a critical factor in any large-scale linkage disequilibrium (LD) study. The use of unrelated individuals for such studies is promising, but in contrast to family designs, unrelated samples do not facilitate detection of genotyping errors, which have been shown to be of great importance for LD and linkage studies and may be even more important in genotyping collaborations across laboratories. Here we employ some of the most commonly-used analysis methods to examine the relative accuracy of haplotype estimation using families vs unrelateds in the presence of genotyping error. The results suggest that even slight amounts of genotyping error can significantly decrease haplotype frequency and reconstruction accuracy, that the ability to detect such errors in large families is essential when the number/complexity of haplotypes is high (low LD/common alleles). In contrast, in situations of low haplotype complexity (high LD and/or many rare alleles) unrelated individuals offer such a high degree of accuracy that there is little reason for less efficient family designs. Moreover, parent-child trios, which comprise the most popular family design and the most efficient in terms of the number of founder chromosomes per genotype but which contain little information for error detection, offer little or no gain over unrelated samples in nearly all cases, and thus do not seem a useful sampling compromise between unrelated individuals and large families. The implications of these results are discussed in the context of large-scale LD mapping projects such as the proposed genome-wide haplotype map.

Prospects for whole genome linkage disequilibrium mapping in domestic dog breeds

Linkage disequilibrium (LD) mapping is commonly used as a fine mapping tool in human genome mapping and has been used with some success for initial disease gene isolation in certain isolated inbred human populations. An understanding of the population history of domestic dog breeds suggests that LID mapping could be routinely utilized in this species for initial genome-wide scans. Such an approach offers significant advantages over traditional linkage analysis. Here, we demonstrate, using canine copper toxicosis in the Bedlington terrier as the model, that LID mapping could be reasonably expected to be a useful strategy in low-resolution, genome-wide scans in pure-bred dogs. Significant LID was demonstrated over distances up to 33.3 cM. It is very unlikely, for a number of reasons discussed, that this result could be extrapolated to the rest of the genome. It is, however, consistent with the expectation given the population structure of canine breeds and, in this breed at least, with the hypothesis that it may be possible to utilize LID in a genome-wide scan. In this study, LD mapping confirmed the location of the copper toxicosis in Bedlington terrier gene (CT-BT) and was able to do so in a population that was refractory to traditional linkage analysis.