Sequence variation and disease in the wake of the draft human genome.

The sequencing phase of the human genome project will soon be over. In its wake, repertoires of sequence polymorphisms among the human population are being sampled and a battery of functional genomics projects, from gene and protein expression studies to whole proteome interaction experiments, are generating vast quantities of data. Now that the data, or the means to generate data, are available it is the application of this information in enhancing our understanding of biology that represents the next formidable challenge. Two prominent issues should be considered. First, existing data must be analysed using the best methods available. The prediction of enzymatic activity for bestrophin, whose gene is mutated in Best macular dystrophy, is described in this review. This is an example of the experimentally testable hypotheses that can result from such detailed and exhaustive analyses. Secondly, the torrents of data from high-throughput studies will need to be made more accessible to all using web-based resources that integrate and digest complementary data types. The internet sites that showcase the human genome sequence are blazing a new trail. Ultimately, the success of genome sequencing and functional genomics will be measured not by the quantity and accuracy of raw data generated, but how rapidly they can be harnessed to span the divide between genotype and phenotype.     

A practical guide to orient yourself in the labyrinth of genome databases

The identification of genes involved in human inherited disorders has been revolutionized by the resources produced by the Human Genome Project. In particular, the generation of >1 000 000 human expressed sequence tags (ESTs) has led to the partial identification of a significant percentage of all human genes. In the next 7 years, we will witness another revolution when sequencing of the human genome is complete. The generation of large amounts of genomic data must be accompanied by parallel efforts to make the information easily accessible. Efforts towards this goal have already started, but retrieval of information from genomic databases still remains an arduous task. With practical examples, we will try to show how the currently available information can be exploited usefully, in particular to identify candidate genes for human diseases.      

Comparative genome mapping in the sequence-based era: early experience with human chromosome 7

The success of the ongoing Human Genome Project has resulted in accelerated plans for completing the human genome sequence and the earlier-than-anticipated initiation of efforts to sequence the mouse genome. As a complement to these efforts, we are utilizing the available human sequence to refine human-mouse comparative maps and to assemble sequence-ready mouse physical maps. Here we describe how the first glimpses of genomic sequence from human chromosome 7 are directly facilitating these activities. Specifically, we are actively enhancing the available human-mouse comparative map by analyzing human chromosome 7 sequence for the presence of orthologs of mapped mouse genes. Such orthologs can then be precisely positioned relative to mapped human STSs and other genes. The chromosome 7 sequence generated to date has allowed us to more than double the number of genes that can be placed on the comparative map. The latter effort reveals that human chromosome 7 is represented by at least 20 orthologous segments of DNA in the mouse genome. A second component of our program involves systematically analyzing the evolving human chromosome 7 sequence for the presence of matching mouse genes and expressed-sequence tags (ESTs). Mouse-specific hybridization probes are designed from such sequences and used to screen a mouse bacterial artificial chromosome (BAC) library, with the resulting data used to assemble BAC contigs based on probe-content data. Nascent contigs are then expanded using probes derived from newly generated BAC-end sequences. This approach produces BAC-based sequence-ready maps that are known to contain a gene(s) and are homologous to segments of the human genome for which sequence is already available. Our ongoing efforts have thus far resulted in the isolation and mapping of >3,800 mouse BACs, which have been assembled into >100 contigs. These contigs include >250 genes and represent approximately 40% of the mouse genome that is homologous to human chromosome 7. Together, these approaches illustrate how the availability of genomic sequence directly facilitates studies in comparative genomics and genome evolution.     

Reverse vaccinology: developing vaccines in the era of genomics

The sequence of microbial genomes made all potential antigens of each pathogen available for vaccine development. This increased by orders of magnitude potential vaccine targets in bacteria, parasites, and large viruses and revealed virtually all their CD4(+) and CD8(+) T cell epitopes. The genomic information was first used for the development of a vaccine against serogroup B meningococcus, and it is now being used for several other bacterial vaccines. In this review, we will first summarize the impact that genome sequencing has had on vaccine development, and then we will analyze how the genomic information can help further our understanding of immunity to infection or vaccination and lead to the design of better vaccines by diving into the world of T cell immunity.     

Genes,mutations, and human inherited disease at the dawn of the age of personalized genomics

The number of reported germline mutations in human nuclear genes, either underlying or associated with inherited disease, has now exceeded 100,000 in more than 3,700 different genes. The availability of these data has both revolutionized the study of the morbid anatomy of the human genome and facilitated “personalized genomics.” With ～300 new “inherited disease genes” (and ～10,000 new mutations) being identified annually, it is pertinent to ask how many “inherited disease genes” there are in the human genome, how many mutations reside within them, and where such lesions are likely to be located? To address these questions, it is necessary not only to reconsider how we define human genes but also to explore notions of gene “essentiality” and “dispensability.” Answers to these questions are now emerging from recent novel insights into genome structure and function and through complete genome sequence information derived from multiple individual human genomes. However, a change in focus toward screening functional genomic elements as opposed to genes sensu stricto will be required if we are to capitalize fully on recent technical and conceptual advances and identify new types of disease‐associated mutation within noncoding regions remote from the genes whose function they disrupt. Hum Mutat 31:631–655, 2010. © 2010 Wiley‐Liss, Inc.     

The advent of personal genome sequencing

Rapid technological advances are decreasing DNA sequencing costs and making it practical to undertake complete human genome sequencing on a large scale for the first time. Disease studies that involve sequencing hundreds of patient genomes are underway. The all-inclusive sequencing price per genome is expected to reach $1000 over the next few years and will likely decline further in the following years. This dramatic price decline will herald widespread personal genome sequencing and lead to significant improvements in human health and reduced health care costs. Key to realizing these benefits will be medical genomics' and systems biology's success in providing increasing contextual interpretation of biological and medical effects of the detected sequence variants in a genome. Given the substantial potential benefits and the manageability of the health and discrimination risks involved with the possible misuse of this information, we propose that governments and insurance companies support or even require personal genome sequencing. Critical to the widespread acceptance of personal genome sequencing, however, will be the need to educate physicians and the public about the realistic benefits and risks of such an analysis to prevent overinterpretation and misuse of this valuable information.     

Evolutionary genomics of Entamoeba

Entamoeba histolytica is a human pathogen that causes amoebic dysentery and leads to significant morbidity and mortality worldwide. Understanding the genome and evolution of the parasite will help explain how, when and why it causes disease. Here we review current knowledge about the evolutionary genomics of Entamoeba: how differences between the genomes of different species may help explain different phenotypes, and how variation among E. histolytica parasites reveals patterns of population structure. The imminent expansion of the amount genome data will greatly improve our knowledge of the genus and of pathogenic species within it.     

Reconstructing contiguous regions of an ancestral genome

This article analyzes mammalian genome rearrangements at higher resolution than has been published to date. We identify 3171 intervals, covering approximately 92% of the human genome, within which we find no rearrangements larger than 50 kilobases (kb) in the lineages leading to human, mouse, rat, and dog from their most recent common ancestor. Combining intervals that are adjacent in all contemporary species produces 1338 segments that may contain large insertions or deletions but that are free of chromosome fissions or fusions as well as inversions or translocations >50 kb in length. We describe a new method for predicting the ancestral order and orientation of those intervals from their observed adjacencies in modern species. We combine the results from this method with data from chromosome painting experiments to produce a map of an early mammalian genome that accounts for 96.8% of the available human genome sequence data. The precision is further increased by mapping inversions as small as 31 bp. Analysis of the predicted evolutionary breakpoints in the human lineage confirms certain published observations but disagrees with others. Although only a few mammalian genomes are currently sequenced to high precision, our theoretical analyses and computer simulations indicate that our results are reasonably accurate and that they will become highly accurate in the foreseeable future. Our methods were developed as part of a project to reconstruct the genome sequence of the last ancestor of human, dogs, and most other placental mammals.     

Assessing the enrichment performance in targeted resequencing experiments

Target enrichment strategies are a very common approach to sequence a predefined part of an individual's genome using second-generation sequencing technologies. While highly dependent on the technology and the target sequences selected, the performance of the various assays is also variable between samples and is influenced by the way how the libraries are handled in the laboratory. Here, we show how to find detailed information about the enrichment performance using a novel software package called NGSrich, which we developed as a part of a whole-exome resequencing pipeline in a medium-sized genomics center. Our software is suitable for high-throughput use and the results can be shared using HTML and a web server. Finally, we have sequenced exome-enriched DNA libraries of 18 human individuals using three different enrichment products and used our new software for a comparative analysis of their performance.     

Studying genotype-phenotype relationships: cardiovascular disease as an example

Cardiovascular disease is a multifactorial disorder resulting from the complex interaction of a plethora of both environmental and genetic factors. The "candidate gene" approach aims at identifying the genes contributing to cardiovascular disease assessing their entire polymorphic spectrum and the consequences of their combination in appropriate large association studies. In view of the sequence data available for the entire human genome, candidate sequences will more easily be traced and located in their chromosomal context. More powerful sequencing devices are available and will hopefully give reliable and reproducible data not only on the nucleotide sequence diversity in different populations throughout candidate regions of the human genome. The choice and the assessment of disease-related or intermediate phenotypes, especially in clinical settings, will be again more crucial in the future when the knowledge of gene sequence variation significantly increases. Identification of relevant genes and genetic variants involved in the different pathophysiological steps leading to cardiovascular disease may considerably improve our understanding of the mechanisms of the disease course. This may help to identify high-risk individuals and groups or subgroups in whom specific therapeutic interventions are indicated or necessary, leading to an individually adapted clinical management.     

Toward a genome-wide systems biology analysis of host-pathogen interactions in group A Streptococcus

Genome-wide analysis of microbial pathogens and molecular pathogenesis processes has become an area of considerable activity in the last 5 years. These studies have been made possible by several advances, including completion of the human genome sequence, publication of genome sequences for many human pathogens, development of microarray technology and high-throughput proteomics, and maturation of bioinformatics. Despite these advances, relatively little effort has been expended in the bacterial pathogenesis arena to develop and use integrated research platforms in a systems biology approach to enhance our understanding of disease processes. This review discusses progress made in exploiting an integrated genome-wide research platform to gain new knowledge about how the human bacterial pathogen group A Streptococcus causes disease. Results of these studies have provided many new avenues for basic pathogenesis research and translational research focused on development of an efficacious human vaccine and novel therapeutics. One goal in summarizing this line of study is to bring exciting new findings to the attention of the investigative pathology community. In addition, we hope the review will stimulate investigators to consider using analogous approaches for analysis of the molecular pathogenesis of other microbes.     

Sequencing of a new target genome: the Pediculus humanus humanus (Phthiraptera: Pediculidae) genome project

The human body louse, Pediculus humanus humanus (L.), and the human head louse, Pediculus humanus capitis, belong to the hemimetabolous order Phthiraptera. The body louse is the primary vector that transmits the bacterial agents of louse-borne relapsing fever, trench fever, and epidemic typhus. The genomes of the bacterial causative agents of several of these aforementioned diseases have been sequenced. Thus, determining the body louse genome will enhance studies of host-vector-pathogen interactions. Although not important as a major disease vector, head lice are of major social concern. Resistance to traditional pesticides used to control head and body lice have developed. It is imperative that new molecular targets be discovered for the development of novel compounds to control these insects. No complete genome sequence exists for a hemimetabolous insect species primarily because hemimetabolous insects often have large (2000 Mb) to very large (up to 16,300 Mb) genomes. Fortuitously, we determined that the human body louse has one of the smallest genome sizes known in insects, suggesting it may be a suitable choice as a minimal hemimetabolous genome in which many genes have been eliminated during its adaptation to human parasitism. Because many louse species infest birds and mammals, the body louse genome-sequencing project will facilitate studies of their comparative genomics. A 6-8X coverage of the body louse genome, plus sequenced expressed sequence tags, should provide the entomological, evolutionary biology, medical, and public health communities with useful genetic information.     

The nature of the hepatitis B virus and its mode of replication

Conclusion Hepatitis B research until recently was confined to a further understanding of virus immunopathology and sequelae. The application of molecular biology techniques to the hepatitis B virus genome has added a new and important dimension to the investigation of the processes accompanying replication of the virus, as has been outlined in this review. It is anticipated that the availability of monoclonal antibodies to hepatitis B antigens will complement this newly acquired information allowing a clearer picture of hepatitis B virus replication to emerge whereby accurate predictions from the genome sequence will be combined with the identification of virus specific products using sera of single specificity. It has often been stressed that the virology of hepatitis B is unique; this concept has now been swept aside to reveal the hepatitis B virus as one of a family of variously related viruses with similar morphology, proteins, and genome structure. These newly described agents are pathogens of widely differing animal species including American woodchucks [88], prairie dogs [78], ducks [95] and squirrels [96]. There is also a preliminary report that one agent of non-A, non-B hepatitis of man is related to hepatitis B virus [19] but this remains to be confirmed. An expansion of research into the pathogenesis and properties of these similar viruses will inevitably further our understanding of human hepatitis B virus infection.     

Traversing the biological complexity in the hierarchy between genome and CAD endpoints in the population at large

An emerging challenge facing those who are concerned about the efficacy of public health programs is to understand how information from the DNA revolution might be used to improve our ability to predict the initiation, progression and severity of a common disease having a complex multifactorial etiology. In the course of research to evaluate the role of information about DNA, combinations of genome types and environmental exposures that predispose to disease will be identified. Such information is expected to be useful in efforts to identify individuals and families at higher risk of disease and to predict their responses to a proposed therapy. This paper begins with a discussion of the features of a realistic biological model for the study of a common multifactorial disease. We present evidence for the complexity in the relationship between genome type variation and variation in risk of coronary artery disease (CAD) and review the preliminary results of our studies to determine whether information about genome type variation can improve our ability to predict the distribution of CAD among individuals in the population at large. Such studies make it apparent that new analytical strategies are necessary to deal with the plethora of genome type information available for the evaluation of risk of a common disease like CAD. This shift in the research paradigm will build upon new strategies to understand the organization of natural systems that are coming from outside the mainstream of genetic research.     

Comparative DNA sequence analysis of wheat and rice genomes

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.     

Human genome project: a federator program of genomic medicine

The Human Genome Project improves our understanding of the molecular genetics basis of the inherited and complex diseases such as diabetes, schizophrenia, and cancer. Information from the human genome sequence is essential for several antenatal and neonatal screening programmes. The new genomic tools emerging from this project have revolutionized biology and medicine and have transformed our understanding of health and the provision of healthcare. Its implications pervade all areas of medicine, from disease prediction and prevention to the diagnosis and treatment of all forms of disease. Increasingly, it will be possible to drive predisposition testing into clinical practice, to develop new treatments or to adapt available treatments more specifically to an individual's genetic make-up. This genomic information should transform the traditional medications that are effective for every members of the population to personalized medicine and personalized therapy. The pharmacogenomics could give rise to a new generation of highly effective drugs that treat causes, not just symptoms.     

Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis

Lysosomal storage diseases are due to inherited deficiencies in various enzymes involved in basic metabolic processes. As with other genetic diseases, accurate structure data for these enzymatic proteins should help in better understanding the molecular effects of mutations identified in patients with the corresponding lysosomal diseases; however, no such three-dimensional (3D) structure data are available for many lysosomal enzymes. Thus, we herein intend to illustrate for an audience of molecular geneticists how structure information can nonetheless be obtained via a bioinformatics approach in the case of five human lysosomal glycoside hydrolases. Indeed, using the two-dimensional hydrophobic cluster analysis method to decipher the sequence information available in data banks for the large group of glycoside hydrolases (clan GH-A) to which these human lysosomal enzymes belong, we could deduce structure predictions for their catalytic domains and propose explanations for the molecular effects of mutations described in patients. In addition, in the case of human beta-glucuronidase for which experimental 3D data have been reported, we also show here that bioinformatics methods relying on the available 3D structure information can be used to obtain further insights into the effects of various mutations described in patients with Sly disease. In a broader perspective, our work stresses that, in the context of a rapid increase in protein sequence information through genome sequencing, bioinformatics approaches might be highly useful for generating structure-function predictions based on sequence-structure interrelationships.