Contemplating the End of the Beginning

The past year has brought unprecedented public attention to biomedical research, with a particularly intense focus on the Human Genome Project and the completion of a first-generation ∼3-billion-basepair human genome sequence. Much of this attention related to the competition between the two parallel, yet separate, efforts of the publicly-funded International Human Genome Sequencing Consortium and the private company Celera Genomics. Despite the apparent rancor between the groups, two celebratory events notably punctuate the past year: the joint media announcement in late June 2000 that both groups had generated a “working draft” sequence of the human genome and the two landmark scientific publications in February 2001 that describe the efforts of each project (International Human Genome Sequencing Consortium 2001; Venter et al. 2001). Numerous grandiose cliches and metaphors have been used to convey the magnitude of these accomplishments and their associated implications for biomedical research and clinical medicine. Here we add one more to this list. Our choice for capturing the essence of contemporary human genome analysis is an analogy to a mountain climbing expedition, one where significant progress has been made to provide a spectacular view of the genetic landscape. But this is not an expedition that is complete, with uncertain— yet exciting—genomic terrain ahead. Indeed, the Human Genome Project is now firmly at the “base camp” of the expedition to elucidate the human genetic blueprint and to begin to understand its content. Nevertheless, this is a milestone of tremendous significance and excitement. Here we outline some of the key lessons learned during the initial analysis of the human genome sequence. We highlight a few of the many remaining questions in understanding the genome’s structure and function, with most answers likely becoming available later in the expedition. Finally, we preview the anticipated climb to the final summit and the ascent to a complete and finished sequence.

Chromosome 21 is the smallest human chromosome and represents a model for physical mapping of the entire human genome. Three copies of this autosome cause Down syndrome, the most frequent genetic disorder associated with significant mental retardation. Five years ago, in the framework of the international human genome project, and as an integrated part of the chromosome 21 community, a consortium, of academic groups from Japan and Germany was formed to map and sequence this chromosome. Several whole-genome and chromosome-specific DNA libraries were constructed for mapping purposes. Using a combination of nested-deletion [1] and shotgun sequencing approaches, our center determined and analyzed over 17 million bases of high-quality data. In total, over 33.5 million basepairs of DNA, distributed over four contigs, were sequenced with very high accuracy [2]. The largest of these contigs is nearly 25.5Mb long. Only three small clone gaps remain, which together comprise approximately 100kb. Thus, we achieved a coverage of 99.7% of 21q. In addition, 211,116 bp from the short arm were also sequenced. Analysis of the chromosome revealed far fewer genes than previously estimated. Instead of 800-1000 expected genes, we found only 225 genes, of which 127 are known genes and 98 are predicted genes. In addition, 59 pseudogenes were identified. The completion of the 21q sequence provides a unique resource for understanding the molecular pathophysiology of Down syndrome, as well as all other monogenic and complex disorders that map to this chromosome, including Alzheimer’s disease, leukemia, autoimmune disease, epilepsy and manic-depressive psychosis. It also stands as a structural framework from which the complete molecular architecture of the chromosome can be determined. With the announcement of the first “working draft” of the human genome by the international human genome sequencing consortium this past June, there has been a great interest in the total number of genes in the human genome[3]. Some of the current estimates range from as low as 34,000 genes to as high as 140,000 genes. The low gene-density of chromosome 21 was quite unexpected. Most striking is a 7-Mb region near the region near the centromere that contains only one gene. This region is much larger than the whole genome of some species, such as Escherichia coli, yet evolutionary processes permitted the existence of such a gene-poor SNA segment. This finding leads us to propose that there are additional large gene-less regions in other human/mammalian chromosomes. By combining the gene numbers of chromosomes 21 and 22 [4] (770genes; 2-3% of human genome) and assuming that together they represent the average gene content of the human genome, we estimate that the total number of human genes may be close to 40,000. As part of the international human genome sequencing project, we are also sequencing parts of chromosomes 11 and 18. Our high-throughput sequence production line generates about 10 Mb per day. In order to handle such a high volume of output, we have developed a system for automated data assembly, annotation and release. All the data is released through our website and through the DNA Database of Japan immediately according to the policies established by the international consortium. In addition to chromosomes 11, 18 and 21, we are also interested in the structure of the entire human genome. In collaboration with the University of Tokyo, we have developed a database called HGREP [5] (Human Genome Reconstruction Project) that contains working draft and finished sequences which cover more than 85% of the human genomic sequences. Sequence entries are aligned along the chromosomes based on sequence similarities to STS markers, BAC-end and other entry sequences. Further, biological features, such as genes, gene functions, repeats and CpG islands, are fully annotated on these entries. To identify evolutionally conserved and biologically important information in the genome, we are taking the strategy of comparative genomic sequence analysis between human and other related genomes such as those of rodents and primates. We are currently sequencing several regions of the mouse, including some regions that are counterpart to human chromosome 21, and are preparing genomic resources for sequencing of the chimpanzee. Through comparative methods, we are also trying to understand telomeric regions, structures at the end of chromosomes that play an important role in several biological functions and have been associated with human diseases like an oncogenesis and a cellular aging. The structure and function of all genes and their regulatory regions can only be fully understood by looking at how they interact under different circumstances and in several different backgrounds. Determining which genes and elements are human-specific will lead to a deeper understanding of who we are and will advance medical sciences drastically.

2025 marks the twenty-fifth anniversary of the completion of a working draft of the 3-Gb human genome sequence and its availability in public databases to promote research into human health and disease for the benefit of all. The sequence was produced by the International Human Genome Sequencing Consortium, which comprised sequencing centers from six countries who together undertook the largest collaborative biological project to date. Under the leadership of Sir John Sulston, the United Kingdom played a significant role in the project through the Sanger Centre (now the Wellcome Sanger Institute), which was founded in 1992 with support from the Wellcome Trust, a charitable foundation funding medical research. The Sanger Centre contributed approximately one-third of the final human genome sequence generated by the Human Genome Project and, along with the European Bioinformatics Institute, developed Ensembl, one of the major databases providing free access to genomic data and annotation for biomedical research. As a result of a chance meeting, I came to work at the Sanger Centre (and later the Wellcome Sanger Institute) from 1992 to 2007, initially as a scientific administrator and later as the Human Genome Project manager and head of sequencing. Over that period, the Sanger Centre became one of the largest genome sequencing centers in the world and began its transition to become a world-leading center in genomics research to advance biology and health.

The 1000 Genomes Project: paving the way for personalized genomic medicine.

In this chapter, we focus on an institution that represents the exclusivity of the International Human Genome Sequencing Consortium (IHGSC) while also contributing to the ‘funnelling effect’ that shaped it: the Sanger Institute. After situating its origins in the sequencing of the worm Caenorhabditis elegans, we portray the Sanger Institute as a driver in the formation of the IHGSC and as an advocate for the concentration of its membership into a few high-throughput sequencing centres. The intensification of this genome centre model, alongside competition with the corporate sequencing effort led by Celera Genomics, shaped the formation of the IHGSC. As a result, the large-scale production of sequence data became an increasingly differentiated and exclusive practice undertaken by its members. Conversely, the smaller laboratories that used this data in their own genetics research were sidelined from the determination of the human reference sequence.

I t is more than a year since the complete sequence of the human genome was publicly announced (International Human Genome Sequencing Consortium, 2001).The impact of this milestone on medical research is already apparent.Scientists are geared up to adopt new experimental approaches in the race to combat human disease.New information flowing from the human genome project should fuel a revolution in medical research with wide-ranging implications for human health, longevity and employability.There are very good reasons to collect and use all of this information for the benefit of mankind, to improve health care and sustain life.It remains for society to dictate the level of protection placed on personal genetic information.Checks and balances will need to be implemented sooner rather than later.However, the time may come when the genetic blueprint of an individual will be viewed no differently to any other personal information.The potential to predict disease risk by genetic screening is considered here along with the health and medical benefits which should flow from new approaches to medical research fuelled by completion of the human genome project.

The goal of the International Human Genome Project is to generate a reference sequence of the human genome with an accuracy of <1 error in 10,000 bases. The approach chosen by the International Human Genome Sequencing Consortium is a hierarchical mapping and sequencing strategy similar to that described for other large genomes, including Saccharomyces cerevisiae, Caenorhabditis elegans, and Arabidopsis thaliana (1–3). A physical map of the human genome has been assembled on the basis of "fingerprints" generated by restriction digestion of individual bacterial clones and mapping of individual clones to chromosomes using landmarks from existing genetic maps. At Washington University, St. Louis, more than 300,000 clones from the whole human genome bacterial artificial chromosome (BAC) libraries RPCI-11 and RPCI-13 from P. de Jong (4) were fingerprinted, and the resulting maps of these overlapping clones have been integrated with clone maps of individual chromosomes generated by other centers (5). Sets of minimal overlapping clones have been selected for sequencing from the resulting maps.

The February 15th, 2001 announcement of the draft human genome sequence was the culmination of a momentous undertaking. The analyses of this sequence (International Human Genome Sequencing Consortium 2001; Venter et al. 2001) predicted a surprisingly modest 31,000 genes for Homo sapiens (although this number has yet to be finalized), as compared with estimates as high as 140,000 genes just a few years ago (Fields et al. 1994). Given this, we narrowly top the list for the eukaryotic genomes that have been completely sequenced (Table 1). Even though sequencing the human genome may be merely a first pass at a deeper understanding of our biology, one fact stands out as demanding an immediate explanation: Why do humans have so few genes? The assumptions and chauvinism implicit in this question—that humans are vastly more complex than the other fully sequenced eukaryotes and should therefore have a commensurately larger suite of genes—are difficult to argue clearly and may be even more difficult to justify biologically (McShea 1996). Still, it is hard to deny our intuitive perception that the number of genes in a genome should be roughly correlated with complexity and that organismal complexity can be ranked as yeast nematodes flies humans (we reserve judgment on the relative position of the “green fly,” Arabidopsis ). However, the number of genes in the genomes of these organisms does not match our naive expectation. This disjunction between the number of genes and organismal complexity, what we call the “G-value paradox,” parallels the finding during the 1950s that the physical size of genomes does not correlate with organismal complexity, a relationship known as the C-value paradox (Cavalier-Smith 1985; Appendix and Table 1). The finding that much of the genome contains noncoding repeats and “junk” DNA seemed to resolve the C-value paradox. Implicitly, this resolution rested on the assumption that once noncoding DNA was taken into account, the total number of genes would then correlate with organismal complexity (Cavalier-Smith 1985). However, the published G values of the completely sequenced eukaryotes make it clear that we have not yet resolved the C-value paradox—it has merely given way to the G-value paradox. Just as the discovery of noncoding DNA seemed to resolve the C-value paradox, so a few simple observations may in time resolve the G-value paradox. These observations all attempt to give more value to each of our genes and thus to give us a more accurate genomic predictor of organismal complexity by identifying the true measure of information encoded by a genome, the “I-value” (Appendix; the concept of “information” with respect to genes is itself highly debated, see Oyama 1985 and Sarkar 1998, but a philosophical resolution to this issue is beyond the scope of this article). Some of the observations we discuss here have been offered as the answer to explaining our modest number of genes (Davidson 2001; Hanke et al. 1999; Szathmary et al. 2001), whereas some have been invoked in combination (International Human Genome Sequencing Consortium 2001; Petsko 2001). These observations indicate that the evolution of organismal complexity will typically involve changes in the genome that are subtler than simply adding genes. The C-value paradox was resolved by a plea to the G value; a resolution of the G-value paradox may be offered by a plea to the I value. However, what if no measure of genomic information content, no matter how precise, correctly predicts organismal complexity? Our last observation will attempt to undermine the basic assumption that organismal complexity somehow corresponds to even a refined measure of genomic complexity.

The ‘omics’ of cancer

At the First (1996) and Second (1997) International Strategy Meetings on Human DNA Sequencing, the attendees discussed and strongly endorsed the concept that human genomic DNA sequence produced by large-scale DNA sequencing centers funded by the Human Genome Project should release data as rapidly as possible. In the case of unfinished data, it was suggested that sequence assemblies of 1–2 kb in size should be released within 24 hr of generation. Finished data should be submitted to the public sequence databases on a similarly rapid time scale. Most of the funding agencies engaged in supporting the Human Genome Project have adopted policies that reflect the importance of rapid dissemination of genomic sequence data. Similarly the nucleotide sequence databases have made provision for the acceptance of the unfinished data as well as, of course, finished sequence data. At the 1998 meeting, the question was raised about similar data dissemination practices for genomic sequence data from other organisms. The attendees unanimously agreed that this was also critical and adopted the following statement as individual scientists. ‘‘As extensive determination of the genomic DNA sequence of several organisms proceeds, it is increasingly clear that sequence information has enormous and immediate scientific value, even prior to its final assembly and completion. Delaying the release of either unfinished or finished genomic DNA sequence data serves no useful purpose and actually has the effect of slowing the progress of research. Therefore, the attendees at the Third International Strategy Meeting on Human Genome Sequencing (Bermuda, Feb. 27–28, 1998) agreed unanimously to support, as individual scientists, the view that all publicly funded large-scale DNA sequencing projects, regardless of the organism, should deposit data immediately into the public domain, following the same guidelines that have previously been adopted by this group for human genomic sequence [see D.R. Bentley, Science 274: 533–534 (1996); http:// hugo.gdb.org/bermuda.htm]. The scientists attending this meeting will continue to adhere to these principles and urge all other scientists and policymaking groups involved in large-scale sequencing to adopt them as well.’’ This statement does not yet reflect, nor is it binding on, the policy of any funding agency. The attendees at the Third International Strategy Meeting on Human Genome Sequencing were Mark Adams, David Bentley, Bruce Birren, Martin Bobrow, Elbert Branscomb, Graham Cameron, Anthony V. Carrano, Ellson Chen, Francis Collins, David Cox, Mike Davies, Pieter de Jong, Richard Durbin, Glen Evans, Asao Fujiyama, Richard Gibbs, Eric Green, Phil Green, Mark Guyer, LaDeana Hillier, Ursula Hurtenbach, Elke Jordan, Jill Kent, Fumihiko Kikuchi, Steven Koonin, Jane Lamerdin, Eric Lander, Chris Martin, Richard McCombie, John McPherson, Michael Morgan, Richard Myers, James Ostell, Ari Patrinos, Jane Peterson, Bruce Roe, Jane Rogers, Andre Rosenthal, Yoshiyuki Sakaki, Barbara Skene, Edwin Southern, Jilly Steward, John Sulston, Mark Vaudin, Jeffrey Wadsworth, Robert Waterson, James Watson, Jean Weissenbach, Richard Wilson, and MarieLaure Yaspo.

Human genome project Europe

Killer Immunoglobulin-like Receptors (KIR) comprise a diverse, highly polymorphic family of cell-surface glycoproteins that are principally expressed by Natural Killer (NK) cells. These innate immune lymphocytes fulfill vital functions in human reproduction and immune responses to viral infection. KIR3DL2 is an inhibitory NK cell receptor that recognizes a common epitope of the HLA-A3 and HLA-A11 class I glycoproteins of the major histocompatibility complex. KIR3DL2 also binds exogenous DNA containing the CpG motif. This interaction causes internalization of the KIR-DNA. Exogenous CpG-DNA typically activates NK cells, but the specificity of KIR3DL2-DNA binding and internalization is unclear. We hypothesized that KIR3DL2 binds exogenous DNA in a sequence-specific manner that differentiates pathogen DNA from self-DNA. In testing this hypothesis, we surveyed octameric CpG-DNA sequences in the human genome, and in reference genomes of all bacteria, fungi, viruses, and parasites, with focus on medically relevant species. Among all pathogens, the nucleotides flanking CpG motifs in the genomes of parasitic worms that infect humans are most divergent from those in the human genome. We cultured KIR3DL2+NKL cells with the commonest CpG-DNA sequences in either human or pathogen genomes. DNA uptake was negatively correlated with the most common CpG-DNA sequences in the human genome. These CpG-DNA sequences induced inhibitory signaling in KIR3DL2+NKL cells. In contrast, KIR3DL2+NKL cells lysed more malignant targets and produced more IFNγ after culture with CpG-DNA sequences prevalent in parasitic worms. By applying functional immunology to evolutionary genomics, we conclude that KIR3DL2 allows NK cells to differentiate self-DNA from pathogen DNA.

A review of Michael Ashburner's book Drosophila .

Reproductive Options for Individuals at Risk for Transmission of a Genetic Disorder

The sequencing of the human genome has revealed the existence of a large number of new members of the various cytokine families and has raised the perennial issue of how cytokines function to benefit human evolution. Usually duplication or redundancy of a gene suggests that it functions to assist the survival of the species. In the case of the proinflammatory cytokines, their role in the survival of the host is less clear since this class of cytokines is clearly implicated in host defense mechanisms against infection as well as the pathogenic processes of several diseases. The interleukin-1 (IL-1) and tumor necrosis factor (TNF) families of proinflammatory cytokines have been studied in great detail for their roles in disease; however, studies on their roles in host defense against infection are limited to a few organisms and models. However, it remains likely that these cytokines contribute to host defense mechanisms using the very same molecular and cellular pathways involved in chronic inflammation, tissue destruction, and tissue remodeling. How does one reconcile the existence of two opposing outcomes for the host—one clearly not in the interest of survival and another vital to the host in order to survive even a minor invasion by a microbe? In each case, identical molecular mechanisms are at work. For example, the processes for emigration of neutrophils into tissues to engulf microorganisms via an increase in endothelial adhesion molecules and chemokine production are the same for the host whether in infected tissue or an inflamed joint. On closer examination, the production and activity of proinflammatory cytokines reveal several levels of regulation, which may explain these seemingly discordant roles for the host. For routine infection or injury, cytokines such as IL-1β and TNF-α are transiently expressed and secreted, their activities are modulated by coproduction of naturally occurring antiinflammatory cytokines, and the levels of production fall rapidly. Thus, IL-1β and TNF-α turn on and off rapidly during infection. In contrast, the production of these same cytokines in chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease fails to cease. Cytokine production in host defense is regulated, whereas in chronic disease it is dysregulated.