Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history.

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.

Validation of mRNA/EST-based gene predictions in human Xp11.4 revealed differences to the organization of the orthologous mouse locus.

Careful manual annotation of the human reference sequence provides a solid basis for the identification of disease-associated genes. Toward this end, we focused on a medically relevant 2.6-Mb region of the human chromosome Xp11.4 between markers DXS9851 and DXS9751 and identified 16 transcription units according to the Vertebrate Genome Annotation (Vega) rules. In order to validate these annotations, we performed a comprehensive RT-PCR expression analysis and a human-mouse comparison. This revealed, despite the high overall genomic conservation of the region, remarkable differences of the gene content between human and mouse. Whereas 12 of 16 annotations were confirmed by RT-PCR in human tissues, for only seven genes mouse orthologs could be identified and found to be expressed. This indicates that a comprehensive and experimentally supported annotation effort of the human genome simultaneously highlights regions with striking differences in gene organization to other species and may indicate evolutionary events specific to the human lineage demanding further functional analyses.

The DNA sequence of the human X chromosome.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

Genomic sequence of the class II region of the canine MHC: comparison with the MHC of other mammalian species.

The domestic dog, Canis familiaris, is an excellent model species in which to study complex inherited diseases, having over 200 recognized breeds, each of which represents a closed gene pool. Overlapping canine genomic BAC clones were sequenced to obtain 711,521 bp of the canine classical and extended MHC class II regions. Analysis and annotation of this sequence reveals that it contains 45 loci, of which 29 are predicted to be functionally expressed. Comparison of the DLA class II sequence with those of the cat, human, and mouse highlights regions of syntenic conservation and species-specific gene rearrangement and duplication and gives an insight into the evolution of the DR region in the order Carnivora. Elucidation of functionally important dog class II genes and the identification of 23 microsatellite markers spanning this region will contribute significantly to the study of canine diseases that have an immune component.

Identification of mammalian microRNA host genes and transcription units.

To derive a global perspective on the transcription of microRNAs (miRNAs) in mammals, we annotated the genomic position and context of this class of noncoding RNAs (ncRNAs) in the human and mouse genomes. Of the 232 known mammalian miRNAs, we found that 161 overlap with 123 defined transcription units (TUs). We identified miRNAs within introns of 90 protein-coding genes with a broad spectrum of molecular functions, and in both introns and exons of 66 mRNA-like noncoding RNAs (mlncRNAs). In addition, novel families of miRNAs based on host gene identity were identified. The transcription patterns of all miRNA host genes were curated from a variety of sources illustrating spatial, temporal, and physiological regulation of miRNA expression. These findings strongly suggest that miRNAs are transcribed in parallel with their host transcripts, and that the two different transcription classes of miRNAs ('exonic' and 'intronic') identified here may require slightly different mechanisms of biogenesis.

Organization and evolution of a gene-rich region of the mouse genome: a 12.7-Mb region deleted in the Del(13)Svea36H mouse.

Del(13)Svea36H (Del36H) is a deletion of approximately 20% of mouse chromosome 13 showing conserved synteny with human chromosome 6p22.1-6p22.3/6p25. The human region is lost in some deletion syndromes and is the site of several disease loci. Heterozygous Del36H mice show numerous phenotypes and may model aspects of human genetic disease. We describe 12.7 Mb of finished, annotated sequence from Del36H. Del36H has a higher gene density than the draft mouse genome, reflecting high local densities of three gene families (vomeronasal receptors, serpins, and prolactins) which are greatly expanded relative to human. Transposable elements are concentrated near these gene families. We therefore suggest that their neighborhoods are gene factories, regions of frequent recombination in which gene duplication is more frequent. The gene families show different proportions of pseudogenes, likely reflecting different strengths of purifying selection and/or gene conversion. They are also associated with relatively low simple sequence concentrations, which vary across the region with a periodicity of approximately 5 Mb. Del36H contains numerous evolutionarily conserved regions (ECRs). Many lie in noncoding regions, are detectable in species as distant as Ciona intestinalis, and therefore are candidate regulatory sequences. This analysis will facilitate functional genomic analysis of Del36H and provides insights into mouse genome evolution.

Complete MHC haplotype sequencing for common disease gene mapping.

The future systematic mapping of variants that confer susceptibility to common diseases requires the construction of a fully informative polymorphism map. Ideally, every base pair of the genome would be sequenced in many individuals. Here, we report 4.75 Mb of contiguous sequence for each of two common haplotypes of the major histocompatibility complex (MHC), to which susceptibility to >100 diseases has been mapped. The autoimmune disease-associated-haplotypes HLA-A3-B7-Cw7-DR15 and HLA-A1-B8-Cw7-DR3 were sequenced in their entirety through a bacterial artificial chromosome (BAC) cloning strategy using the consanguineous cell lines PGF and COX, respectively. The two sequences were annotated to encompass all described splice variants of expressed genes. We defined the complete variation content of the two haplotypes, revealing >18,000 variations between them. Average SNP densities ranged from less than one SNP per kilobase to >60. Acquisition of complete and accurate sequence data over polymorphic regions such as the MHC from large-insert cloned DNA provides a definitive resource for the construction of informative genetic maps, and avoids the limitation of chromosome regions that are refractory to PCR amplification.

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants.

Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions. Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively. Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR. The cDNAs were able to complement their respective E. coli auxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy. The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol. This implies that there must be transporters for pathway intermediates. KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures. We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E. coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue.

Gene annotation: prediction and testing.

Fifty years after the publication of DNA structure, the whole human genome sequence will be officially finished. This achievement marks the beginning of the task to catalogue every human gene and identify each of their function expression patterns. Currently, researchers estimate that there are about 30,000 human genes and approximately 70% of these can be automatically predicted using a combination of ab initio and similarity-based programs. However, to experimentally investigate every gene's function, the research community requires a high-quality annotation of alternative splicing, pseudogenes, and promoter regions that can only be provided by manual intervention. Manual curation of the human genome will be a long-term project as experimental data are continually produced to confirm or refine the predictions, and new features such as noncoding RNAs and enhancers have not been fully identified. Such a highly curated human gene-set made publicly available will be a great asset for the experimental community and for future comparative genome projects.

Meeting highlights: genome informatics.

We bring you the highlights of the second Joint Cold Spring Harbor Laboratory and Wellcome Trust 'Genome Informatics' Conference, organized by Ewan Birney, Suzanna Lewis and Lincoln Stein. There were sessions on in silico data discovery, comparative genomics, annotation pipelines, functional genomics and integrative biology. The conference included a keynote address by Sydney Brenner, who was awarded the 2002 Nobel Prize in Physiology or Medicine (jointly with John Sulston and H. Robert Horvitz) a month later.

Interaction of a PDZ Protein Domain with a Synthetic Library of All Human Protein C Termini.

New interaction partners of a PDZ protein domain were identified through use of a library made up of all known human protein C-terminal peptides ("P" in the schematic representation). The library was displayed on a cellulose membrane by positional resolution of inverted peptides.