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

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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

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

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

Haplotype Alignment of the Region Presenting Differing Variation Rates

<p>The alignment covers the centromeric side of the DR–DQ 158-kb DNA segment (left half, low variation) and the adjacent DNA segment (increased variation). Coordinates refer to Chromosome 6 build NCBI35. Rows represent the allelic state for 26 single chromosomes with the same <i>DRB1*1501-DQA1*0102-DQB1*0602</i> (DR15–DQ6) haplotype at successive SNPs which are represented by columns (A, red; C, blue; G, orange; and T, green). Identity is interrupted at a position perfectly matching with a recombination hotspot coordinate [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b005" target="_blank">5</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b053" target="_blank">53</a>] represented as hotspot number 2 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-g004" target="_blank">Figure 4</a>.</p

Positional Distributions of Variations between PGF and QBL and COX and QBL

<div><p>(A) Shows the distribution for PGF and QBL and (B) shows COX and QBL. MHC sequences were divided into 10-kb bins, and variations were calculated in each bin. Results are expressed as variations per 1 kb. Red and blue plots relate to SNP and DIP variations respectively. The sequence is interrupted by five gaps, shown as green vertical bars, where BACs encompassing these regions could not be identified from the clone library, which by comparison with PGF comprise a total of approximately 317 kb. The lengths and gene content of these gaps were as follows, from left to right: 159 kb including <i>OR2U1P</i> to <i>OR12D2;</i> 51 kb containing <i>HCP5;</i> 26 kb containing <i>C6orf26, C6orf27,</i> and the three exons of 3′ end of <i>MSH5;</i> 53 kb containing <i>CREBL1, FKBPL,</i> and six exons of the 5′ end of <i>TNXB;</i> and 27 kb containing <i>HLA-DOB</i>. These gaps do not represent large genomic deletions within the QBL haplotype since exonic sequence from selected genes within these regions were successfully amplified from QBL genomic DNA and sequenced to confirm their identity. The grey shaded area at the telomeric end of the map represents sequence for which overlap was not obtained and was therefore outside the area that was compared.</p><p>Boundaries of the class I, II, and III regions are shown. The positions of <i>RFP</i> and <i>KIFC1</i> that define the ends of the MHC haplotype sequencing project are indicated. Landmark genes are labelled in blue. Regions 1 and 2 are the RCCX module and the <i>HLA-DRB</i> region, respectively. The <i>HLA-DRB3</i> and <i>HLA-DQB3</i> region, which shows little variation between COX and QBL haplotypes, is shaded in orange.</p></div