High resolution HLA analysis reveals independent class I haplotypes and amino-acid motifs protective for multiple sclerosis.

We investigated association between HLA class I and class II alleles and haplotypes, and KIR loci and their HLA class I ligands, with multiple sclerosis (MS) in 412 European American MS patients and 419 ethnically matched controls, using next-generation sequencing. The DRB1*15:01~DQB1*06:02 haplotype was highly predisposing (odds ratio (OR) = 3.98; 95% confidence interval (CI) = 3-5.31; p-value (p) = 2.22E-16), as was DRB1*03:01~DQB1*02:01 (OR = 1.63; CI = 1.19-2.24; p = 1.41E-03). Hardy-Weinberg (HW) analysis in MS patients revealed a significant DRB1*03:01~DQB1*02:01 homozyote excess (15 observed; 8.6 expected; p = 0.016). The OR for this genotype (5.27; CI = 1.47-28.52; p = 0.0036) suggests a recessive MS risk model. Controls displayed no HW deviations. The C*03:04~B*40:01 haplotype (OR = 0.27; CI = 0.14-0.51; p = 6.76E-06) was highly protective for MS, especially in haplotypes with A*02:01 (OR = 0.15; CI = 0.04-0.45; p = 6.51E-05). By itself, A*02:01 is moderately protective, (OR = 0.69; CI = 0.54-0.87; p = 1.46E-03), and haplotypes of A*02:01 with the HLA-B Thr80 Bw4 variant (Bw4T) more so (OR = 0.53; CI = 0.35-0.78; p = 7.55E-04). Protective associations with the Bw4 KIR ligand resulted from linkage disequilibrium (LD) with DRB1*15:01, but the Bw4T variant was protective (OR = 0.64; CI = 0.49-0.82; p = 3.37-04) independent of LD with DRB1*15:01. The Bw4I variant was not associated with MS. Overall, we find specific class I HLA polymorphisms to be protective for MS, independent of the strong predisposition conferred by DRB1*15:01

A specific amino acid motif of HLA-DRB1 mediates risk and interacts with smoking history in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease in which genetic risk has been mapped to HLA, but precise allelic associations have been difficult to infer due to limitations in genotyping methodology. Mapping PD risk at highest possible resolution, we performed sequencing of 11 HLA genes in 1,597 PD cases and 1,606 controls. We found that susceptibility to PD can be explained by a specific combination of amino acids at positions 70-74 on the HLA-DRB1 molecule. Previously identified as the primary risk factor in rheumatoid arthritis and referred to as the "shared epitope" (SE), the residues Q/R-K/R-R-A-A at positions 70-74 in combination with valine at position 11 (11-V) is highly protective in PD, while risk is attributable to the identical epitope in the absence of 11-V. Notably, these effects are modified by history of cigarette smoking, with a strong protective effect mediated by a positive history of smoking in combination with the SE and 11-V (P = 10-4; odds ratio, 0.51; 95% confidence interval, 0.36-0.72) and risk attributable to never smoking in combination with the SE without 11-V (P = 0.01; odds ratio, 1.51; 95% confidence interval, 1.08-2.12). The association of specific combinations of amino acids that participate in critical peptide-binding pockets of the HLA class II molecule implicates antigen presentation in PD pathogenesis and provides further support for genetic control of neuroinflammation in disease. The interaction of HLA-DRB1 with smoking history in disease predisposition, along with predicted patterns of peptide binding to HLA, provide a molecular model that explains the unique epidemiology of smoking in PD

Standard reference sequences for submission of HLA genotyping for the 18th International HLA and Immunogenetics Workshop

The International human leukocyte antigen (HLA) and Immunogenetics Workshops (IHIWs) have fostered international collaborations of researchers and experts in the fields of HLA, histocompatibility and immunology. These IHIW collaborations have comprised many projects focused on achieving a variety of specific goals. The international and collaborative nature of these projects necessitates the collection and analysis of complex data generated in multiple laboratories, often using multiple methods of acquisition. Collection and storage of these data in a consistent way adds value to IHIW projects, which can be extended to future work. DNA-based genotyping data, especially HLA genotyping data, can be transmitted in the form of a Histoimmunogenetics Markup Language (HML) document. HML facilitates clear communication of a genotype and supporting metadata, such as, sequencing platform, laboratory assays, consensus sequence, and interpretation. Sequence information can be reported relative to known reference sequences, which add meaning and context to genotypes. Selecting the correct reference sequence for a given allele sequence is nuanced, and guidelines have emerged through collaborative community efforts such as Data Standards Hackathons. Here, we describe the guidelines established for the selection of reference sequences to be used in transmission of HLA (and MICA/MICB) genotyping data for the 18th IHIW.Transplantation and autoimmunit

The amphioxus genome and the evolution of the chordate karyotype

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approx520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies

Isolation of a 97-kb Minimal Essential MHC B Locus from a New Reverse-4D BAC Library of the Golden Pheasant

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC

A hybrid BAC physical map of potato: a framework for sequencing a heterozygous genome

<p>Abstract</p> <p>Background</p> <p>Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH).</p> <p>Results</p> <p>First, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps <it>in silico </it>anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map.</p> <p>Conclusions</p> <p>The AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.</p

Genome-wide BAC-end sequencing of Cucumis melo using two BAC libraries

<p>Abstract</p> <p>Background</p> <p>Although melon (<it>Cucumis melo </it>L.) is an economically important fruit crop, no genome-wide sequence information is openly available at the current time. We therefore sequenced BAC-ends representing a total of 33,024 clones, half of them from a previously described melon BAC library generated with restriction endonucleases and the remainder from a new random-shear BAC library.</p> <p>Results</p> <p>We generated a total of 47,140 high-quality BAC-end sequences (BES), 91.7% of which were paired-BES. Both libraries were assembled independently and then cross-assembled to obtain a final set of 33,372 non-redundant, high-quality sequences. These were grouped into 6,411 contigs (4.5 Mb) and 26,961 non-assembled BES (14.4 Mb), representing ~4.2% of the melon genome. The sequences were used to screen genomic databases, identifying 7,198 simple sequence repeats (corresponding to one microsatellite every 2.6 kb) and 2,484 additional repeats of which 95.9% represented transposable elements. The sequences were also used to screen expressed sequence tag (EST) databases, revealing 11,372 BES that were homologous to ESTs. This suggests that ~30% of the melon genome consists of coding DNA. We observed regions of microsynteny between melon paired-BES and six other dicotyledonous plant genomes.</p> <p>Conclusion</p> <p>The analysis of nearly 50,000 BES from two complementary genomic libraries covered ~4.2% of the melon genome, providing insight into properties such as microsatellite and transposable element distribution, and the percentage of coding DNA. The observed synteny between melon paired-BES and six other plant genomes showed that useful comparative genomic data can be derived through large scale BAC-end sequencing by anchoring a small proportion of the melon genome to other sequenced genomes.</p

BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.)

Background: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with 250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.Daniela Schulte, Ruvini Ariyadasa, Bujun Shi, Delphine Fleury, Chris Saski, Michael Atkins, Pieter deJong, Cheng-Cang Wu, Andreas Graner, Peter Langridge and Nils Stei

Genomic and gene expression profiling of minute alterations of chromosome arm 1p in small-cell lung carcinoma cells

Genetic alterations occurring on human chromosome arm 1p are common in many types of cancer including lung, breast, neuroblastoma, pheochromocytoma, and colorectal. The identification of tumour suppressors and oncogenes on this arm has been limited by the low resolution of current technologies for fine mapping. In order to identify genetic alterations on 1p in small-cell lung carcinoma, we developed a new resource for fine mapping segmental DNA copy number alterations. We have constructed an array of 642 ordered and fingerprint-verified bacterial artificial chromosome clones spanning the 120 megabase (Mb) 1p arm from 1p11.2 to p36.33. The 1p arm of 15 small-cell lung cancer cell lines was analysed at sub-Mb resolution using this arm-specific array. Among the genetic alterations identified, two regions of recurrent amplification emerged. They were detected in at least 45% of the samples: a 580 kb region at 1p34.2–p34.3 and a 270 kb region at 1p11.2. We further defined the potential importance of these genomic amplifications by analysing the RNA expression of the genes in these regions with Affymetrix oligonucleotide arrays and semiquantitative reverse transcriptase–polymerase chain reaction. Our data revealed overexpression of the genes HEYL, HPCAL4, BMP8, IPT, and RLF, coinciding with genomic amplification