Fine-scale structural variation of the human genome.

Inversions, deletions and insertions are important mediators of disease and disease susceptibility. We systematically compared the human genome reference sequence with a second genome (represented by fosmid paired-end sequences) to detect intermediate-sized structural variants >8 kb in length. We identified 297 sites of structural variation: 139 insertions, 102 deletions and 56 inversion breakpoints. Using combined literature, sequence and experimental analyses, we validated 112 of the structural variants, including several that are of biomedical relevance. These data provide a fine-scale structural variation map of the human genome and the requisite sequence precision for subsequent genetic studies of human disease.

Parameters for reliable results in genetic association studies in common disease.

It is increasingly apparent that the identification of true genetic associations in common multifactorial disease will require studies comprising thousands rather than the hundreds of individuals employed to date. Using 2,873 families, we were unable to confirm a recently published association of the interleukin 12B gene in 422 type I diabetic families. These results emphasize the need for large datasets, small P values and independent replication if results are to be reliable.

Linkage and association with type 1 diabetes on chromosome 1q42.

Type 1 diabetes is a complex disorder with multiple genetic loci and environmental factors contributing to disease etiology. In the current study, a human type 1 diabetes candidate region on chromosome 1q42 was mapped at high marker density in a panel of 616 multiplex type 1 diabetic families. To facilitate the identification and evaluation of candidate genes, a physical map of the 7-cM region surrounding the maximum logarithm of odds (LOD) score (2.46, P = 0.0004) was constructed. Genes were identified in the 500-kb region surrounding the marker yielding the peak LOD score and evaluated for polymorphism by resequencing. Single-nucleotide polymorphisms (SNPs) identified in these genes as well as other anonymous markers were tested for allelic association with type 1 diabetes by both family-based and case-control methods. A haplotype formed by common alleles at three adjacent markers (D1S225, D1S2383, and D1S251) was preferentially transmitted to affected offspring in type 1 diabetic families (nominal P = 0.006). These findings extend the evidence supporting the existence of a type 1 diabetes susceptibility locus on chromosome 1q42 and identify a candidate region amenable to positional cloning efforts.

Closing gaps in the human genome with fosmid resources generated from multiple individuals.

The human genome sequence has been finished to very high standards; however, more than 340 gaps remained when the finished genome was published by the International Human Genome Sequencing Consortium in 2004. Using fosmid resources generated from multiple individuals, we targeted gaps in the euchromatic part of the human genome. Here we report 2,488,842 bp of previously unknown euchromatic sequence, 363,114 bp of which close 26 of 250 euchromatic gaps, or 10%, including two remaining euchromatic gaps on chromosome 19. Eight (30.7%) of the closed gaps were found to be polymorphic. These sequences allow complete annotation of several human genes as well as the assignment of mRNAs. The gap sequences are 2.3-fold enriched in segmentally duplicated sequences compared to the whole genome. Our analysis confirms that not all gaps within 'finished' genomes are recalcitrant to subcloning and suggests that the paired-end-sequenced fosmid libraries could prove to be a rich resource for completion of the human euchromatic genome.

Recurrent duplication-driven transposition of DNA during hominoid evolution.

The underlying mechanism by which the interspersed pattern of human segmental duplications has evolved is unknown. Based on a comparative analysis of primate genomes, we show that a particular segmental duplication (LCR16a) has been the source locus for the formation of the majority of intrachromosomal duplications blocks on human chromosome 16. We provide evidence that this particular segment has been active independently in each great ape and human lineage at different points during evolution. Euchromatic sequence that flanks sites of LCR16a integration are frequently lineage-specific duplications. This process has mobilized duplication blocks (15-200 kb in size) to new genomic locations in each species. Breakpoint analysis of lineage-specific insertions suggests coordinated deletion of repeat-rich DNA at the target site, in some cases deleting genes in that species. Our data support a model of duplication where the probability that a segment of DNA becomes duplicated is determined by its proximity to core duplicons, such as LCR16a.

High-throughput genotyping of intermediate-size structural variation.

The contribution of large-scale and intermediate-size structural variation (ISV) to human genetic disease and disease susceptibility is only beginning to be understood. The development of high-throughput genotyping technologies is one of the most critical aspects for future studies of linkage disequilibrium (LD) and disease association. Using a simple PCR-based method designed to assay the junctions of the breakpoints, we genotyped seven simple insertion and deletion polymorphisms ranging in size from 6.3 to 24.7 kb among 90 CEPH individuals. We then extended this analysis to a larger collection of samples (n=460) by application of an oligonucleotide extension-ligation genotyping assay. The analysis showed a high level of concordance ( approximately 99%) when compared with PCR/sequence-validated genotypes. Using the available HapMap data, we observed significant LD (r2=0.74-0.95) between each ISV and flanking single nucleotide polymorphisms, but this observation is likely to hold only for similar simple insertion/deletion events. The approach we describe may be used to characterize a large number of individuals in a cost-effective manner once the sequence organization of ISVs is known.

A genome-wide survey of structural variation between human and chimpanzee.

Structural changes (deletions, insertions, and inversions) between human and chimpanzee genomes have likely had a significant impact on lineage-specific evolution because of their potential for dramatic and irreversible mutation. The low-quality nature of the current chimpanzee genome assembly precludes the reliable identification of many of these differences. To circumvent this, we applied a method to optimally map chimpanzee fosmid paired-end sequences against the human genome to systematically identify sites of structural variation > or = 12 kb between the two species. Our analysis yielded a total of 651 putative sites of chimpanzee deletion (n = 293), insertions (n = 184), and rearrangements consistent with local inversions between the two genomes (n = 174). We validated a subset (19/23) of insertion and deletions using PCR and Southern blot assays, confirming the accuracy of our method. The events are distributed throughout the genome on all chromosomes but are highly correlated with sites of segmental duplication in human and chimpanzee. These structural variants encompass at least 24 Mb of DNA and overlap with > 245 genes. Seventeen of these genes contain exons missing in the chimpanzee genomic sequence and also show a significant reduction in gene expression in chimpanzee. Compared with the pioneering work of Yunis, Prakash, Dutrillaux, and Lejeune, this analysis expands the number of potential rearrangements between chimpanzees and humans 50-fold. Furthermore, this work prioritizes regions for further finishing in the chimpanzee genome and provides a resource for interrogating functional differences between humans and chimpanzees.

A functional variant of IRS1 is associated with type 1 diabetes in families from the US and UK.

A collection of 767 multiplex type 1 diabetes families from the US and UK were tested for linkage to the IRS1 gene and for allelic association with a specific variant of IRS1, G972R. Pedigree disequilibrium testing revealed preferential transmission of the 972R allele to affected offspring in these families (P = 0.02). Linkage analyses conditioning on status at IRS1 position 972 suggest the possibility of interaction with an unidentified locus on chromosome 8.

Mutations and molecular variants of the NBS1 gene in non-Hodgkin lymphoma.

Non-Hodgkin lymphomas (NHLs) are characterized by chromosomal translocations that juxtapose loci encoding lymphoid antigen receptors with cellular proto-oncogenes. These translocations are thought to arise from inaccurate processing of DNA breaks created during physiologic recombination of the antigen receptor genes in lymphocytes. The inherited disorders ataxia-telangiectasia and Nijmegen breakage syndrome are caused by mutations in the ATM and NBS1 genes, respectively, and are characterized by generalized genomic instability and a high incidence of lymphoid cancers. Lymphoid cells from patients with either disorder frequently have chromosomal translocations involving T-cell-receptor or immunoglobulin loci. To investigate the potential role of the NBS1 gene in the pathogenesis of NHL, we screened tumor DNA samples from 91 sporadic cases of NHL and genomic DNA from 154 control individuals for mutations in all 16 exons of the NBS1 gene and in flanking intronic sequences. One NHL case with a truncating mutation in NBS1 and a second NHL case with a putative missense mutation were detected. Neither mutation was observed among controls. Three additional putative missense mutations were observed only in the normal control samples. A panel of six common polymorphisms spanning the NBS1 gene was genotyped and provided no evidence for loss of heterozygosity in the NHL cases with mutations or in the NHL population overall. These results suggest that mutations in NBS1 do not play a major role in the development of NHL in the United States.