Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.     

Distribution and functional impact of DNA copy number variation in the rat

The abundance and dynamics of copy number variants (CNVs) in mammalian genomes poses new challenges in the identification of their impact on natural and disease phenotypes. We used computational and experimental methods to catalog CNVs in rat and found that they share important functional characteristics with those in human. In addition, 113 one-to-one orthologous genes overlap CNVs in both human and rat, 80 of which are implicated in human disease. CNVs are nonrandomly distributed throughout the genome. Chromosome 18 is a cold spot for CNVs as well as evolutionary rearrangements and segmental duplications, suggesting stringent selective mechanisms underlying CNV genesis or maintenance. By exploiting gene expression data available for rat recombinant inbred lines, we established the functional relationship of CNVs underlying 22 expression quantitative trait loci. These characteristics make the rat an excellent model for studying phenotypic effects of structural variation in relation to human complex traits and disease.     

Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer

Epimutations in the germline, such as methylation of the MLH1 gene, may contribute to hereditary cancer syndrome in human, but their transmission to offspring has never been documented. Here we report a family with inheritance, in three successive generations, of germline allele-specific and mosaic hypermethylation of the MSH2 gene, without evidence of DNA mismatch repair gene mutation. Three siblings carrying the germline methylation developed early-onset colorectal or endometrial cancers, all with microsatellite instability and MSH2 protein loss. Clonal bisulfite sequencing and pyrosequencing showed different methylation levels in different somatic tissues, with the highest level recorded in rectal mucosa and colon cancer tissue, and the lowest in blood leukocytes. This mosaic state of germline methylation with different tissue distribution could act as the first hit and provide a mechanism for genetic disease inheritance that may deviate from the mendelian pattern and be overlooked in conventional leukocyte-based genetic diagnosis strategy.     

Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA

Genetic integrity is crucial to normal cell function, and mutations in genes required for DNA replication and repair underlie various forms of genetic instability and disease, including cancer. One structural feature of intact genomes is runs of homopolymeric dC/dG. Here we describe an unusual mutator phenotype in Caenorhabditis elegans characterized by deletions that start around the 3' end of polyguanine tracts and terminate at variable positions 5' from such tracts. We observed deletions throughout genomic DNA in about half of polyguanine tracts examined, especially those containing 22 or more consecutive guanine nucleotides. The mutator phenotype results from disruption of the predicted gene F33H2.1, which encodes a protein with characteristics of a DEAH helicase and which we have named dog-1 (for deletions of guanine-rich DNA). Nematodes mutated in dog-1 showed germline as well as somatic deletions in genes containing polyguanine tracts, such as vab-1. We propose that DOG-1 is required to resolve the secondary structures of guanine-rich DNA that occasionally form during lagging-strand DNA synthesis.     

Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution.

Human endogenous retroviruses (HERVs), which are remnants of past retroviral infections of the germline cells of our ancestors, make up as much as 8% of the human genome and may even outnumber genes. Most HERVs seem to have entered the genome between 10 and 50 million years ago, and they comprise over 200 distinct groups and subgroups. Although repeated sequence elements such as HERVs have the potential to lead to chromosomal rearrangement through homologous recombination between distant loci, evidence for the generality of this process is lacking. To gain insight into the expansion of these elements in the genome during the course of primate evolution, we have identified 23 new members of the HERV-K (HML-2) group, which is thought to contain the most recently active members. Here we show, by phylogenetic and sequence analysis, that at least 16% of these elements have undergone apparent rearrangements that may have resulted in large-scale deletions, duplications and chromosome reshuffling during the evolution of the human genome.     

Karyotypic abnormalities create discordance of germline genotype and cancer cell phenotypes

The nature of mendelian inheritance assumes that all tissues in which a phenotype of interest is expressed have a uniform diploid karyotype, which is often not the case in cancer cells. Owing to nonrandom gains of chromosomes, trisomies are present in many cases of leukemia and other malignances. We used polymorphisms in the genes encoding thiopurine S-methyltransferase (TPMT), gamma-glutamyl hydrolase (GGH) and the reduced folate carrier (SLC19A1) to assess the nature of chromosomal acquisition and its influence on genotype-phenotype concordance in cancer cells. TPMT and GGH activities in somatic cells were concordant with germline genotypes, whereas activities in leukemia cells were determined by chromosomal number and whether the acquired chromosomes contained a wild-type or variant allele. Leukemia cells that had acquired an additional chromosome containing a wild-type TPMT or GGH allele had significantly lower accumulation of thioguanine nucleotides or methotrexate polyglutamates, respectively. Among these genes, there was a comparable number of acquired chromosomes with wild-type and variant alleles. Therefore, chromosomal gain can alter the concordance of germline genotype and cancer cell phenotypes, indicating that allele-specific quantitative genotyping may be required to define cancer pharmacogenomics unequivocally.     

Evidence of en bloc duplication in vertebrate genomes

It has been 30 years since it was first proposed that the vertebrate genome evolved through several rounds of genome-wide duplications (polyploidizations). Despite rapid advances in genetics, including sequencing of the complete genomes of several divergent species, this hypothesis has not been tested rigorously and is still a matter of debate. If polyploidizations occurred during chordate evolution, there should be a network of paralogous regions in the present-day jawed vertebrate (Gnathostomata) genomes. Here we present an investigation of the major histocompatibility complex (MHC) paralogous regions, which we accomplished by characterizing the corresponding region in amphioxus by identifying nine anchor genes and sequencing both the anchor genes and the regions that flank them (a total of 400 kb). Phylogenetic analysis of 31 genes (including the anchor genes) in these regions shows that duplications occurred after the divergence of cephalochordates and vertebrates but before the Gnathostomata radiation. The distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events. Our analysis represents the first step towards demonstrating that the human ancestral genome has undergone polyploidization. Moreover, reconstruction of the pre-duplicated region indicates that one of the duplicated regions retains the ancestral organization.     

Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR

Somatic activating mutations in EGFR identify a subset of non-small cell lung cancer that respond to tyrosine kinase inhibitors. Acquisition of drug resistance is linked to a specific secondary somatic mutation, EGFR T790M. Here we describe a family with multiple cases of non-small cell lung cancer associated with germline transmission of this mutation. Four of six tumors analyzed showed a secondary somatic activating EGFR mutation, arising in cis with the germline EGFR mutation T790M. These observations implicate altered EGFR signaling in genetic susceptibility to lung cancer.     

Germline BAP1 mutations predispose to malignant mesothelioma

Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma, and because mesothelioma clustering is observed in some families, we searched for genetic predisposing factors. We discovered germline mutations in the gene encoding BRCA1 associated protein-1 (BAP1) in two families with a high incidence of mesothelioma, and we observed somatic alterations affecting BAP1 in familial mesotheliomas, indicating biallelic inactivation. In addition to mesothelioma, some BAP1 mutation carriers developed uveal melanoma. We also found germline BAP1 mutations in 2 of 26 sporadic mesotheliomas; both individuals with mutant BAP1 were previously diagnosed with uveal melanoma. We also observed somatic truncating BAP1 mutations and aberrant BAP1 expression in sporadic mesotheliomas without germline mutations. These results identify a BAP1-related cancer syndrome that is characterized by mesothelioma and uveal melanoma. We hypothesize that other cancers may also be involved and that mesothelioma predominates upon asbestos exposure. These findings will help to identify individuals at high risk of mesothelioma who could be targeted for early intervention.     

Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome

We identified complex genomic rearrangements consisting of intermixed duplications and triplications of genomic segments at the MECP2 and PLP1 loci. These complex rearrangements were characterized by a triplicated segment embedded within a duplication in 11 unrelated subjects. Notably, only two breakpoint junctions were generated during each rearrangement formation. All the complex rearrangement products share a common genomic organization, duplication-inverted triplication-duplication (DUP-TRP/INV-DUP), in which the triplicated segment is inverted and located between directly oriented duplicated genomic segments. We provide evidence that the DUP-TRP/INV-DUP structures are mediated by inverted repeats that can be separated by >300 kb, a genomic architecture that apparently leads to susceptibility to such complex rearrangements. A similar inverted repeat-mediated mechanism may underlie structural variation in many other regions of the human genome. We propose a mechanism that involves both homology-driven events, via inverted repeats, and microhomologous or nonhomologous events.     

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.     

Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS; also known as poikiloderma congenitale) is a rare, autosomal recessive genetic disorder characterized by abnormalities in skin and skeleton, juvenile cataracts, premature ageing and a predisposition to neoplasia. Cytogenetic studies indicate that cells from affected patients show genomic instability often associated with chromosomal rearrangements causing an acquired somatic mosaicism. The gene(s) responsible for RTS remains unknown. The genes responsible for Werner and Bloom syndromes (WRN and BLM, respectively) have been identified as homologues of Escherichia coli RecQ, which encodes a DNA helicase that unwinds double-stranded DNA into single-stranded DNAs. Other eukaryotic homologues thus far identified are human RECQL, Saccharomyces cerevisiae SGS1 and Schizosaccharomyces pombe rqh1. We recently cloned two new human helicase genes, RECQL4 at 8q24.3 and RECQL5 at 17q25, which encode members of the RecQ helicase family. Here, we report that three RTS patients carried two types of compound heterozygous mutations in RECQL4. The fact that the mutated alleles were inherited from the parents in one affected family and were not found in ethnically matched controls suggests that mutation of RECQL4 at human chromosome 8q24.3 is responsible for at least some cases of RTS.     

Evidence for co-evolution of gene order and recombination rate

There is increasing evidence in eukaryotic genomes that gene order is not random, even allowing for tandem duplication. Notably, in numerous genomes, genes of similar expression tend to be clustered. Are there other reasons for clustering of functionally similar genes? If genes are linked to enable genetic, rather than physical clustering, then we also expect that clusters of certain genes might be associated with blocks of reduced recombination rates. Here we show that, in yeast, essential genes are highly clustered and this clustering is independent of clustering of co-expressed genes and of tandem duplications. Adjacent pairs of essential genes are preferentially conserved through evolution. Notably, we also find that clusters of essential genes are in regions of low recombination and that larger clusters have lower recombination rates. These results suggest that selection acts to modify both the fine-scale intragenomic variation in the recombination rate and the distribution of genes and provide evidence for co-evolution of gene order and recombination rate.