Nonreciprocal chromosomal translocations in renal cancer involve multiple DSBs and NHEJ associated with breakpoint inversion but not necessarily with transcription.

Chromosomal translocations and other abnormalities are central to the initiation of cancer in all cell types. Understanding the mechanism is therefore important to evaluate the evolution of cancer from the cancer initiating events to overt disease. Recent work has concentrated on model systems to develop an understanding of the molecular mechanisms of translocations but naturally occurring events are more ideal case studies since biological selection is absent from model systems. In solid tumours, nonreciprocal translocations are most commonly found, and accordingly we have investigated the recurrent nonreciprocal t(3;5) chromosomal translocations in renal carcinoma to better understand the mechanism of these naturally occurring translocations in cancer. Unexpectedly, the junctions of these translocations can be associated with site-specific, intrachromosomal inversion involving at least two double strand breaks (DSB) in cis and rejoining by nonhomologous end joining or micro-homology end joining. However, these translocations are not necessarily associated with transcribed regions questioning accessibility per se in controlling these events. In addition, intrachromosomal deletions also occur. We conclude these naturally occurring, nonreciprocal t(3;5) chromosomal translocations occur after complex and multiple unresolved intrachromosomal DSBs leading to aberrant joining with concurrent interstitial inversion and that clonal selection of cells is the critical element in cancer development emerging from a plethora of DSBs that may not always be pathogenic.

Engineering de novo reciprocal chromosomal translocations associated with Mll to replicate primary events of human cancer.

The etiology of human tumors often involves chromosomal translocations. Models that emulate translocations are essential to understanding the determinants of frank malignancy, those dictating the restriction of translocations to specific lineages, and as a basis for development of rational therapeutic methods. We demonstrate that developmentally regulated Cre-loxP-mediated interchromosomal recombination between the Mll gene, whose human counterpart is involved in a spectrum of leukemias, and the Enl gene creates reciprocal chromosomal translocations that cause myeloid tumors. There is a rapid onset and high penetrance of leukemogenesis in these translocator mice, and high proportions of cells carrying chromosomal translocations can be found in bone marrow as early as 12 days after birth. This de novo strategy is a direct recapitulation of naturally occurring human cancer-associated translocations.

Homozygous disruption of PDZD7 by reciprocal translocation in a consanguineous family: a new member of the Usher syndrome protein interactome causing congenital hearing impairment.

A homozygous reciprocal translocation, 46,XY,t(10;11),t(10;11), was detected in a boy with non-syndromic congenital sensorineural hearing impairment. Both parents and their four other children were heterozygous translocation carriers, 46,XX,t(10;11) and 46,XY,t(10;11), respectively. Fluorescence in situ hybridization of region-specific clones to patient chromosomes was used to localize the breakpoints within bacterial artificial chromosome (BAC) RP11-108L7 on chromosome 10q24.3 and within BAC CTD-2527F12 on chromosome 11q23.3. Junction fragments were cloned by vector ligation and sequenced. The chromosome 10 breakpoint was identified within the PDZ domain containing 7 (PDZD7) gene, disrupting the open reading frame of transcript PDZD7-C (without PDZ domain) and the 5'-untranslated region of transcript PDZD7-D (with one PDZ and two prolin-rich domains). The chromosome 11 breakpoint was localized in an intergenic segment. Reverse transcriptase-polymerase chain reaction analysis revealed PDZD7 expression in the human inner ear. A murine Pdzd7 transcript that is most similar in structure to human PDZD7-D is known to be expressed in the adult inner ear and retina. PDZD7 shares sequence homology with the PDZ domain-containing genes, USH1C (harmonin) and DFNB31 (whirlin). Allelic mutations in harmonin and whirlin can cause both Usher syndrome (USH1C and USH2D, respectively) and congenital hearing impairment (DFNB18 and DFNB31, respectively). Protein-protein interaction assays revealed the integration of PDZD7 in the protein network related to the human Usher syndrome. Collectively, our data provide strong evidence that PDZD7 is a new autosomal-recessive deafness-causing gene and also a prime candidate gene for Usher syndrome.

Differences in DNA methylation patterns and expression of the CCRK gene in human and nonhuman primate cortices.

Changes in DNA methylation patterns during embryo development and differentiation processes are linked to the transcriptional plasticity of our genome. However, little is known about the evolutionary conservation of DNA methylation patterns and the evolutionary impact of epigenetic differences between closely related species. Here we compared the methylation patterns of CpG islands (CGIs) in the promoter regions of seven genes in humans and chimpanzees. We identified a block of CpGs in the cell cycle-related kinase (CCRK) gene that is more methylated in the adult human cortex than in the chimpanzee cortex and, in addition, it exhibits considerable intraspecific variation both in humans and chimpanzees. The species-specifically methylated region (SMR) lies between the almost completely methylated 5' region and the completely demethylated 3' region of the presumed CCRK CGI promoter. It is part of an Alu-Sg1 repeat that has been integrated into the promoter region in a common ancestor of humans and New World monkeys. This SMR is relatively hypomethylated in the rhesus monkey cortex and more or less completely methylated in the baboon cortex, indicating extraordinary methylation dynamics during primate evolution. The mRNA expression level of CCRK has also changed during the course of primate evolution. CCRK is expressed at much higher levels in human and baboon cortices, which display an average SMR methylation of 70% and 100%, respectively, than in chimpanzee and rhesus macaque cortices with an average SMR methylation of 35% and 40%, respectively. The observed evolutionary dynamics suggests a possibility that CCRK has been important for evolution of the primate brain.

The Ews-ERG fusion protein can initiate neoplasia from lineage-committed haematopoietic cells.

The EWS-ERG fusion protein is found in human sarcomas with the chromosomal translocation t(21;22)(q22;q12), where the translocation is considered to be an initiating event in sarcoma formation within uncommitted mesenchymal cells, probably long-lived progenitors capable of self renewal. The fusion protein may not therefore have an oncogenic capability beyond these progenitors. To assess whether EWS-ERG can be a tumour initiator in cells other than mesenchymal cells, we have analysed Ews-ERG fusion protein function in a cellular environment not typical of that found in human cancers, namely, committed lymphoid cells. We have used Ews-ERG invertor mice having an inverted ERG cDNA cassette flanked by loxP sites knocked in the Ews intron 8, crossed with mice expressing Cre recombinase under the control of the Rag1 gene to give conditional, lymphoid-specific expression of the fusion protein. Clonal T cell neoplasias arose in these mice. This conditional Ews gene fusion model of tumourigenesis shows that Ews-ERG can cause haematopoietic tumours and the precursor cells are committed cells. Thus, Ews-ERG can function in cells that do not have to be pluripotent progenitors or mesenchymal cells.

De novo t(12;17)(p13.3;q21.3) translocation with a breakpoint near the 5' end of the HOXB gene cluster in a patient with developmental delay and skeletal malformations.

A boy with severe mental retardation, funnel chest, bell-shaped thorax, and hexadactyly of both feet was found to have a balanced de novo t(12;17)(p13.3;q21.3) translocation. FISH with BAC clones and long-range PCR products assessed in the human genome sequence localized the breakpoint on chromosome 17q21.3 to a 21-kb segment that lies <30 kb upstream of the HOXB gene cluster and immediately adjacent to the 3' end of the TTLL6 gene. The breakpoint on chromosome 12 occurred within telomeric hexamer repeats and, therefore, is not likely to affect gene function directly. We propose that juxtaposition of the HOXB cluster to a repetitive DNA domain and/or separation from required cis-regulatory elements gave rise to a position effect.

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes.

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.

Interrogation of genomes by molecular copy-number counting (MCC).

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations.

Spontaneous B cell hyperactivity in autoimmune-prone MRL mice.

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.

Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis.

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.

The invertor knock-in conditional chromosomal translocation mimic.

Knock-in models of tumor-specific chromosomal translocations can generate lethal mutations. To circumvent this, a new conditional gene fusion model has been developed (invertor mice) and exemplified with the Ews-ERG fusion oncogene. An ERG segment, flanked by loxP sites, was knocked in to an intron of the Ews gene but in an inverted transcription orientation and lineage-specific Ews-ERG fusion created by Cre-mediated inversion. This invertor method is a completely conditional approach, applicable to any gene fusion, to emulate effects of translocations found in human cancers.

Mouse models for the genetic dissection of atopy.

Atopic disorders are complex traits with a significant contribution of heritability. Several mouse models for atopic phenotypes such as allergen-induced and intrinsic asthma, atopic dermatitis and immediate cutaneous hypersensitivity have been used to systematically dissect the genetics of these diseases. Many of the chromosomal regions identified in genome-wide screens colocalize with linkage regions of autoimmune phenotypes suggesting common regulatory genes in combination (or addition) to disease-specific genes. Moreover, many of these regions correspond to regions found in the human system, thus strengthening the likelihood of a gene contributing to the phenotype. The role of individual candidate genes has been vigorously pursued in genetically modified mice by destruction or overexpression of the candidate gene. The complex interactions of multiple contributing genes may further be elucidated in randomly mutated mouse strains. Thus mouse models can contribute largely to the elucidation of disease genes.