Bee Ling Ng

The complex nature of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes

Objective: To describe the systematic analysis of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes, characterise the structural chromosome rearrangements, map the translocation breakpoints, and report detectable genomic imbalances. Methods: DNA microarrays were used with a resolution of 1 Mb for the detailed genome-wide analysis of the patients. Array CGH was used to screen for genomic imbalance and array painting to map chromosome breakpoints rapidly. These two methods facilitate rapid analysis of translocation breakpoints and screening for cryptic chromosome imbalance. Breakpoints of rearrangements were further refined (to the level of spanning clones) using fluorescence in situ hybridisation where appropriate. Results: Unexpected additional complexity or genome imbalance was found in six of 10 patients studied. The patients could be grouped according to the general nature of the karyotype rearrangement as follows: (A) three cases with complex multiple rearrangements including deletions, inversions, and insertions at or near one or both breakpoints; (B) three cases in which, while the translocations appeared to be balanced, microarray analysis identified previously unrecognised imbalance on chromosomes unrelated to the translocation; (C) four cases in which the translocation breakpoints appeared simple and balanced at the resolution used. Conclusions: This high level of unexpected rearrangement complexity, if generally confirmed in the study of further patients, will have an impact on current diagnostic investigations of this type and provides an argument for the more widespread adoption of microarray analysis or other high resolution genome-wide screens for chromosome imbalance and rearrangement.

Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion

One Two T T cells develop in the thymus, where they proceed through several developmental stages, losing alternative lineage potential as they progress. The molecular regulation of this developmental process, however, is not fully understood (see the Perspective by Di Santo). P. Li et al. (p. 85, published online 10 June), L. Li et al. (p. 89), and Ikawa et al. (p. 93) now identify expression of the zinc finger transcription factor Bcl11b as the earliest checkpoint in T cell development in mice. Genetic deletion of Bcl11b in developing T cells inhibited commitment to the T cell lineage. Under conditions that should have stimulated T lineage differentiation, Bcl11b-deficient T cell progenitors failed to up-regulate genes associated with lineage-committed T cells and maintained stem cell– and progenitor cell–associated gene expression. In both developing and committed T cells, loss of Bcl11b resulted in the generation of cells that resembled natural killer (NK) cells in both phenotype and function. These NK-like cells could be expanded easily in vitro and possessed antitumor cytotoxicity, but they did not exhibit cytotoxicity against normal cells and were not tumorigenic. Because T cells are much easier to obtain from human patients than NK cells, deletion of Bcl11b in T cells may thus provide a source of easy-to-grow NK cells for cell-based antitumor therapies. A transcription factor is essential for maintenance of T cell identity. T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell–associated gene expression. These induced T-to–natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.

Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer

Summary The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations. PaperClip

Human Y Chromosome Base-Substitution Mutation Rate Measured by Direct Sequencing in a Deep-Rooting Pedigree

Summary Understanding the key process of human mutation is important for many aspects of medical genetics and human evolution. In the past, estimates of mutation rates have generally been inferred from phenotypic observations or comparisons of homologous sequences among closely related species [1–3]. Here, we apply new sequencing technology to measure directly one mutation rate, that of base substitutions on the human Y chromosome. The Y chromosomes of two individuals separated by 13 generations were flow sorted and sequenced by Illumina (Solexa) paired-end sequencing to an average depth of 11× or 20×, respectively [4]. Candidate mutations were further examined by capillary sequencing in cell-line and blood DNA from the donors and additional family members. Twelve mutations were confirmed in ∼10.15 Mb; eight of these had occurred in vitro and four in vivo. The latter could be placed in different positions on the pedigree and led to a mutation-rate measurement of 3.0 × 10−8 mutations/nucleotide/generation (95% CI: 8.9 × 10−9–7.0 × 10−8), consistent with estimates of 2.3 × 10−8–6.3 × 10−8 mutations/nucleotide/generation for the same Y-chromosomal region from published human-chimpanzee comparisons [5] depending on the generation and split times assumed.

Prenatal detection of unbalanced chromosomal rearrangements by array CGH

Background: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. Methods: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. Results: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. Conclusions: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

Fibroblast Growth Factor 2-Mediated Translational Control of IAPs Blocks Mitochondrial Release of Smac/DIABLO and Apoptosis in Small Cell Lung Cancer Cells

ABSTRACT The mitochondrial release of cytochrome c and Smac/DIABLO has been implicated in the activation of apoptosis in response to cell stress. Smac promotes cytochrome c-induced activation of caspases by sequestering the inhibitor of apoptosis protein (IAP) family of potent caspase suppressors. Differential release from mitochondria of cytochrome c and Smac can occur, but the underlying mechanism and physiological significance of this are unclear. Here we show that the mechanism by which fibroblast growth factor 2 (FGF-2) protects small cell lung cancer (SCLC) cells from etoposide-induced cell death involves inhibition of Smac release but not of cytochrome c release. This process is MEK dependent and correlates with an increased expression of XIAP and cellular IAP-1, mediated principally through translational regulation. Exogenous expression of XIAP is sufficient to inhibit caspase 9 activation, Smac release, and cell death induced by etoposide. Prevention of the FGF-2-promoted increase in levels of functional IAPs by RNA interference or the cell-permeant Smac amino-terminal peptide blocked FGF-2-induced protection. FGF-2 can thus protect SCLC cells from chemotherapeutic drugs by modulating IAP levels via posttranscriptional regulation, providing a mechanism for postmitochondrial survival signaling by the MEK/mitogen-activated protein kinase pathway.

The RASSF1A Isoform of RASSF1 Promotes Microtubule Stability and Suppresses Tumorigenesis

ABSTRACT The RASSF1A isoform of RASSF1 is frequently inactivated by epigenetic alterations in human cancers, but it remains unclear if and how it acts as a tumor suppressor. RASSF1A overexpression reduces in vitro colony formation and the tumorigenicity of cancer cell lines in vivo. Conversely, RASSF1A knockdown causes multiple mitotic defects that may promote genomic instability. Here, we have used a genetic approach to address the function of RASSF1A as a tumor suppressor in vivo by targeted deletion of Rassf1A in the mouse. Rassf1A null mice were viable and fertile and displayed no pathological abnormalities. Rassf1A null embryonic fibroblasts displayed an increased sensitivity to microtubule depolymerizing agents. No overtly altered cell cycle parameters or aberrations in centrosome number were detected in Rassf1A null fibroblasts. Rassf1A null fibroblasts did not show increased sensitivity to microtubule poisons or DNA-damaging agents and showed no evidence of gross genomic instability, suggesting that cellular responses to genotoxins were unaffected. Rassf1A null mice showed an increased incidence of spontaneous tumorigenesis and decreased survival rate compared with wild-type mice. Irradiated Rassf1A null mice also showed increased tumor susceptibility, particularly to tumors associated with the gastrointestinal tract, compared with wild-type mice. Thus, our results demonstrate that Rassf1A acts as a tumor suppressor gene.

Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1–AHCY and RIF1–PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes)-An atypical species with low diploid number

The chicken is the most extensively studied species in birds and thus constitutes an ideal reference for comparative genomics in birds. Comparative cytogenetic studies indicate that the chicken has retained many chromosome characters of the ancestral avian karyotype. The homology between chicken macrochromosomes (1–9 and Z) and their counterparts in more than 40 avian species of 10 different orders has been established by chromosome painting. However, the avian homologues of chicken microchromosomes remain to be defined. Moreover, no reciprocal chromosome painting in birds has been performed due to the lack of chromosome-specific probes from other avian species. Here we have generated a set of chromosome-specific paints using flow cytometry that cover the whole genome of the stone curlew (Burhinus oedicnemus, Charadriiformes), a species with one of the lowest diploid number so far reported in birds, as well as paints from more microchromosomes of the chicken. A genome-wide comparative map between the chicken and the stone curlew has been constructed for the first time based on reciprocal chromosome painting. The results indicate that extensive chromosome fusions underlie the sharp decrease in the diploid number in the stone curlew. To a lesser extent, chromosome fissions and inversions occurred also during the evolution of the stone curlew. It is anticipated that this complete set of chromosome painting probes from the first Neoaves species will become an invaluable tool for avian comparative cytogenetics.