The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major ...differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.
We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host ...chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications.
SHANK3 is located on chromosome 22q13.3 and encodes a scaffold protein that is found in excitatory synapses opposite the pre-synaptic active zone. SHANK3 is a binding partner of neuroligins, some of ...whose genes contain mutations in a small subset of individuals with autism. In individuals with autism spectrum disorders (ASDs), several studies have found SHANK3 to be disrupted by deletions ranging from hundreds of kilobases to megabases, suggesting that 1% of individuals with ASDs may have these chromosomal aberrations. To further analyse the involvement of SHANK3 in ASD, we screened the International Molecular Genetic Study of Autism Consortium (IMGSAC) multiplex family sample, 330 families, for SNP association and copy number variants (CNVs) in SHANK3. A collection of 76 IMGSAC Italian probands from singleton families was also examined by multiplex ligation-dependent probe amplification for CNVs. No CNVs or SNP associations were found within the sample set, although sequencing of the gene was not performed. Our data suggest that SHANK3 deletions may be limited to lower functioning individuals with autism.
Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for ...the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive ‘passaging’. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.
Physical mapping of transgenic insertions by Fluorescence in situ Hybridization (FISH) is a reliable and cost-effective technique. Chromosomal assignment is commonly achieved either by concurrent ...G-banding or by a multi-color FISH approach consisting of iteratively co-hybridizing the transgenic sequence of interest with one or more chromosome-specific probes at a time, until the location of the transgenic insertion is identified.
Here we report a technical development for fast chromosomal assignment of transgenic insertions at the single cell level in mouse and rat models. This comprises a simplified 'single denaturation mixed hybridization' procedure that combines multi-color karyotyping by Multiplex FISH (M-FISH), for simultaneous and unambiguous identification of all chromosomes at once, and the use of a Quantum Dot (QD) conjugate for the transgene detection.
Although the exploitation of the unique optical properties of QD nanocrystals, such as photo-stability and brightness, to improve FISH performance generally has been previously investigated, to our knowledge this is the first report of a purpose-designed molecular cytogenetic protocol in which the combined use of QDs and standard organic fluorophores is specifically tailored to assist gene transfer technology.
Human artificial chromosomes (HAC) are small functional extrachromosomal elements, which segregate correctly during each cell division. In human cells, they are mitotically stable, however when the ...HAC are transferred to murine cells they show an increased and variable rate of loss. In some cell lines the HAC are lost over a short period of time, while in others the HAC become stable without acquiring murine DNA.
In this study, we linked the loss rate to the position of the HAC in the murine cell nucleus with respect to the chromocenters. HAC that associated preferentially with the chromocenter displayed a lower loss rate compared to the HAC that are less frequently associated. The chromocenter acts as a hub for the deposition of heterochromatic markers, controlling centromeric and pericentromeric DNA replication timing and chromosome segregation. The HAC which localized more frequently outside the chromocenters bound variable amounts of histone H3 tri-methylated at lysine 9, and the high level of intraclonal variability was associated with an increase in HAC segregation errors and delayed DNA replication timing.
This is a novel result indicating that HAC segregation is closely linked to the position in the murine nucleus and gives important insight for HAC gene expression studies in murine cells and establishing murine models of human genetic disease.
PURPOSE. To characterize anatomically and functionally the retinal degeneration observed in a transgenic mouse line (OPN1LW-EGFP) expressing enhanced green fluorescent protein (EGFP) in a ...subpopulation of cone photoreceptors, and to map the location of the transgenic insertion. METHODS. An anatomic comparison of cone survival was carried out between wild type (WT) and transgenic mice at three postnatal time points (P80, P140, and P245). Retinal function was assessed at P245 by ERG and included an ultraviolet flicker stimulus to isolate S-cone function. Chromosomal mapping by FISH and high-resolution mapping on DNA fibers (Fiber-FISH) were performed to identify the location of the transgenic insertion. RESULTS. GFP expression was largely absent in S-cones. Cone numbers were significantly reduced in OPN1LW-EGFP mice at all time points compared to WT, with cone loss independent of GFP expression. Anatomic loss correlated with a functional deficit in dark- and light-adapted ERG responses, including a reduction in UV-flicker response, confirming the degeneration of S-cones. The phenotype of heterozygote mice was slightly less severe than in homozygotes, consistent with a dominantly inherited cone dystrophy. The transgenic insertion mapped to a specific region on chromosome 10 orthologous with loci for progressive bifocal chorioretinal atrophy and North Carolina macular dystrophy on human chromosome 6. CONCLUSIONS. Cone loss is global in OPN1LW-EGFP mice and is independent of GFP expression. The mechanism underlying the degeneration remains elusive; however, disruption of loci associated with dominantly inherited retinal degenerations in humans makes this mouse of great interest.
We report on a young female, who presents with a severe speech and language disorder and a balanced de novo complex chromosomal rearrangement, likely to have resulted from a chromosome 7 ...pericentromeric inversion, followed by a chromosome 7 and 11 translocation.
Using molecular cytogenetics, we mapped the four breakpoints to 7p21.1-15.3 (chromosome position: 20,954,043-21,001,537, hg19), 7q31 (chromosome position: 114,528,369-114,556,605, hg19), 7q21.3 (chromosome position: 93,884,065-93,933,453, hg19) and 11p12 (chromosome position: 38,601,145-38,621,572, hg19). These regions contain only non-coding transcripts (ENSG00000232790 on 7p21.1 and TCONS_00013886, TCONS_00013887, TCONS_00014353, TCONS_00013888 on 7q21) indicating that no coding sequences are directly disrupted. The breakpoint on 7q31 mapped 200 kb downstream of FOXP2, a well-known language gene. No splice site or non-synonymous coding variants were found in the FOXP2 coding sequence. We were unable to detect any changes in the expression level of FOXP2 in fibroblast cells derived from the proband, although this may be the result of the low expression level of FOXP2 in these cells.
We conclude that the phenotype observed in this patient either arises from a subtle change in FOXP2 regulation due to the disruption of a downstream element controlling its expression, or from the direct disruption of non-coding RNAs.
Autism spectrum disorder is a genetically complex and clinically heterogeneous neurodevelopmental disorder. A recent study by the Autism Genome Project (AGP) used 1M single-nucleotide polymorphism ...arrays to show that rare genic copy number variants (CNVs), possibly acting in tandem, play a significant role in the genetic aetiology of this condition. In this study, we describe the phenotypic and genomic characterisation of a multiplex autism family from the AGP study that was found to harbour a duplication of exons 31-44 of the Duchenne/Becker muscular dystrophy gene DMD and also a rare deletion involving exons 1-9 of TRPM3. Further characterisation of these extremely rare CNVs was carried out using quantitative PCR, fluorescent in situ hybridisation, long-range PCR amplification and sequencing of junction fragments. The maternal chrX:32,097,213-32,321,945 tandem duplication and paternal chr9:72,480,413-73,064,196 deletion (NCBI build 36 coordinates) were transmitted to both affected boys, potentially signifying a multi-hit mechanism. The DMD reading frame rule predicts a Becker phenotype, characterised by later onset and milder symptoms. When last evaluated, neither child had developed signs of muscular dystrophy. These data are consistent with a degree of comorbidity between autism and muscular dystrophy and suggest that genomic background as well as the position of the mutation within the DMD gene may impact on the neurological correlates of Duchenne/Becker muscular dystrophy. Finally, communicating unexpected findings such as these back to families raises a number of ethical questions, which are discussed.
Digenic causes of human disease are rarely reported. Insulin via its receptor, which is encoded by INSR, plays a key role in both metabolic and growth signaling pathways. Heterozygous INSR mutations ...are the most common cause of monogenic insulin resistance. However, growth retardation is only reported with homozygous or compound heterozygous mutations. We describe a novel translocation t(7,19)(p15.2;p13.2) cosegregating with insulin resistance and pre- and postnatal growth deficiency. Chromosome translocations present a unique opportunity to identify modifying loci; therefore, our objective was to determine the mutational mechanism resulting in this complex phenotype.
Breakpoint mapping was performed by fluorescence in situ hybridization (FISH) on patient chromosomes. Sequencing and gene expression studies of disrupted and adjacent genes were performed on patient-derived tissues. RESULTS Affected individuals had increased insulin, C-peptide, insulin-to-C-peptide ratio, and adiponectin levels consistent with an insulin receptoropathy. FISH mapping established that the translocation breakpoints disrupt INSR on chromosome 19p15.2 and CHN2 on chromosome 7p13.2. Sequencing demonstrated INSR haploinsufficiency accounting for elevated insulin levels and dysglycemia. CHN2 encoding beta-2 chimerin was shown to be expressed in insulin-sensitive tissues, and its disruption was shown to result in decreased gene expression in patient-derived adipose tissue.
We present a likely digenic cause of insulin resistance and growth deficiency resulting from the combined heterozygous disruption of INSR and CHN2, implicating CHN2 for the first time as a key element of proximal insulin signaling in vivo.
PDF
