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K H Andy Choo

Publications and source records attributed to K H Andy Choo.

At least 19 recordsLinked to original sources
PubMed2006

Mouse telocentric sequences reveal a high rate of homogenization and possible role in Robertsonian translocation.

The telomere and centromere are two specialized structures of eukaryotic chromosomes that are essential for chromosome stability and segregation. These structures are usually characterized by large tracts of tandemly repeated DNA. In mouse, the two structures are often located in close proximity to form telocentric chromosomes. To date, no detailed sequence information is available across the mouse telocentric regions. The antagonistic mechanisms for the stable maintenance of the mouse telocentric karyotype and the occurrence of whole-arm Robertsonian translocations remain enigmatic. We have identified large-insert fosmid clones that span the telomere and centromere of several mouse chromosome ends. Sequence analysis shows that the distance between the telomeric T2AG3 and centromeric minor satellite repeats range from 1.8 to 11 kb. The telocentric regions of different mouse chromosomes comprise a contiguous linear order of T2AG3 repeats, a highly conserved truncated long interspersed nucleotide element 1 repeat, and varying amounts of a recently discovered telocentric tandem repeat that shares considerable identity with, and is inverted relative to, the centromeric minor satellite DNA. The telocentric domain as a whole exhibits the same polarity and a high sequence identity of >99% between nonhomologous chromosomes. This organization reflects a mechanism of frequent recombinational exchange between nonhomologous chromosomes that should promote the stable evolutionary maintenance of a telocentric karyotype. It also provides a possible mechanism for occasional inverted mispairing and recombination between the oppositely oriented TLC and minor satellite repeats to result in Robertsonian translocations.

Animals↗
PubMed2006

Permissive transcriptional activity at the centromere through pockets of DNA hypomethylation.

DNA methylation is a hallmark of transcriptional silencing, yet transcription has been reported at the centromere. To address this apparent paradox, we employed a fully sequence-defined ectopic human centromere (or neocentromere) to investigate the relationship between DNA methylation and transcription. We used sodium bisulfite PCR and sequencing to determine the methylation status of 2,041 CpG dinucleotides distributed across a 6.76-Mbp chromosomal region containing a neocentromere. These CpG dinucleotides were associated with conventional and nonconventional CpG islands. We found an overall hypermethylation of the neocentric DNA at nonconventional CpG islands that we designated as CpG islets and CpG orphans. The observed hypermethylation was consistent with the presence of a presumed transcriptionally silent chromatin state at the neocentromere. Within this neocentric chromatin, specific sites of active transcription and the centromeric chromatin boundary are defined by DNA hypomethylation. Our data demonstrate, for the first time to our knowledge, a correlation between DNA methylation and centromere formation in mammals, and that transcription and "chromatin-boundary activity" are permissible at the centromere through the selective hypomethylation of pockets of sequences without compromising the overall silent chromatin state and function of the centromere.

Animals↗
PubMed2005

Engineering chromosomes for delivery of therapeutic genes.

The ability to create fully functional human chromosome vectors represents a potentially exciting gene-delivery system for the correction of human genetic disorders with several advantages over viral delivery systems. However, for the full potential of chromosome-based gene-delivery vectors to be realized, several key obstacles must be overcome. Methods must be developed to insert therapeutic genes reliably and efficiently and to enable the stable transfer of the resulting chromosomal vectors to different therapeutic cell types. Research to achieve these outcomes continues to encounter major challenges; however recent developments have reiterated the potential of chromosome-based vectors for therapeutic gene delivery. Here we review the different strategies under development and discuss the advantages and problems associated with each.

Animals↗
PubMed2005

Kiss and break up--a safe passage to anaphase in mitosis and meiosis.

Eukaryotic chromosomes have many challenges to overcome between DNA replication and sister chromatid segregation. If these challenges are not met, cell death or unregulated cell division (cancer) may result. During prophase, chromosomes condense, the nuclear membrane breaks down and cohesins are removed from chromosome arms. In prometaphase, initial spindle attachments are made by sister kinetochores followed by correction of erroneous attachments, centromere oscillation between spindle poles and congression towards the cell's equator. In metaphase, all chromosomes attain stable bipolar spindle attachments and align at the metaphase plate, ready for the metaphase-anaphase transition when all ties between sister chromatids are broken. This review concentrates on recent developments that have revealed the intricacies of these processes. We now know more about how the mechanisms of cohesin removal differ between prophase and the metaphase-anaphase transition, the processes for detection and correction of improper spindle-kinetochore attachments and the concept that tension between sister kinetochores is the driving factor for satisfying the spindle checkpoint. We are also beginning to gain some understanding of the mechanisms behind the co-segregation of sister chromatids at the first meiotic division.

Anaphase↗
PubMed2005

High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs.

Mutation of the human genome ranges from single base-pair changes to whole-chromosome aneuploidy. Karyotyping, fluorescence in situ hybridization, and comparative genome hybridization are currently used to detect chromosome abnormalities of clinical significance. These methods, although powerful, suffer from limitations in speed, ease of use, and resolution, and they do not detect copy-neutral chromosomal aberrations--for example, uniparental disomy (UPD). We have developed a high-throughput approach for assessment of DNA copy-number changes, through use of high-density synthetic oligonucleotide arrays containing 116,204 single-nucleotide polymorphisms, spaced at an average distance of 23.6 kb across the genome. Using this approach, we analyzed samples that failed conventional karyotypic analysis, and we detected amplifications and deletions across a wide range of sizes (1.3-145.9 Mb), identified chromosomes containing anonymous chromatin, and used genotype data to determine the molecular origin of two cases of UPD. Furthermore, our data provided independent confirmation for a case that had been misinterpreted by karyotype analysis. The high resolution of our approach provides more-precise breakpoint mapping, which allows subtle phenotypic heterogeneity to be distinguished at a molecular level. The accurate genotype information provided on these arrays enables the identification of copy-neutral loss-of-heterozygosity events, and the minimal requirement of DNA (250 ng per array) allows rapid analysis of samples without the need for cell culture. This technology overcomes many limitations currently encountered in routine clinical diagnostic laboratories tasked with accurate and rapid diagnosis of chromosomal abnormalities.

Chromosome Aberrations↗
PubMed2005

Spef1, a conserved novel testis protein found in mouse sperm flagella.

We describe the cloning and characterisation of Spef1, a novel testis-specific gene. Spef1 has evolutionary orthologues in a wide range of species including mammals, other vertebrates, Drosophila, and protozoans with motile cilia or flagella. A second homologue of the gene, Spef2, is also present in several species, suggesting that these genes form part of a novel gene family. The Spef1 protein has two conserved domains, one of which is more strongly conserved in both homologues of the gene. Expression analysis of Spef1 in mice shows that it is expressed predominantly in adult testis, suggesting a role in spermatogenesis. Using an antibody generated to recombinant Spef1, we demonstrate a specific pattern of Spef1 localisation in the seminiferous epithelium of adult mouse testis. Further immunohistochemical analysis using electron microscopy shows Spef1 to be present in the tails of developing and epididymal sperm, internal to the fibrous sheath and around the outer dense fibres of the sperm flagellum.

Alternative Splicing↗
PubMed2005

Unstable Robertsonian translocations der(13;15)(q10;q10): heritable chromosome fission without phenotypic effect in two kindreds.

Robertsonian translocations (RTs) are amongst the most common chromosome abnormalities, but being essentially balanced are not usually associated with phenotypic abnormality. Despite being dicentric, RTs are almost always transmitted stably through cell division without chromosome breakage. We have investigated spontaneous fission of der(13;15)(q10;q10) chromosomes in eight individuals from two unrelated kindreds with a view to assessing clinical significance and to seek an explanation for the peculiar heritable instability displayed by these chromosomes. In Family 1, fission products were observed in five members in three generations. The instability was observed in cells derived from chorionic villus and lymphocytes. In Family 2, the same phenomenon was observed in amniocytes from two separate pregnancies and maternal blood lymphocytes. Detailed FISH analysis of these RTs showed them to be dicentric with an unremarkable pericentromeric structure. Notably, combined immunofluoresence and FISH analysis showed the presence of the centromere-specific proteins CENP-A and CENP-E, consistent with functional dicentricity in >75% of cells analyzed. The fission products are, therefore, presumed to be the result of sporadic, bipolar kinetochore attachment, anaphase bridging with resultant inter-centromeric breakage in a small proportion of mitoses. None of the eight carriers shows phenotypic abnormality and therefore, for prenatal counseling purposes, there appears to be no increased specific risk associated with this phenomenon.

Autoantigens↗
PubMed2005

Mosaic monosomy of a neocentric ring chromosome maps brachyphalangy and growth hormone deficiency to 13q31.1-13q32.3.

We describe a boy with moderate intellectual disability associated with distinctive hand malformations (hypoplastic and angel-shaped middle phalanges) and partial growth hormone (GH) deficiency associated with mosaic deletion of 13q31.1-13q32.3. The deleted segment was mapped to a 20-Mb region bounded by BACs RP11-1143C2 and RP11-139C1, narrowing the previously described locus for hand malformations at this region and suggesting that a locus for GH deficiency is also present at this location. The deleted segment contains at least three candidate genes, glypican-5, FARP1 and SOX21, that may be contributing to the phenotype in this boy. In a significant proportion (approximately 50%) of cells, the deleted region is present as a supernumerary ring chromosome stabilized by the formation of a neocentromere at 13q31-q32, within a region with a known propensity for neocentromere formation. The ring chromosome appears to be prone to low-level misdivision and loss in vitro which, in vivo, must be countered by selection for the balanced karyotype because the level of mosaicism has remained stable over 13 years.

Abnormalities, Multiple↗
PubMed2005

Molecular distinction between true centric fission and pericentric duplication-fission.

Centromere (centric) fission, also known as transverse or lateral centric misdivision, has been defined as the splitting of one functional centromere of a metacentric or submetacentric chromosome to produce two derivative centric chromosomes. It has been observed in a range of organisms and has been ascribed an important role in karyotype evolution; however, the underlying mechanisms remain unknown. We have investigated four cases of apparent centric fission in humans. Two cases show a missing chromosome 22 or 18 that is replaced by two centric ring products, a third case shows two chromosome-10-derived telocentric chromosomes, whereas a fourth case involves the formation of two chromosome-18-derived isochromosomes. In all four cases, results of gross cytogenetic and fluorescence in situ hybridisation analyses were consistent with a simple centric fission event. However, detailed molecular analyses provided evidence in support of centromere duplication as a predisposing mechanism for the observed chromosomal breakage in two of the cases. Results for the third case are consistent with direct centric fission not involving centromere pre-duplication as the likely mechanism. Insufficient material has precluded the further study of the fourth case. The data provide the first molecular evidence for centromere pre-duplication as a possible mechanism to explain the classically assumed simple "centric fission" events in clinical cytogenetics, karyotype evolution and speciation.

Fluorescent Antibody Technique↗
PubMed2005

Increased chromosome instability but not cancer predisposition in haploinsufficient Bub3 mice.

Mitotic spindle checkpoint proteins have been shown to play a crucial role in the accurate segregation of chromosomes during cell division. Bub3 is a member of a group of mitotic checkpoint proteins that are essential for this process. To investigate the role of Bub3 in chromosome segregation and cancer development, we analyzed haploinsufficient cells in mice. Heterozygous Bub3 embryonic fibroblasts displayed increased aneuploidy and premature sister-chromatid separation. In addition, when challenged with the microtubule disruptor nocodazole, the cells showed a slight increase in chromatid breakage and a decrease in the mitotic index. No substantial differences were observed between wild-type and Bub3 heterozygous mice in the frequency or the rate at which tumors appeared. Crossing Bub3(+/-) mice onto a heterozygous tumor-suppressor background of Trp53 or Rb1 similarly revealed no substantial differences in either the number or the rate at which tumors appeared. These results suggest that haploinsufficiency of Bub3 causes a slight increase in chromosome instability but is not clearly associated with a noticeable rise in the probability of tumor formation in the animal, possibly because of a partially functional mitotic checkpoint, or cells exhibiting chromosome instability could have activated the apoptosis pathway and thus escaped tumor induction and detection.

Aneuploidy↗
PubMed2005

BAC-based PCR fragment microarray: high-resolution detection of chromosomal deletion and duplication breakpoints.

The introduction of molecular techniques in conjunction with classical cytogenetic methods has in recent years greatly improved the diagnostic potential for chromosomal abnormalities. In particular, microarray-comparative genomic hybridization (CGH) based on the use of BAC clones promises a sensitive strategy for the detection of DNA copy-number changes on a genomewide scale, offering a resolution as high as >30,000 "bands" (as defined by the number of BACs within the currently highest-density BAC array) [Ishkanian et al., 2004]. We have tested the possibility of further increasing this resolution using PCR fragments generated from individual BAC clones. Using this approach, we have efficiently defined the proximal and distal breakpoints in two cytogenetic cases, one duplication and one deletion, to within 5-20 kb. The results support the potential use of BAC-based PCR fragments to further improve the resolution of the microarray-CGH strategy by an order of magnitude.

Chromosome Aberrations↗
PubMed2005

Detection of cryptic subtelomeric chromosome abnormalities and identification of anonymous chromatin using a quantitative multiplex ligation-dependent probe amplification (MLPA) assay.

The need to detect clinically significant segmental aneuploidies beyond the range of light microscopy demands the development of new cost-efficient, sensitive, and robust analytical techniques. Multiplex ligation-dependent probe amplification (MLPA) has already been shown to be particularly effective and flexible for measuring copy numbers in a multiplex format. Previous attempts to develop a reliable MLPA to assay all chromosome subtelomeric regions have been confounded by unforeseen copy number variation in some genes that are very close to the telomeres in healthy individuals. We addressed this shortcoming by substituting all known polymorphic probes and using two complementary multiplex assays to minimize the likelihood of false results. We developed this new quantitative MLPA strategy for two important diagnostic applications. First, in a group of cases with high clinical suspicion of a chromosome abnormality but normal, high-resolution karyotypes, MLPA detected subtelomeric abnormalities in three patients. Two were de novo terminal deletions (del(4p) and del(1p)), and one was a derivative chromosome 1 from a maternal t(1p;17p). The range of these segmental aneuploidies was 1.8-6.6 Mb, and none were visible on retrospective microscopy. Second, in a group of six patients with apparently de novo single-chromosome abnormalities containing anonymous chromatin, MLPA identified two cases with simple intrachromosomal duplications: dup(6p) and dup(8q). Three cases showed derivative chromosomes from translocations involving the distal regions of 9q and 4q, 5p and 11q, and 6q and 3p. One case showed a nonreciprocal, interchromosomal translocation of the distal region of 10p-7p. All abnormalities in both groups were confirmed by fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs). This quantitative MLPA technique for subtelomeric assays is compared with previously described alternative techniques.

Adolescent↗
PubMed2004

Analysis of mitotic and expression properties of human neocentromere-based transchromosomes in mice.

Human neocentromeres are functional centromeres that are devoid of the typical human centromeric alpha-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model.

Animals↗
PubMed2004

Variable and hierarchical size distribution of L1-retroelement-enriched CENP-A clusters within a functional human neocentromere.

Human neocentromeres are fully functional centromeres that arise epigenetically from non-centromeric precursor sequences that are devoid of alpha-satellite DNA. Using chromatin immunoprecipitation (ChIP) and BAC-array analysis, we have previously described a 330 kb binding domain for CENP-A (a histone H3 variant that confers centromere-specific nucleosomal property) at the 10q25 neocentromere found on a chromosome 10-derived marker chromosome mardel(10). For the further detailed analysis of the CENP-A-associated chromatin, we have generated a high-resolution genomic array consisting of PCR fragments with an average size of 8 kb, providing an approximately 20-fold increment in analytical resolution. ChIP and PCR-array analysis reveals seven distinct CENP-A-binding clusters within the 330 kb domain, demonstrating the interspersion of CENP-A-associated nucleosomal blocks within the neocentromeric chromatin. Independent ChIP-PCR analysis verified this distribution profile and indicated that histone H3-containing nucleosomes directly intervene the CENP-A-binding clusters. The CENP-A-binding clusters are uneven in size, with the central cluster (>50 kb) being significantly larger than the flanking ones (10-30 kb), and the flanking clusters arranged in an interesting hierarchical and symmetrical configuration of alternating larger and smaller sizes around the central cluster. In silico sequence analysis indicates an approximately 2.5-fold increase in the prevalence of L1 retroelements within the CENP-A-binding clusters when compared with the non-CENP-A-binding regions. These results provide insight into the possible role of retroelements in determining the positioning of CENP-A binding at human neocentromeres, and that a hierarchical and symmetrical arrangement of CENP-A-binding clusters of varying sizes may be an important structural requirement for mammalian kinetochore assembly and/or to provide stability to withstand polar microtubule forces.

Animals↗
PubMed2004

Effects of scaffold/matrix alteration on centromeric function and gene expression.

We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nmtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 microg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo IIalpha, CENP-A, CENP-C, and HP1alpha, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by approximately 20 and 50%, respectively, in the size of the enhanced S/MAR domain at the alpha-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.

Animals↗
PubMed2004

Human centromere repositioning "in progress".

Centromere repositioning provides a potentially powerful evolutionary force for reproductive isolation and speciation, but the underlying mechanisms remain ill-defined. An attractive model is through the simultaneous inactivation of a normal centromere and the formation of a new centromere at a hitherto noncentromeric chromosomal location with minimal detrimental effect. We report a two-generation family in which the centromeric activity of one chromosome 4 has been relocated to a euchromatic site at 4q21.3 through the epigenetic formation of a neocentromere in otherwise cytogenetically normal and mitotically stable karyotypes. Strong epigenetic inactivation of the original centromere is suggested by retention of 1.3 megabases of centromeric alpha-satellite DNA, absence of detectable molecular alteration in chromosome 4-centromereproximal p- and q-arm sequences, and failure of the inactive centromere to be reactivated through extensive culturing or treatment with histone deacetylase inhibitor trichostatin A. The neocentromere binds functionally essential centromere proteins (CENP-A, CENP-C, CENP-E, CENP-I, BUB1, and HP1), although a moderate reduction in CENP-A binding and sister-chromatid cohesion compared with the typical centromeres suggests possible underlying structural/functional differences. The stable mitotic and meiotic transmissibility of this pseudodicentric-neocentric chromosome in healthy individuals and the ability of the neocentric activity to form in a euchromatic site in preference to a preexisting alphoid domain provide direct evidence for an inherent mechanism of human centromere repositioning and karyotype evolution "in progress." We discuss the wider implication of such a mechanism for meiotic drive and the evolution of primate and other species.

Autoantigens↗
PubMed2004

Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

Charcot-Marie-Tooth Disease↗