34 research outputs found
Somatic Genomic Variations in Extra-Embryonic Tissues
In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism
BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer
Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid
cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, RET and neurotrophic tyrosine kinase
receptor type I (NTRK1) by intra- and interchromosomal rearrangements has been suggested as a cause of the disease. However,
many phenotypically similar tumors do not carry an activated RET or NTRK-1 gene or express abnormal ret or NTRK-
1 transcripts. Thus, we hypothesize that other cellular RTK-type genes are aberrantly expressed in these tumors. Using fluorescence
in situ hybridization-based methods, we are studying karyotype changes in a relatively rare subgroup of PTCs,
i.e., tumors that arose in children following the 1986 nuclear accident in Chernobyl, Ukraine. Here, we report our technical
developments and progress in deciphering complex chromosome aberrations in case S48TK, an aggressively growing PTC
cell line, which shows an unusual high number of unbalanced translocations
Rapid mapping of chromosomal breakpoints: from blood to BAC in 20 days
Structural chromosome aberrations and associated segmental or chromosomal aneusomies are major causes of
reproductive failure in humans. Despite the fact that carriers of reciprocal balanced translocation often have no other clinical
symptoms or disease, impaired chromosome homologue pairing in meiosis and karyokinesis errors lead to over-representation
of translocations carriers in the infertile population and in recurrent pregnancy loss patients. At present, clinicians have no
means to select healthy germ cells or balanced zygotes in vivo, but in vitro fertilization (IVF) followed by preimplantation
genetic diagnosis (PGD) offers translocation carriers a chance to select balanced or normal embryos for transfer. Although a
combination of telomeric and centromeric probes can differentiate embryos that are unbalanced from normal or unbalanced
ones, a seemingly random position of breakpoints in these IVF-patients poses a serious obstacle to differentiating between normal
and balanced embryos, which for most translocation couples, is desirable. Using a carrier with reciprocal translocation
t(4;13) as an example, we describe our state-of-the-art approach to the preparation of patient-specific DNA probes that span or
'extent' the breakpoints. With the techniques and resources described here, most breakpoints can be accurately mapped in a matter
of days using carrier lymphocytes, and a few extra days are allowed for PGD-probe optimization. The optimized probes will
then be suitable for interphase cell analysis, a prerequisite for PGD since blastomeres are biopsied from normally growing day
3 – embryos regardless of their position in the mitotic cell cycle. Furthermore, routine application of these rapid methods
should make PGD even more affordable for translocation carriers enrolled in IVF programs
Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA
<p>Abstract</p> <p>Background</p> <p>Fluorescence <it>in situ </it>hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance.</p> <p>Results</p> <p>We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus.</p> <p>Conclusion</p> <p>The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.</p
BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer.
Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, RET and neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- and interchromosomal rearrangements has been suggested as a cause of the disease. However, many phenotypically similar tumors do not carry an activated RET or NTRK-1 gene or express abnormal ret or NTRK-1 transcripts. Thus, we hypothesize that other cellular RTK-type genes are aberrantly expressed in these tumors. Using fluorescence in situ hybridization-based methods, we are studying karyotype changes in a relatively rare subgroup of PTCs, i.e., tumors that arose in children following the 1986 nuclear accident in Chernobyl, Ukraine. Here, we report our technical developments and progress in deciphering complex chromosome aberrations in case S48TK, an aggressively growing PTC cell line, which shows an unusual high number of unbalanced translocations
Rapid mapping of chromosomal breakpoints: from blood to BAC in 20 days.
Structural chromosome aberrations and associated segmental or chromosomal aneusomies are major causes of reproductive failure in humans. Despite the fact that carriers of reciprocal balanced translocation often have no other clinical symptoms or disease, impaired chromosome homologue pairing in meiosis and karyokinesis errors lead to over-representation of translocations carriers in the infertile population and in recurrent pregnancy loss patients. At present, clinicians have no means to select healthy germ cells or balanced zygotes in vivo, but in vitro fertilization (IVF) followed by preimplantation genetic diagnosis (PGD) offers translocation carriers a chance to select balanced or normal embryos for transfer. Although a combination of telomeric and centromeric probes can differentiate embryos that are unbalanced from normal or unbalanced ones, a seemingly random position of breakpoints in these IVF-patients poses a serious obstacle to differentiating between normal and balanced embryos, which for most translocation couples, is desirable. Using a carrier with reciprocal translocation t(4;13) as an example, we describe our state-of-the-art approach to the preparation of patient-specific DNA probes that span or 'extent' the breakpoints. With the techniques and resources described here, most breakpoints can be accurately mapped in a matter of days using carrier lymphocytes, and a few extra days are allowed for PGD-probe optimization. The optimized probes will then be suitable for interphase cell analysis, a prerequisite for PGD since blastomeres are biopsied from normally growing day 3--embryos regardless of their position in the mitotic cell cycle. Furthermore, routine application of these rapid methods should make PGD even more affordable for translocation carriers enrolled in IVF programs
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Molecular cytogenetic characterization of a human thyroid cancercell line
The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the twelve years following the accident, the average incidence of childhood PTCs (chPTC) increased over one hundred-fold compared to the rate of about 1 tumor incidence per 10{sup 6} children per year prior to 1986. To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed ''BAC-FISH'' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences