Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics.

DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY: +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with 60Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7:13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.

Heterogeneities in the distribution of (GACA)n simple repeats in the karyotypes of primates and mouse.

Tandemly organized simple repetitive sequences are widespread in all eukaryotes. The organization of the simple tetrameric (GACA)n sequences at chromosomal loci has been investigated using in situ hybridization with chemically pure oligonucleotide probes. Both biotin- and digoxigenin-attached (GACA)4 probes reveal specific hybridization signals over the short arms of all acrocentric autosomes in man. In the other examined primates the NOR-bearing autosomes could be detected by in situ hybridization with (GACA)4, and a major concentration of the GACA simple repeats could be observed on the Y chromosome in the gibbon and mouse: the hybridization site in the gibbon Y chromosome coincides particularly with the silver-stainable NOR. In the past, accumulations of (GACA)n sequences were demonstrated mainly on vertebrate sex chromosomes. Therefore, the organization of GACA simple sequences is discussed in the context of their evolutionary potential accumulation and the possible linkage with the primate rDNA loci.

Isolation of fetal trophoblast cells from peripheral blood of pregnant women.

Fetal trophoblast cells were isolated from maternal peripheral blood by means of murine monoclonal antibodies of high specificity and affinity for human syncytiotrophoblast and nonvillous cytotrophoblast cells. The cells were isolated in sufficient numbers to allow polymerase chain reaction (PCR) amplification of the Y-chromosome-specific DNA sequence from the peripheral blood of thirteen pregnant women. The fetal sex predicted by PCR analysis of the isolated trophoblast cells accorded with that ascertained by karyotyping of chorionic villus samples in eleven of twelve women studied in early pregnancy and with the sex of the baby on delivery in one woman studied at 34 weeks' gestation. Isolation of these fetal cells could allow noninvasive diagnosis of a wide range of inherited disorders.

Determinaçäo no primeiro trimestre do sexo fetal pela técnica do DNA recombinante / Fetal sex determination in the first trimester by recombinant DNA technique

O sexo de três fetos foi determinado no primeiro trimestre através da técnica do DNA recombinante. Foram utilizadas as sondas DYZ, e DYZ, ambas específicas para o cromossomo Y. O DNA fetal foi obtido por biopsia do vilo coriônico. O processo utilizado foi o de "southern blotting", que consiste, basicamente, em digestäo enzimática do DNA, e sua transferência para filtro de nitrocelulose onde é realizada a hibridizaçäo com a sonda específica. O método é seguro e rápido

Chimerism studies using in situ hybridization for the Y chromosome after T cell-depleted bone marrow transplantation.

In situ hybridization for the Y chromosome (Y-ISH) was used to monitor engraftment in 10 patients with hematological malignancies who had received T cell-depleted marrow transplants from sex-mismatched donors, seven of whom were only partially HLA-matched. In the three patients who engrafted, as the peripheral counts rose, the percentage of host peripheral blood and marrow mononuclear cells decreased steadily, although host cells (less than 1%) could still be detected as late as day 252. The percentage of host granulocytes fell rapidly to less than 0.2%. Seven patients did not achieve full engraftment by day 28. Those with a low percentage of host cells (less than 1%) improved with observation or treatment with steroids, while those with a high or increasing percentage of host cells did not improve even after treatment with GM-CSF or with repeat marrow infusion without reconditioning. In one patient with graft failure, the residual host cells were predominantly CD8+ CD57+ and CD3+ CD56+, phenotypes consistent with non-MHC-restricted cytotoxic T cells. Lack of full engraftment in recipients of T cell-depleted marrow is not always associated with autologous reconstitution and does not always require retransplantation. Y-ISH may be useful for monitoring patients at high risk for graft failure in order to detect adverse trends in mixed chimerism that will alter therapy early after transplantation.

Detection of engraftment and chimerism after bone marrow transplantation by in situ hybridization using a Y-chromosome specific probe.

After bone marrow transplantation (BMT), the recipient and donor cells must be distinguished from each other to document and characterize successful engraftment. In addition to dot blot and Southern blot analyses, we have performed in situ hybridization in two sex-mismatched cases using a Y-chromosome specific DNA probe (PHY10). In situ hybridization showed that greater than 95% of the peripheral mononuclear cells had clusters of grains indicative of male cell origin in a recipient girl (case 1), and no cells had clusters of grains in another recipient boy (case 2) at the time of engraftment and 3 months after BMT. In situ hybridization using the PHY10 probe appears to facilitate identification of individual cells of male and female origin, and it requires only 20 hr to obtain the results. The technique provides a powerful new method for the documentation of engraftment and the detection of mixed hematopoietic chimerism in peripheral blood and bone marrow cell compartments after BMT.

In situ hybridization in hematopathology.

In situ hybridization (ISH) is one of the molecular techniques that has applications in diagnostic hematopathology. This procedure allows the detection of DNA or RNA in intact cells from various preparations, including cytology specimens and routinely fixed paraffin-embedded tissues. ISH is therefore analogous to detecting proteins (antigens) in intact cells with immunohistochemistry. The purpose of this article is to review the basic concepts and principles of ISH and to briefly discuss the important technical details of this procedure. Examples of potential applications of ISH in hematopathology are then discussed, including detection of Epstein-Barr virus, Y chromosome, and oncogene activation.

Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification.

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.

Generation of a panel of somatic cell hybrids containing unselected fragments of human chromosome 10 by X-ray irradiation and cell fusion: application to isolating the MEN2A region in hybrid cells.

We have used X-ray irradiation and cell fusion to generate somatic cell hybrids containing fragments of human chromosome 10. Our experiments were directed towards isolating the region of the MEN2A gene in hybrids and to use those as the source of DNA for cloning and mapping new markers from near the MEN2A locus. A number of hybrid clones containing human sequences that are tightly linked to the MEN2A gene were identified. Some 25% of our hybrids, however, proved to contain more than one human chromosome 10-derived fragment or showed evidence of deletions and/or rearrangements. A detailed analysis of the human content of X-ray irradiation hybrids is required to assess the integrity and number of human fragments retained. Despite retention of multiple human-derived fragments, these hybrids will prove useful as cloning and mapping resources.

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei.

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.

Clinical applications of flow karyotyping in myelocytic leukemia by stimulation of different subpopulations of cells in blood or bone marrow samples.

Examples are presented in which normal as well as abnormal chromosome distributions could be obtained from the same individual by means of bivariate flow karyotyping. Selective stimulation of T-lymphocytes obtained by E-rosetting from the blood of a patient with acute myelocytic leukemia resulted in a normal flow karyogram. The specific stimulation of myelocytic leukemia cells with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL-3) yielded flow karyograms displaying the leukemia-associated chromosome abnormalities. The resulting flow karyograms could be used to discriminate between homolog differences, which appear normally in virtually every individual, and leukemia-associated chromosomal aberrations. In the case of a female chronic myelocytic leukemia patient who received bone marrow form an HLA-identical male donor, specific stimulation of various subsets of cells enabled to discriminate between leukemic host cells and non-leukemic donor cells. Both the leukemia-specific translocations and sex chromosomes were used as markers to analyse the flow karyograms obtained from the same sample.

[Familial Yqs chromosome in 4 generations]. / Yqs familiar en cuatro generaciones.

A structural aberration of the Y chromosome (Yqs) was identified in a 4-year-old child who visited a physician for right unilateral cryptorchidism. The structure of this chromosome was determined through bands C, G and NOR. The family cytogenetic study allowed for the detection of the Yqs in up to four generations. A comparison is made of the different phenotypes associated with this marker in the medical literature, concluding that this is probably due to another polymorphism of the Y chromosome.

Variability of euchromatic and heterochromatic regions of Y chromosome in two types of cancer patients.

Heteromorphism of Y chromosome was studied in head and neck cancer patients and leukemia patients. The results were compared with similar data obtained for healthy men. It was observed that, compared to the controls, mean lengths of Y chromosome were nonsignificantly higher for leukemia patients and lower for head and neck cancer patients. The euchromatic region of Y chromosome (Y-eu) remained comparable in the controls and the leukemia patients, whereas it was smaller in patients with head and neck malignancies. The heterochromatic region (Y-het) was more or less analogous in controls and head and neck cancer patients, however, it was significantly larger in patients with leukemia (P less than 0.02).

Rapid determination of fetal sex by deoxyribonucleic acid amplification of Y chromosome-specific sequences.

Rapid fetal sex tests use either dot blot hybridization to Y chromosome-specific (male) repeat sequences or polymerase chain reaction deoxyribonucleic acid amplification of these Y-specific sequences. We have performed 35 fetal sex determinations, 16 by dot blot alone, 13 by polymerase chain reaction alone, and 6 by dot blot and polymerase chain reaction on samples of fetal blood, amniotic fluid, and chorionic villi. All results have been confirmed by karyotyping. Dot blots have given false-positive "male" results three times. In contrast, the polymerase chain reaction has correctly determined fetal sex in every case, even when the dot blot was in error. The Y-specific polymerase chain reaction has been applied to fetal deoxyribonucleic DNA with a chromosome 15; Y translocation to identify the origin of the translocated material. Thus the polymerase chain reaction appears to be a reliable method to rapidly determine fetal sex that also can be used diagnostically to identify translocated Y-chromosomal material.

A group of non-Y encoded Drosophila hydei primary spermatocyte nuclear glycoproteins exhibits epitopes depending on formation of Y chromosomal giant lampbrush loops.

In wild-type Drosophila hydei (genotype X/Y) four different primary spermatocyte nuclear glycoproteins, classified as non-Y encoded because of their occurrence in X/O genotypes, were demonstrated to possess a few epitopes that depended on formation of the Y chromosomal giant lampbrush loops threads (th; Mr 55,000 proteins) or pseudonucleolus (ps; Mr 38,000, 58,000 and 98,000 proteins). The epitopes reacted with lectins and/or antibodies in vitro lectin-/immunoreplica of primary spermatocyte total nuclear protein), and were lacking in mutants not possessing the respective loops. Those dependent on ps reacted with human sera. Epitopes restricted to proteins from th-forming spermatocytes reacted with lectin Con A (specific for D-Man and/or D-Glc) and antibodies directed against mouse immunoglobulins (AIA). In situ experiments (immunofluorescence microscopy of primary spermatocyte nuclei) revealed antibody cross-reactions with the respective loops. The reagents stained the distal (fused) sections and proximal (compact) parts of ps (human sera) or the proximal (compact) parts of th (AIA). Reaction with the latter loops was significantly repressed after absorption of AIA with the L-Fuc carbohydrate unit, classifying the AIA as fucosyl specific, and the epitopes along th as L-Fuc carbohydrate units.

Position effect variegation in Drosophila melanogaster: relationship between suppression effect and the amount of Y chromosome.

Position effect variegation results from chromosome rearrangements which translocate euchromatic genes close to the heterochromatin. The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing property of the D. melanogaster Y chromosome. We have determined the extent to which different cytologically characterized Y chromosome deficiencies and Y fragments suppress three V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes. We find that: (1) chromosomes which are cytologically different and yet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 60-80% of the entire Y, after which the effect reaches a plateau. These findings suggest suppression is a function of the amount of Y heterochromatin present in the genome and is not attributable to any discrete Y region.