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.

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells.

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.

Heterogeneity of clinical severity and molecular lesions in Aicardi syndrome.

All patients with Aicardi syndrome are female or have a 47,XXY karyotype. This finding, along with a report of an Aicardi syndrome patient with an Xp22/autosome translocation, led to the hypothesis that Aicardi syndrome might be caused by an X-linked dominant, male-lethal mutation on the short arm of the X chromosome. To study this hypothesis, we investigated X chromosome inactivation patterns in peripheral lymphocytes from seven patients. We used two methods: methylation-sensitive restriction enzyme analysis and segregation of the active X chromosome in somatic cell hybrids. We found that three of seven cytogenetically normal girls with Aicardi syndrome had profoundly skewed X-inactivation in their lymphocytes, supporting the concept that Aicardi syndrome is X linked. Three of the five girls with the greatest degree of psychomotor retardation and the poorest seizure control had skewed X-inactivation. In contrast, the two highest-functioning children had random X-inactivation. We screened DNA using eight polymorphic probes from the Xp22 region but were unable to identify a deletion in any of the seven patients. Nonrandom X-inactivation in lymphocytes and possibly other tissues in some, but not all, patients with Aicardi syndrome may reflect heterogeneity of their molecular lesions.

Distribution of rDNA and 28S, 18S, and 5S rRNA in micronuclei containing a single chromosome.

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S-28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S-28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a-e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S-28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S-28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.

Molecular analysis of no-on-transient A, a gene required for normal vision in Drosophila.

Mutation of the Drosophila melanogaster gene no-on-transient A (nonA) results in reduced visual acuity, behavior abnormalities, and an electrophysiological defect for which the mutant is named. We mapped the nonA gene genetically to a 20 kb interval within the 14C1,2 region of the X chromosome, isolated this chromosomal region, and used P element-mediated transformation to delimit the nonA gene to a 9 kb region. Analysis of cDNA clones indicates that this region encodes alternatively spliced transcripts encoding protein products of approximately 77 kd that differ only in their C-terminal 35 amino acids. Analysis of mutations generated in vitro in this transcription unit confirm that these transcripts are the products of the nonA gene.

Genetic counselling in Duchenne and Becker muscular dystrophy is problematic when carrier studies give controversial results.

DNA-analysis with flanking and intragenic markers gave confusing results in 7 out of 74 (9.5%) Finnish families with Duchenne or Becker muscular dystrophy. In five families a sister or maternal aunt of the patient had elevated serum creatine kinase (CK) activity, although DNA-analysis indicated a low risk for carriership. In one family the two affected brothers had different pERT87 alleles. In one family the intragenic deletion found in a patient was not present in his mother, who was an obligatory carrier. Deletions were detected with cDNA probes in the probands in five of the families, but the controversy regarding carriership still remained. It is necessary to combine all available data from pedigree analysis, CK measurements, and DNA studies whenever carrier studies are performed, but it appears that major problems in counselling and prenatal diagnosis will still remain in a proportion of the families.

New polymorphic DNA marker close to the fragile site FRAXA.

DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.

Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome.

Phosphorylase kinase is a glycogenolytic enzyme in several animal tissues. Within the last few years all four subunits of the enzyme have been cloned. The beta, gamma, and delta subunits are known to be autosomal. We have mapped the alpha subunit of phosphorylase kinase, recently cloned by Zander et al. (1988), in an interspecific mouse pedigree and localized it on the X chromosome, where it maps between the X-linked zinc finger protein and phosphoglycerate kinase genes, close to the latter. In man and mouse several X-linked disorders of this enzyme have been described. Although the X-linked phosphorylase kinase deficiency in mice may be caused by a mutation in the structural gene for the alpha subunit, mapped here, the existence of a separate regulatory locus, important in the normal expression or function of the enzyme in muscle, still remains a possibility.

Fragile-X mental retardation in a large five-generation family. A clinical and cytogenetic study.

A number of problems concerning both clinical and genetic or cytogenetic aspects of the fragile-X syndrome remain unsolved. In the present work, a large 5-generation fragile-X family has been clinically and cytogenetically investigated. The results of our study indicated that an unusually high proportion of affected males was present in the family examined; all fragile-X-positive males were profoundly retarded and showed the phenotype typically described for this syndrome; moreover, we observed a variability of penetrance within the pedigree and all fragile-X-positive females in the 4th and 5th generation had some degree of mental impairment. A multistep mutation model has been proposed in order to explain some of these findings.

Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping.

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.

Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots.

We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Y-sequence positive patients agrees with the variable nature of the X-Y interchange in these individuals as determined by the use of Y-specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X-Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X-Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.

[Linkage studies in Emery-Dreifuss muscular dystrophy]. / Etudes de liaison dans la dystrophie musculaire d'Emery-Dreifuss.

We report linkage studies between Emery-Dreifuss muscular dystrophy (EDMD) and polymorphic probes from the long arm of chromosome X in two pedigrees. The results don't show significant linkage but are consistent with previous localisation of EDMD in Xq28. Further studies will be necessary to apply molecular biology to genetic counselling and prenatal diagnosis of this disease.

A rare case of de novo structural rearrangement of X chromosome diagnosed by amniocentesis.

A dicentric X chromosome was found in a female fetus during cytogenetic studies performed on amniotic cells. Blood samples from the parents showed normal karyotypes and the pregnancy was terminated. The mechanism for the formation of this 'de novo' rearrangement is discussed.

SIMREP: a program simulating differential DNA replication.

During endoreplication, different organisms in different taxa carry out DNA syntheses without nuclear division. The result of such endocycles is either a polyploid nucleus or a polytene architecture of the chromosomes. Since not all sequences of the genome may be reduplicated simultaneously and to the same extent, endocycles provide an opportunity for primary cell differentiation at the DNA level as a result of DNA amplification or underreplication. We have designed a numerical model which simulates differential endoreplications. The program SIMREP is written in TURBO PASCAL 4.0 and can be executed on a PC/XT/AT with MS-DOS greater than or equal to 2.0. It uses diploid DNA contents derived from meiotic or mitotic nuclei together with data on amounts of DNA present after a given number of endocycles. SIMREP can be applied to genomes containing arbitrary numbers of chromosomes (maximum N = 24) to model details of their replication behaviour. It is also useful in analysing differential replication of single genes. The application of SIMREP is illustrated with two examples. (1) Female and male specific types of underreplication were found in the chironomid midge Prodiamesa olivacea. The heterosomes which appear homomorphic in metaphases were identified by their differential polytenization. (2) The Y chromosome of Drosophila nasutoides was assessed to ascertain whether its replication is regulated in parallel with, or independently from the large chromosome pair 4.

Prenatal diagnosis of a fetus with an extra idic(X)(q27).

A fetus with an extra idic(X)(q27) was ascertained during prenatal diagnosis. The derived X and one normal X chromosome were late replicating. Due to lack of previous experience, genetic counselling presented obvious difficulties and the fetal phenotype could be only tentatively predicted.

Selection and separation of X- and Y- chromosome-bearing mammalian sperm.

Preselection of the gender of offspring is a subject that has held man's attention since the beginning of recorded history. Most scientific hypotheses for producing the desired sex of offspring address separation of X- and Y-bearing sperm, and most have had limited, if any success. Eight of these hypotheses and their experimental verifications are discussed here. Three hypotheses are based on physical characteristics of sperm, one on supposed differences in size and shape, another on differences in density, and a third on differences in surface charge. There has been no experimental verification of differences based on size and shape, and the results from attempts to verify separation of X- and Y-bearing sperm based on density have been mixed. Electrophoresis may provide a method for separating X- and Y-bearing sperm, but it is currently unproven and would be of little practical utility, since sperm motility is lost. A fourth hypothesis employs H-Y antigen to select preimplantation embryos. This method reliably produces female offspring, but does not permit the selection of male offspring and does not work on sperm. There are two applications of the theory that X- and Y-bearing sperm should be separable by flow fractionation. Flow fractionation using thermal convection, counter-streaming sedimentation, and galvanization is highly promoted by its originator but has not gained wide acceptance due to lack of independent confirmation. Flow fractionation by laminar flow is said to provide up to 80% enrichment of both X- and Y-bearing sperm; however, this method also has not been confirmed by other workers or tested in breeding trials. The sixth theory discussed is that of separation through Sephadex gel filtration. This method may provide enrichment of X-bearing sperm, but, again, other experimenters have not been able to adequately confirm the enrichment. The best-known approach to sperm separation is that employing albumin centrifugation, yet even with this method, not all researchers have been able to confirm a final fraction rich in Y sperm, and trials in animals have given contradictory results. The most reliable method for separating X- and Y-bearing sperm is use of flow cytometric and flow sorting techniques. These techniques routinely separate fractions with a purity greater than 80% and can be above 90%. Unfortunately, these methods do not always work for human samples. Furthermore, as with electrophoretic approaches, the methods identify and separate only chemically fixed sperm and provide limited biological applications.(ABSTRACT TRUNCATED AT 400 WORDS)

Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration.

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.

Cytochrome b-245 and its involvement in the molecular pathology of chronic granulomatous disease.

Cytochrome b-245 is an integral, and probably the terminal, component of the microbicidal oxidase electron transport chain of phagocytic cells. Current knowledge of the biochemistry and cell and molecular biology of this molecule is described. The molecular basis of chronic granulomatous disease, in which defective electron transport down this chain predisposes to infection and impaired digestion by phagocytes, is explained in terms of anomalies of the cytochrome b and related molecules.

Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient.

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.

Carrier detection in typical and atypical X-linked agammaglobulinemia.

We have recently demonstrated that B cells from obligate carriers of typical X-linked agammaglobulinemia (XLA) exhibit nonrandom X chromosome inactivation. The active X is always the X that does not carry the gene defect. To determine if this were also true in carriers of atypical XLA and to provide carrier detection for all women at risk of being carriers of XLA, we developed a technique that permits analysis of X chromosome inactivation in cells from any woman. This technique combines the production of somatic cell hybrids that selectively retain the active X chromosome with the use of X-linked restriction fragment length polymorphisms that permit the distinction of the two X chromosomes. Three obligate carriers of typical XLA and four women whose sons might be considered to have atypical or sporadic XLA were studied. B cell hybrids from all seven women demonstrated exclusive use a single X as the active X. In addition, B cell hybrids from four of eight women at 25% or 50% risk of being carriers exhibited nonrandom X chromosome inactivation, indicating that these women were also carriers of X-linked forms of hypogammaglobulinemia. These results illustrate a technique that can be used both to help define XLA and to provide carrier detection for all women at risk of being carriers of this disorder.