A molecular study of X isochromosomes: parental origin, centromeric structure, and mechanisms of formation.

Fourteen individuals with an i(Xq) or idic(Xq) were studied using RFLP analysis in order to determine both parental origin and extent of heterozygosity of the isochromosome and to search for the presence of short-arm material. In five cases the isochromosome was paternally derived, while nine patients had a maternal i(Xq). The analysis of heterozygosity of the nine maternally derived isochromosomes by using Xq markers showed heterozygosity in two cases, suggesting an origin from two homologous X chromosomes. Homozygosity was found at all informative loci in seven cases, which therefore are probably the product of either centromere misdivision or sister-chromatid exchange. Presence of Xp markers was seen both in the three i(Xq) chromosomes which appeared dicentric by cytogenetic analysis and in three additional cytogenetically monocentric cases. Mean parental ages were greater for the maternally derived cases as compared with the paternally derived cases.     

Indirect immunofluorescence of inactive centromeres as indicator of centromeric function

Summary Two previous single case reports from the literature showed the presence or absence of centromeric antigens at the site of the inactive centromeres in one (X;X) and in one (9;11) dicentric chromosome. We studied nine different dicentric chromosomes using anticentromeric antibodies and immunofluorescence techniques. In the four autosomal dicentrics the inactive centromere was consistently positive while the dicentrics composed of two X chromosomes were either positive or negative; one case of (X;Y) dicentric was negative. The results indicate that the X chromosome mode of replication may be involved in the suppression of immunofluorescence at the site of the inactive centromere and that one centromere of the dicentric chromosome may lose its function but conserve some of its antigenic properties. This indicates that not all these antigens play a rôle in the microtubules-centromere interaction.     

Parental origin of the extra chromosomes in polysomy X

Five polymorphic index markers were analyzed by polymerase chain reaction (PCR) to ascertain the parental origin of the extra X chromosomes in seven polysomic cases (one 49,XXXXX, three 49,XXXXY, two 48,XXXY, and one 48, XXYY). All four X chromosomes in 49, X polysomies were maternal in origin and the extra X chromosomes in 48 X polysomies were paternal. In each case the multiple X chromosomes were contributed by a single parent. Taken together with previously reported cases, these data support a single mechanism of sequential nondisjunction during either maternal or paternal gametogenesis as the cause of higher order sex chromosome polysomy.     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.     

Chromosome engineering: generation of mono- and dicentric isochromosomes in a somatic cell hybrid system

The most common isochromosome found in humans involves the long arm of the X, i(Xq), and is associated with a subset of Turner syndrome cases. To study the formation and behavior of isochromosomes in a more tractable experimental system, we have developed a somatic cell hybrid model system that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp). Simultaneous positive and negative counterselection of a mouse/human somatic cell hybrid containing a human X chromosome, selecting for retention of the UBE1 locus in Xp but against the HPRT locus in Xq, results in a variety of abnormalities of the X chromosome involving deletions of Xq. We have generated 70 such ”Pushmi-Pullyu” hybrids derived from seven independent X chromosomes. Cytogenetic analysis of these hybrids using fluorescence in situ hybridization showed i(Xp) chromosomes in ∼19% of the hybrids. Southern blot and polymerase chain reaction analyses of the Pushmi-Pullyu hybrids revealed a distribution of breakpoints along Xq. The distance between the centromeres of the dicentric i(Xp)s generated ranged from ∼2 Mb to ∼20 Mb. To examine centromeric activity in these dicentric i(Xp)s, we used indirect immunofluorescence with antibodies to centromere protein E (CENP-E). CENP-E was detected at only one of the centromeres of a dicentric i(Xp) with ∼2–3 Mb of Xq DNA. In contrast, CENP-E was detected at both centromeres of a dicentric i(Xp) with ∼14 Mb of Xq DNA. Two other dicentric i(Xp) chromosomes were heterogeneous with respect to centromeric activity, suggesting that centromeric activity and chromosome stability of dicentric chromosomes may be more complicated than previously thought. The Pushmi-Pullyu model system presented in this study may provide a tool for examining the structure and function of mammalian centromeres. Received: 15 December 1998; in revised form: 2 March 1999 / Accepted: 5 April 1999     

Cd bands and centromeric function in dicentric chromosomes

Summary The Cd technique was applied to two cases of dicentric attached X chromosomes (XpXp and XqXq) and to cells from an established cell line of tumor origin (MaNo9) in which dicentrics with two active centromeres were present and dicentrics with one active and one inactive centromere. It was confirmed that the Cd technique discriminates between active and latent centromeres, and it was demonstrated that true dicentrics and dicentrics with one latent centromere can co-exist in the same cells. This indicates that the mechanism of centromere inactivation is a phenomenon that is specific to each chromosome and not generalized at the level of the individual cell.     

The origin and behavior of two isodicentric bisatellited chromosomes.

Karyotyping revealed three cell lines in a boy with mental retardation and few other abnormalities. Thirty cells exhibited a normal karyotype, and 54 had an extra acrocentric chromosome of E group size with satellites on the long and short arms. The remaining 20 cells each had, in addition to the first marker (M1), a second tiny bisatellited chromosome (M2). C-banding demonstrated that both markers were dicentric. G-, C-, and Q-banding and satellite association data were consistent with the markers having originated from chromosome 15 material. We propose that M1 was formed from a meiotic breakage and a chromatid fusion in the proximal long arms of an acrocentric pair. This would have produced a symmetrical isodicentric chromosomes, plus one or two acentric fragments. M2 then could have resulted from a dicentric bridge-break-synthesis-reunion phenomenon. This model of abnormal meiotic exchange can be generalized to encompass the formation of other dicentric isochromosome cases of isochromosome X.     

Isodicentric Y chromosome: cytogenetic,molecular and clinical studies and review of the literature

Dicentrics are among the most common structural abnormalities of the human Y chromosome. Predicting the phenotypic consequences of different duplications and deletions of dicentric Y chromosomes is usually complicated by varying degrees of mosaicism (45,X cell lines), which may, in some cases, remain undetected. Molecular studies in patients with dicentric Y chromosomes have been few, and only two studies have attempted to determine the presence of SRY (the putative testis-determining factor gene). We report an 18-year-old female with short stature, amenorrhea, hirsutism, hypoplastic labia minora, and clitoromegaly who has a 45,X/46,X,idic(Y)(p11.32)/47,X,idic(Y)(p11.32),idic(Y) (p11.32) karyotype. Southern analysis using Y-specific probes (Y97, 2D6, 1F5, pY3.4) and polymerase chain reaction (PCR) analysis using primers for ZFY and SRY were positive for all loci tested, indicating that almost all of the Y chromosome was present. Our findings and an extensive review of the literature emphasize the importance of molecular analyses of abnormal Y chromosomes before any general conclusions can be reached concerning the relative effects of the Y-chromosome abnormality and mosaicism on sexual differentiation.     

Unstable Chromosome Aberrations Do Not Accumulate in Normal Human Fibroblast after Fractionated X-Irradiation

We determined the frequencies of dicentric chromosomes per cell in non-dividing confluent normal human fibroblasts (MRC-5) irradiated with a single 1 Gy dose or a fractionated 1 Gy dose (10X0.1 Gy, 5X0.2 Gy, and 2X0.5 Gy). The interval between fractions was between 1 min to 1440 min. After the completion of X-irradiation, the cells were incubated for 24 hours before re-plating at a low density. Then, demecolcine was administrated at 6 hours, and the first mitotic cells were collected for 42 hours. Our study demonstrated that frequencies of dicentric chromosomes in cells irradiated with a 1 Gy dose at different fractions were significantly reduced if the fraction interval was increased from 1 min to 5 min (p<0.05, χ²-test). Further increasing the fraction interval from 5 up to 1440 min did not significantly affect the frequency of dicentric chromosomes. Since misrejoining of two independent chromosome breaks introduced in close proximity gives rise to dicentric chromosome, our results indicated that such circumstances might be quite infrequent in cells exposed to fractionated X-irradiation with prolonged fraction intervals. Our findings should contribute to improve current estimation of cancer risk from chronic low-dose-rate exposure, or intermittent exposure of low-dose radiation by medical exposure.     

Dicentric chromosomes and 20q11.2 amplification in MDS/AML with apparent monosomy 20

FISH analysis of 41 previously karyotyped cases of MDS and AML with apparent monosomy of chromosome 20 revealed a variety of dicentric abnormalities involving chromosome 20. These usually, but not always, involved a breakpoint in the long arm of chromosome 20 and loss of the common deleted region at 20q12. Not one case of true monosomy 20 was confirmed. We found evidence for dicentric chromosome formation in 21 of 24 unbalanced translocations containing chromosome 20 and that were studied in more detail. Subsequent loss of one of the centromeres had occurred in eight of these 24 cases, and was more frequent than centromere inactivation as a means of resolving the inherent instability of a dicentric chromosome. In the three cases with dicentric chromosomes from which proximal 20q had been excised along with the 20 centromere, the excised segment was retained, and in two of these it was amplified. Proximal 20q was clearly retained in all but three cases, and present in three or more copies in 17 of 41 cases. The retention and amplification of proximal 20q provides support for the hypothesis that there is an oncogene located in this region of 20q that is activated in cases of MDS/AML with del(20q). Apparent monosomy 20 in MDS/AML should be treated as evidence of unidentified chromosome 20 abnormalities, and familiarity with the typical G-banded morphology of these derivatives can help with their identification. The reported incidence of dicentric chromosomes is clearly an under-estimate but is increasing in myeloid disorders as more cases are studied with methods allowing their detection.     

Deletion of specific sequences or modification of centromeric chromatin are responsible for Y chromosome centromere inactivation

Summary Stable dicentric chromosomes behave as monocentrics because one of the centromeres is inactive. The cause of centromere inactivation is unknown; changes in centromere chromatin conformation and loss of centromeric DNA elements have been proposed as possible mechanisms. We studied the phenomenon of inactivation in two Y centromeres, having as a control genetically identical active Y centromeres. The two cases have the following karyotypes: 45,X/46,X,i(Y)(q12) and 46,XY/ 47,XY,+t(X;Y)(p22.3;p11.3). The analysis of the behaviour of the active and inactive Y chromosome centromeres after Da-Dapi staining, CREST immunofluorescence, and in situ hybridization with centromeric probes leads us to conclude that, in the case of the isochromosome, a true deletion of centromeric chromatin is responsible for its stability, whereas in the second case, stability of the dicentric (X;Y) is the result of centromere chromatin modification.     

Analysis of two 47,XXX males reveals X-Y interchange and maternal or paternal nondisjunction

Summary Two cases of 47,XXX males were studied, one of which has been published previously (Bigozzi et al. 1980). Analysis of X-linked restriction fragment length polymorphisms revealed that in this case, one X chromosome was of paternal and two were of maternal origin, whereas in the other case, two X chromosomes were of paternal and one of maternal origin. Southern blot analysis with Y-specific DNA probes demonstrated the presence of Y short arm sequences in both XXX males. In one case, the results obtained pointed to a paracentric inversion on Yp of the patient's father. In situ hybridization indicated that the Y-specific DNA sequences were localized on Xp22.3 in one of the three X chromosomes in both cases. The presence of Y DNA had no effect on random X inactivation. It is concluded that both XXX males originate from aberrant X-Y interchange during paternal meiosis, with coincident nondisjunction of the X chromosome during maternal meiosis in case 1, and during paternal meiosis II in case 2.     

Parental origin and mechanism of formation of polysomy X: an XXXXX case and four XXXXY cases determined with RFLPs

Summary The parental origin and mechanism of formation of polysomy X were studied in five cases (one case of 49,XXXXX; four cases of 49,XXXXY), using various X-linked restriction fragment length polymorphisms as genetic markers. Segregation and densitometric analyses on the polymorphic DNA fragments revealed that, in all five cases, the additional X chromosomes are of maternal origin and the mechanism of formation is most probably a result of three non-disjunctions during maternal meiotic divisions: once at the first meiosis and simultaneously twice at the second meiosis. The identical origin and the identical mechanism of formation among the five cases are unlikely to be coincidental and suggest a common cause in the mothers of the five cases.     

Karyotypic characteristics of the murine myeloma line sp2/0-Ag14

Using methods of G- and C-banding, a study was made of the karyotype of mouse myeloma cell line sp2/0-Ag14. The number of chromosomes varies from 58 to 65, the modal class being 61-62. 50 per cent of chromosomes are rearranged. Normal chromosomes 6, 12 and X were not detected in either examined cell of this line. Among the marker chromosomes there are an isochromosome (19/19), three dicentric markers and one marker with two interstitial C-bands. There is a specific marker t (12; 15) of mouse plasmacytomas in the karyotype sp2/0-Ag14. A possible association of specific translocations and segregations of the normal chromosomes with the phenotype of line sp2/0-Ag14 is discussed. The results obtained may be useful for cytogenetic analysis of hybridomas.     

Determining the origin of human X isochromosomes by use of DNA sequence polymorphisms and detection of an apparent i(Xq) with Xp sequences

Summary The parental origin of five X isochromosomes were determined using 11 DnA markers. The isochromosome was derived from a maternal X chromosome in three cases and from a paternal X chromosome in two. Unexpected heterozygosity was detected for the proximal Xp region in one individual in whom the i(Xq) chromosome was paternally derived. This was confirmed by in situ hybridisation. A mode of formation of isochromosomes by breakage and reunion between the sister chromatids of the arms of an X chromosome is proposed to account for this. Sister chromatid breakage and reunion can be considered as a significant mechanism for the origin of i(Xq) chromosomes.     

Meiotic and Mitotic Behavior of Dicentric Chromosomes in SACCHAROMYCES CEREVISIAE

Meiotic recombination between a circular and a linear chromosome in Saccharomyces cerevisiae has been investigated. The circle was a haploid-viable derivative of chromosome III constructed by joining regions near the two chromosome ends via a recombinant DNA construction: (HMR/MAT-URA3-pBR322-MAT/HML) and was also deleted for MAL2 (which therefore uniquely marks a linear chromosome III). Recombination along chromosome III was measured for eight intervals spanning the entire length of the circular derivative. Only 25% of all tetrads from a ring/rod diploid contained four viable spores. These proved to be cases in which there was either no recombination along chromosome III or in which there were two-strand double crossovers or higher order crossovers that would not produce a dicentric chromosome.--At least half of the tetrads with three viable spores included one Ura+ Mal+ spore that was genetically highly unstable. The Ura+ Mal+ spore colonies gave rise to as many as seven genetically distinct, stable ("healed") derivatives, some of which had lost either URA3 or MAL2. Analysis of markers on chromosome III suggests that dicentric chromosomes frequently do not break during meiosis but are inherited intact into a haploid spore. In mitosis, however, the dicentric chromosome is frequently broken, giving rise to a variety of genetically distinct derivatives. We have also shown that dicentric ring chromosomes exhibit similar behavior: at least half the time they are not broken during meiosis but are broken and healed during mitosis.--The ring/rod diploid can also be used to determine the frequency of sister chromatid exchange (SCE) along an entire yeast ring chromosome. We estimate that an unequal number of SCE events occurs in approximately 15% of all cells undergoing meiosis. In contrast, the mitotic instability (and presumably SCE events) of a ring chromosome is low, occurring at a rate of about 1.2 X 10(-3) per cell division.     

Chromosome abnormalities in tuberous sclerosis

Summary In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.     

Four new cases of dicentric Y chromosomes

Summary Dicentric Y chromosomes are rare in man. Four new cases of dicentric Y chromosomes are described. The cases of the literature so far reported are reviewed. Among the cases, a wide range of variation in phenotype, external genitalia, histology, and chromosomal findings was observed. The relationship of the clinical picture and structural abnormalities of the Y chromosomes is discussed.