Evidence for structural heterogeneity from molecular cytogenetic analysis of dicentric Robertsonian translocations.

Most Robertsonian translocations are dicentric, suggesting that the location of chromosomal breaks leading to their formation occur in the acrocentric short arm. Previous cytogenetic and molecular cytogenetic studies have shown that few Robertsonian translocations retain ribosomal genes or beta-satellite DNA. Breakpoints in satellite III DNA, specifically between two chromosome 14-specific subfamilies, pTRS-47 and pTRS-63, have been indicated for most of the dicentric 14q21q and 13q14q translocations that have been studied. We have analyzed the structure of 36 dicentric translocations, using several repetitive DNA probes that localize to the acrocentric short arm. The majority of the translocations retained satellite III DNA, while others proved variable in structure. Of 10 14q21q translocations analyzed, satellite III DNA was undetected in 1; 6 retained one satellite III DNA subfamily, pTRS-47; and 3 appeared to contain two 14-specific satellite III DNA sub-families, pTRS-47 and pTRS-63. In 10/11 translocations involving chromosome 15, the presence of satellite III DNA was observed. Our results show that various regions of the acrocentric short arm, and, particularly, satellite III DNA sequences, are involved in the formation of Robertsonian translocations.     

Dicentric Robertsonian translocations in man

Summary The authors studies 17 cases of Robertsonian translocations. In all cases but one C banding showed that a dicentric translocation was involved. Silver staining demonstrated the presence of an NOR between the two centromeres in only one case.     

Molecular cytogenetic evidence for a common breakpoint in the largest inverted duplications of chromosome 15.

Chromosomes from 20 patients were used to delineate the breakpoints of inverted duplications of chromosome 15 (inv dup[15]) that include the Prader-Willi syndrome/Angelman syndrome (PWS/AS) chromosomal region (15q11-q13). YAC and cosmid clones from 15q11-q14 were used for FISH analysis, to detect the presence or absence of material on each inv dup(15). We describe two types of inv dup(15): those that break between D15S12 and D15S24, near the distal boundary of the PWS/AS chromosomal region, and those that share a breakpoint immediately proximal to D15S1010. Among the latter group, no breakpoint heterogeneity could be detected with the available probes, and one YAC (810f11) showed a reduced signal on each inv dup(15), compared with that on normal chromosomes 15. The lack of breakpoint heterogeneity may be the result of a U-type exchange involving particular sequences on either homologous chromosomes or sister chromatids. Parent-of-origin studies revealed that, in all the cases analyzed, the inv dup(15) was maternal in origin.     

Dicentric and monocentric Robertsonian translocations in man

Summary 5 balanced Robertsonian translocations in man were identified by fluorescence studies. Orcein staining showed two distinct centromeres in 4 of these cases (tdic(13;13), tdic(13;14), tdic(15;21), tdic(21;22)) indicating breaks in the short arms of the involved chromosomes. The dicentric translocation chromosomes were rather stable but monocentrics were noticed in each case. Fluorescence- and measurement studies seemed to indicate that an invisible centromere and part of the short arms were present in these monocentric chromosomes. One case, t(14q21q), was monocentric in all metaphase plates but measurement studies were very suggestive of a visible 21 centromere and incorporation of the invisible 14 centromere (and short arm material) in the long arm of the translocated 14 chromosome, indicating that this translocation originally might have been a real dicentric. Heterochromatin staining was carried out in all cases. The tdic(15;21) showed 6 heterochromatin blocks; 2 of these blocks were probably satellites from chromosome No. 21, visible too in fluorescence. The 4 other translocations showed 4 separated blocks. No differences were observed between monocentrics and dicentrics supporting the theory of a preserved, but invisible centromere in monocentrics.

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Zusammenfassung 5 balancierte humane Translokationen vom Robertson-Typ wurden durch Fluorescenzuntersuchungen identifiziert. Die Orceinfärbung zeigte in 4 dieser Fälle 2 distinkte Zentromere (tdic(13;13), tdic(13;14), tdic(15;21), tdic(21;22)). Dieser Fund ließ es als wahrscheinlich erscheinen, daß der Bruch am kurzen Arm der involvierten Chromosomen stattgefunden hatte. Die dizentrischen Translokationschromosomen waren verhältnismäßig stabil. Es wurden doch monozentrische Chromosomen in allen Fällen beobachtet. Eine Kombination von Fluorescenzuntersuchung und Messung der Chromosomen machte es wahrscheinlich, daß auch in diesen monozentrischen Chromosomen ein unsichtbares Zentromer und Teile der kurzen Arme vorhanden sind. Eine (14q21q)-Translokation hatte in allen Metaphasen nur ein Zentromer. Messungen jedoch deuteten an, daß das 21-Zentromer sichtbar war, daß aber das 14-Zentromer und Material der kurzen Arme am langen Arm des translozierten 14-Chromosoms inkorporiert waren. Das Translokationschromosom ist möglicherweise ursprünglich ein dizentrisches Chromosom gewesen. In allen Fällen wurde eine Heterochromatinfärbung ausgeführt. Die tdic(15;21) hatte 6 Heterochromatinblöcke. 2 davon waren wahrscheinlich die Satelliten des Chromosoms Nr. 21, die auch bei der Fluorescenzmikroskopie sichtbar waren. Die 4 anderen Translokationen hatten 4 separate Blöcke. Monozentrische und dizentrische Chromosomen zeigten hier keinen Unterschied, was die Theorie unterstützt, daß die Zentromere im monozentrischen Chromosomen erhalten, aber unsichtbar sind.

     

Common chromatin structures at breakpoint cluster regions may lead to chromosomal translocations found in chronic and acute leukemias

The t(9;22) BCR/ABL fusion is associated with over 90% of chronic myelogenous and 25% of acute lymphocytic leukemia. Chromosome 11q23 translocations in acute myeloid and lymphoid leukemia cells demonstrate myeloid lymphoid leukemia (MLL) fusions with over 40 gene partners, like AF9 and AF4 on chromosomes 9 and 4, respectively. Therapy-related leukemia is associated with the above gene rearrangements following the treatment with topoisomerase II (topo II) inhibitors. BCR, ABL, MLL, AF9 and AF4 have defined patient breakpoint cluster regions. Chromatin structural elements including topo II and DNase I cleavage sites and scaffold attachment sites have previously been shown to closely associate with the MLL and AF9 breakpoint cluster regions, implicating these elements in non-homologous recombination (NHR). In this report, using cell lines and primary cells, chromatin structural elements were analyzed in BCR, ABL and AF4 and, for comparison, in MLL2, which is a homolog to MLL, but not associated with chromosome translocations. Topo II and DNase I cleavage sites associated with all breakpoint cluster regions, whereas SARs associated with ABL and AF4, but not with BCR. No close breakpoint clustering with the topo II/DNase I sites were observed; however, a statistically significant 5′ or 3′ distribution of patient breakpoints to the topo II DNase I sites was found, implicating DNA repair and exonucleases. Although MLL2 was expressed in all cell lines tested, except for the presence of one DNAse I site in the promoter, no other structural elements were found in MLL2. A NHR model presented demonstrates the importance of chromatin structure in chromosome translocations involved with leukemia.     

Molecular evidence of Y-autosomal translocations in owl monkeys

Probe pDP1007, which contains highly conserved DNA sequences from the sex-determining region of the human Y chromosome, cross-hybridized with owl monkey EcoRI restriction fragments of 1.8 kb and 6.6 kb. Southern transfer analysis of owl monkey (karyotype VI)--rodent somatic cell hybrids localized the 1.8-kb fragment on the owl monkey X chromosome and the 6.6-kb fragment, which is male specific, on chromosome 14/Y. Regional in situ chromosome mapping of pDP1007 revealed specific sites of hybridization: the distal short arm of the X chromosome of karyotypes IV, VI, and VII; the small metacentric Y of karyotype IV; the C-band positive region on the short arm of chromosome 17/Y (karyotype VII); and the C-band positive region on the long arm of chromosome 14/Y (karyotype VI). These molecular findings reinforce cytological evidence that Y-chromosomal material has been transferred to autosomes 14 and 17 in owl monkeys of karyotypes VI and VII, respectively, in which there are no independently segregating Y chromosomes.     

Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer.     

Identification of DNA sequences flanking the breakpoint of human t(14q21q) Robertsonian translocations.

We have employed molecular probes and in situ hybridization to investigate the DNA sequences flanking the breakpoint of a group of t(14q21q) Robertsonian translocations. In all the families studied, the probands were patients with Down syndrome who carried a de novo t(14q21q) translocation. The DNA probes used were two alphoid sequences, alphaRI and alphaXT, which are specific for the centromeres of chromosomes 13 and 21 and of chromosomes 14 and 22, respectively; a satellite III sequence, pTRS-47, which is specific for the proximal p11 region of chromosomes 14 and 22; and a newly defined satellite III DNA, pTRS-63, which is specific for the distal p11 region of chromosome 14. The two alphoid probes detected approximately the same amount of autoradiographic signal on the translocated chromosomes as was expected for chromosomes 14 and 21 of the originating parent, suggesting that there has been no loss of these centromeric sequences during the translocation events. Results with the two satellite III probes indicated that the domain corresponding to pTRS-47 was retained in the translocated chromosomes, whereas the domain for pTRS-63 was lost. These results have allowed us to place the translocation breakpoint between the pTRS-47 and pTRS-63 domains within the p11 region of chromosome 14.     

Gamete segregation in female carriers of Robertsonian translocations

Eleven female carriers of either 45,XX,der(13;14) (q10;q10) or 45,XX, der(14;21)(q10;q10) underwent hormonal stimulation with the purpose of producing enough oocytes for in-vitro fertilization and preimplantation genetic diagnosis. Polar body biopsy was performed in those oocytes and FISH with painting probes was applied in their metaphase-like first polar body chromosomes. In this way, unbalanced, normal and balanced oocytes could be distinguished and segregation modes ascertained. der(14;21)(q10;q10) produced 42% unbalanced, 37% normal and 21% balanced oocytes (n = 86) while der(13;14)(q10;q10) generated 33% unbalanced, 51% normal and 16% balanced oocytes (n = 69). In both translocations the number of normal oocytes was significantly higher than the number of balanced oocytes. However, while the frequency of unbalanced events involving chromosome 13 and 14 was similar in der(13;14)(q10;q10), there were significantly more abnormalities involving chromosome 21 than 14 in the der(14;21) (q10;q10) cases. When comparing survival rates to term, trisomies from Robertsonian origin seem to survive more often than those originated by non-disjunction in non-translocation carriers. The meiotic segregation patterns found in female Robertsonian translocations are different from those described in male carriers, with higher rates of unbalanced gametes in females than in males.     

Molecular cytogenetic characterization of 17 rob(13q14q) Robertsonian translocations by FISH, narrowing the region containing the breakpoints.

We have characterized 17 rob(13q14q) Robertsonian translocations, using six molecular probes that hybridize to the repetitive sequences of the centromeric and shortarm regions of the five acrocentric chromosomes by FISH. The rearrangements include six de novo rearrangements and the chromosomally normal parents, five maternally and three paternally inherited translocations, and three translocations of unknown origin. The D21Z1/D13Z1 and D14Z1/D22Z1 centromeric alpha-satellite DNA probes showed all rob(13q14q) chromosomes to be dicentric. The rDNA probes did not show hybridization on any of the 17 cases studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 was retained around the breakpoints in all cases. However, the pTRS-63 satellite III DNA probe specific for chromosome 14 did not show any signals on the translocation chromosomes examined. In 16 of 17 translocations studied, strong hybridization signals on the translocations were detected with the pTRI-6 satellite I DNA probe specific for chromosome 13. All parents of the six de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong positive hybridization signals on each pair of chromosomes 14 and 13, with pTRS-47, pTRS-63, and pTRI-6. Therefore, the translocation breakpoints in the majority of rob(13q14q) are between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13.     

Two new X-autosome Robertsonian translocations in the mouse

The influence of X-autosome Robertsonian (Rb) translocation hemizygosity on meiotic chromosome behaviour was investigated in male mice. Two male fertile translocations [Rb(X.2)2Ad and Rb(X.9)6H] and a male sterile translocation [Rb(X.12)7H] were used. In males of all three Rb translocation types, the acrocentric homologue of the autosome involved in the rearrangement regularly failed at pachytene to pair completely with its partner in the Rb metacentric. The centric end of the acrocentric autosome was found regularly to associate either with the proximal end of the Y chromosome or with the ends of nonhomologous autosomal bivalents; the proportions of cells with such configurations varied between pachytene substages and genotypes. Various other categories of synaptic anomaly, such as nonhomologous synapsis, foldback pairing and interlocks, affected the sex chromosome multivalent in a substantial proportion of cells. In one of the Rb(X.12)7H males screened, an unusual, highly aneuploid spermatocyte that contained trivalent and bivalent configurations was found. Rb translocation hemizygosity did not appear to increase to a significant extent the incidence of X-Y pairing failure at pachytene, although the incidence was elevated at metaphase I in Rb(X.12)7H animals. Overall, a comparison of the frequencies and types of chromosome pairing anomalies did not suggest that these were important factors in the aetiology of infertility in males carrying the Rb(X.12)7H translocation.     

Analysis of translocations observed in three different populations. II. Robertsonian translocations

A sample of 229 Robertsonian translocations was classified into three groups according to the method of their ascertainment (Group I = couples with repeated abortions; Group II = karyotypically unbalanced probands; Group III = balanced translocation heterozygotes). Statistical analysis showed that the distributions of Robertsonian translocations differed significantly from random in all three groups. Additionally, the distributions were significantly different between couples with repeated abortions and karyotypically unbalanced probands and between unbalanced probands and balanced translocation heterozygotes.     

Homozygous Robertsonian translocations in a fetus with 44 chromosomes

Summary We report the unique finding of a human fetus with 44 chromosomes with homozygous 14;21 translocations. This fetus appeared phenotypically normal but the long-term neurodevelopmental outcome had this pregnancy continued could not be predicted. We speculate one 14;21 translocation was inherited from her father and one arose de novo being maternal in origin. A previous sibling with psychomotor retardation has an abnormal chromosome complement of 45,XX,dup(7)(q21pter), t(14;21)(p11;q11). The mother's underlying disease, systemic lupus erythematosis (SLE), and her prior chemotherapy may have contributed to the appearance of these chromosome aberrations. It is interesting that although 14;21 translocations are among the commonest structural chromosome rearrangements in man, there are no previous reports in newborn surveys of a child with 44 chromosomes resulting from the mating of two identical Robertsonian translocation carrier parents.     

Kinetics of oogenesis in mice heterozygous for Robertsonian translocations

The total number of oocytes at different postmating time intervals (18-40 days) was determined in mice homozygous and heterozygous for different Robertsonian (Rb) translocations, of both laboratory and feral origin. The number of oocytes was lower in heterozygous than in homozygous mice throughout the period studied. Independently of the genetic background (i.e. laboratory or feral), structural heterozygosity had a progressive detrimental effect on oocyte numbers: open, or chain diakinetic configurations had a greater detrimental effect than close, or ring, configurations. The genetic background, however, affected the ovarian constitution in terms of the total number of germ cells, which are more numerous in laboratory than in feral mice. The kinetics of oogenesis seems to be faster in feral than in laboratory mice. At the light of the data here presented, and of those already available from the literature on male and female gametogenesis in conditions of structural heterozygosity, it appears that factors other than unsaturation of pairing sites or interference with pachytene X-chromosome inactivation have to be considered. In the wild, the reduced oocyte numbers in Rb heterozygous female can contribute to the retention of isolated populations in contact zones.     

Random acrocentric bivalent associations in human pachytene spermatocytes. Molecular implications in the occurrence of Robertsonian translocations

Acrocentric bivalent associations were studied in 232 human male germ cells at pachytene in order to understand better the preferential involvement of chromosomes 13, 14, and 21 in Robertsonian translocations. The tendency of each acrocentric bivalent to associate with another was not correlated with NOR activity, as measured by silver staining. Good agreement was noticed between their ability to associate and the amount of satellite DNA in human acrocentric chromosomes. The distribution of two-by-two acrocentric bivalent associations was random. In order to reconcile this result with the nonrandom distribution of Robertsonian translocations, a molecular hypothesis is proposed. The model is based on homology of recombinational sites, interspersed at regular interval in satellite DNA, which could increase the probability of accidental unequal crossing-over between two specific acrocentric chromosomes.