Liability to chromosome damage in lymphocytes of "cancer family" subjects: a study of spontaneous and induced chromosomal fragility

Spontaneous chromosomal fragility was detected in seven tumor patients and one healthy member from two families with a high recurrence of cancer. Major chromosome lesions, such as terminal deletions and rearranged chromosomes, were found at levels significantly higher than those reported for control individuals. The prevalence of these aberrations in comparison to minor ones (chromosome gaps and chromatid breaks) in this group of patients seems to indicate that the fragility observed is the end-point of a process of chromosomal instability, which may have already been brought to expression. Study of other parameters of genetic instability in the most unstable karyotypes showed that the chromosome damage observed was neither paralleled by abnormal SCE frequency nor sustained by defective DNA repair mechanisms or expression of inherited or constitutional fragile sites. As all the subjects investigated here had previously been shown to display intraindividual variations in the C-banded region of chromosome 1, it is possible that spontaneous fragility and acquired C-heterochromatin polymorphism may be markers that, combined with chromosomal instability, create genetic predisposition to cancer.     

The constitutional fragility of chromosome 12 in a case of 46,XX,var(12)(qh′,RHG,GAG,CBG)

Summary The constitutional fragility of chromosome no. 12 in a female infant with unspecific clinical signs is described. RHG, GAG, and CBG methods were used to localize the fragile point. The breaks seem to be in 12q1.3, and always within an R band. A possible correlation between the phenotypic modifications and the chromosome variant is discussed.     

Evidence for high frequency of chromosomal mosaicism in spontaneous abortions revealed by interphase FISH analysis.

Numerical chromosomal imbalances are a common feature of spontaneous abortions. However, the incidence of mosaic forms of chromosomal abnormalities has not been evaluated. We have applied interphase multicolor fluorescence in situ hybridization using original DNA probes for chromosomes 1, 9, 13, 14, 15, 16, 18, 21, 22, X, and Y to study chromosomal abnormalities in 148 specimens of spontaneous abortions. We have detected chromosomal abnormalities in 89/148 (60.1%) of specimens. Among them, aneuploidy was detected in 74 samples (83.1%). In the remaining samples, polyploidy was detected. The mosaic forms of chromosome abnormality, including autosomal and sex chromosomal aneuploidies and polyploidy (31 and 12 cases, respectively), were observed in 43/89 (48.3%) of specimens. The most frequent mosaic form of aneuploidy was related to chromosome X (19 cases). The frequency of mosaic forms of chromosomal abnormalities in samples with male chromosomal complement was 50% (16/32 chromosomally abnormal), and in samples with female chromosomal complement, it was 47.4% (27/57 chromosomally abnormal). The present study demonstrates that the postzygotic or mitotic errors leading to chromosomal mosaicism in spontaneous abortions are more frequent than previously suspected. Chromosomal mosaicism may contribute significantly to both pregnancy complications and spontaneous fetal loss.     

A nine-year cytogenetic follow-up of a patient injected with thorotrast

Summary Chromosomal abnormalities in the peripheral lymphocytes of a Thorotrast case were followed up for almost nine years during which four direct bone marrow observations were made. Chromosomal abnormalities, both Cu type (dicentrics, rings, and acentric fragments) and Cs type (reciprocal translocations, inversions, deletions, duplications, and others), were observed with an average frequency of about 7.5% and 8%, respectively. The frequency of chromosomal abnormalities showed no significant changes during the nine years. Formation of clones of cells with identical chromosomal abnormalities was observed both in bone marrow and peripheral lymphocytes. There were clones common to myeloid and lymphoid cell series, which may be regarded as evidence for the presence of pluripotent stem cells in man. One such common clone had a translocation involving chromosomes 2 and 22. Banding analysis revealed a break in chromosome 22 at the q12 or q12/13 band interface. The frequency of the clone remained fairly constant within 5% for several years and the patient showed no indication of leukemia or any other blood disease. The finding seems to suggest a genetic factor relating to the development of chronic myelocytic leukemia (CML), which generally shows the Ph¹ chromosome resulting from a translocation of chromosome 22 with the break at the q11 or q11/12 band interface. The increase in the number of cells lacking the Y chromosome was observed in our final bone marrow sample, but the phenomenon may be attributed to the age of the patient rather than to the direct effect of radiation. Results of the cytogenetic follow-up study seem to indicate the importance of studies in this direction for a better understanding of radiation effects on human beings.     

A detailed analysis of chromosomal changes in heritable and non-heritable retinoblastoma

Summary Full cytogenetic analysis of 27 different retinoblastoma tumors is presented. Gross aneuploidy of chromosome arms 6p and 1q were very common, being observed in 15/27 and 21/27 tumors, respectively. However, we found that chromosome 13 was rarely missing: only 3/27 had a detectable monosomy affecting 13q14. Monosomy of chromosome 13 by small deletion or rearrangement was also not observed in any of 12 retinoblastoma tumor lines analyzed detail at the 300–400 chromosome band level. A novel observation in retinoblastoma was the discovery of non-random translocations at three specific breakpoints, 14q32 (4/12), 17p12 (5/12), and 10q25 (3/12). Genomic rearrangements similar to those described involving C-myc in Burkitt lymphoma 14q+ cells could not be demonstrated in the four 14q+ retinoblastoma lines using molecular techniques, and a probe mapping to the site implicated to have an activating role in lymphoma. These data suggest that there is a target for rearrangement at 14q32 but it is not the same sequence used in some Burkitt lymphomas. Two other breakpoints (2p24 and 8q24) coincided with the mapped position of cellular oncogenes, but also failed to show a molecular rearrangement with the oncogene probes. The breakpoints, 10q25 and 17p12, are constitutional fragile sites which may predispose these regions to act as acceptors of translocations in malignant cells. One line had double minute chromosomes, and was the only one of 16 (6%) tested with the N-myc probe which had an amplification. Different tumors from single patients with multifocal heritable retinoblastoma showed independent karyotype evolution. Unilateral non-heritable tumors exhibited a high level of karyotype stability throughout both in vivo and in vitro growth. The various common patterns of aneuploidy and translocations probably confer an early selective advantage to malignant cells, rather than induce malignant transformation.     

Relationship between chromosome fragility,aneuploidy and severity of the haematological disease in Fanconi anaemia

Fanconi anemia (FA) is a chromosome instability syndrome, characterized by progressive pancytopenia and cancer susceptibility. Other cellular features of FA cells are hypersensitivity to DNA cross-linking agents and accelerated telomere shortening. We have quantified overall genome chromosome fragility and euploidy as well as chromosomes 7 and 8 aneuploidy in peripheral blood lymphocytes from a group of FA patients and age-matched controls that were previously measured for telomere length. The haematology of FA samples were also characterized in terms of whole blood cell, neuthrophil and platelet counts, transfusion dependency, requirement of androgens, cortico-steroids or bone marrow transplantation, and the development of bone marrow clonal cytogenetic abnormalities, myelodysplastic syndrome or acute myeloid leukemia. As expected, a high frequency of spontaneous chromosome breaks was observed in FA patients, especially of chromatid-type. No differences in chromosomes 7 and 8 monosomy, polysomy and non-disjunction were detected between FA patients and controls. The same was true for overall genome haploidy or polyploidy. Interestingly, the spontaneous levels of chromosome fragility but not of numerical abnormalities were correlated to the severity of the haematological disease in FA. None of the variables included in the present investigation (chromosome fragility, chromosome numerical abnormalities and haematological status) were correlated to telomere length.     

Expression of fragile sites in human sperm and lymphocyte chromosomes

Summary Sperm and lymphocyte chromosome studies in a normal, fertile male have shown a high degree of coincidence between chromosome lesions and fragile sites in both types of cells. In this donor we also found that some fragile sites expressed in sperm chromosomes coincided with those expressed in lymphocyte chromosomes. These results indicate that the chromosome lesions expressed in sperm do not occur at random and that they are not technical artifacts. The fragility expression in sperm chromosomes could reflect in vivo conditions. The presence in some sperm metaphases of acentric fragments suggests that chromosome fragility can result in the loss of chromosome fragments or give rise to de novo structural rearrangements. However, the incidence of sperm with chromosomal abnormalities observed in this man was within the normal range.     

Pericentric inversions in man: personal experience and review of the literature

Summary The Leuven cytogenetic centre experience on pericentric inversion in man is discussed with exclusion of the pericentric inversions of the heterochromatic blocks of chromosomes 1 and 9. In a total of 51,500 patients, referred for constitutional chromosome analysis during the period 1970–1985, pericentric inversions were found in 24 index patients. The breakpoints detected in these different pericentric inversions are summarized and compared to those found in previous reports. Bands 2p13, 2q21, 5q31, 6c21, 10q22, and 12q13 were shown to be repeatedly involved in the different studies and, furthermore, breakpoints at bands 2q11, 5p13, 5p15, 5q13, 7q11, 11q25, and 14p11 were present in this study as well as in our previous review on reciprocal autosomal translocations. In 13 familial pericentric inversions, even after exclusion of all inversion carrier probands, a 1.6:1 excess of pericentric inversion carriers versus karyotypically normal progeny was observed. While chromosomally unbalanced offspring represent 3.5% of all chromosomally investigated liveborns of the present study, 7.1% of all liveborn inversion carrier offspring presented with a mental retardation and/or multiple congenital anomalies (MR/MCA) problem. Additional chromosomal abnormalities, i.e. a 21 trisomy and an accessory small ring chromosome were observed in two pericentric inversion carriers. These data and results are discussed and compared to the data available in the literature.     

Cytogenetic investigations in a family with ataxia telangiectasia

Summary Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normla control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7–11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.     

Chromosomal mosaicism in spontaneous abortions: Analysis of 650 cases

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase MFISH and DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy. Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (∼ 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.     

Constitutional chromosome instability and cancer risk

Acquired, clonal chromosome abnormalities are thought to be of pathogenetic importance in human cancer; at the cellular level, neoplasia is best viewed as a genetic disease. It is therefore logical to suggest that cancer risk must somehow be related to individual variations in genomic stability. Those persons whose chromosomes are less stable will, on average, be the ones who are most likely to develop cancer. The testing of this hypothesis shows that, apart from the autosomal recessive chromosome breakage syndromes, only patients with adenomatosis of the colon and rectum have, consistently and by different groups, been found to display elevated spontaneous and clastogen-induced chromosome breakage frequencies. Some evidence indicates a similar tendency in patients with dysplastic nevus syndrome, basal cell carcinoma, cervix cancer, and Kaposi's sarcoma. For several other cancers the data strongly argue against any inherent genomic instability. Although most results thus fail to support constitutional chromosome fragility as a factor of importance in tumorigenesis, conclusive falsification of the hypothesis cannot be said to have been obtained. The possibility remains that variations in chromosome stability and clastogen sensitivity between different cell types, and also difficulties in selecting the most appropriate carcinogens in clastogen-exposure tests, may have masked systematic constitutional differences between patients and controls in the breakage assays.     

Coincidence between fragile site expression and interstitial deletion of chromosome 11 in a case of myelofibrosis

Summary Cytogenetic examination of multiple peripheral blood cultures of a patient with myelofibrosis with myeloid metaplasia revealed the presence of an interstitial deletion of the long arm of chromosome 11, del(11)(q13q21). A folic acid dependent fragile site fra(11)(q13) was found in about 12% of the cells. The possible correlation between constitutional fragile site and acquired chromosomal alteration is discussed briefly.     

inv(9)(p24q13) in three sterile brothers

Only nine non-polymorphic constitutional pericentric inversions of chromosome 9 have been described. We report on a familial inv(9)(p24q13) associated with sterility in three brothers. The mother's chromosomes were normal in blood lymphocytes (n=130); the father was already deceased and his karyotype unknown. However, the presence of any of the maternal chromosomes 9 (as assessed by C-banding) in her carrier children is inconsistent with the assumption of maternal mosaicism. Two single sisters were also carriers. The same rearranged chromosome 9 in the three sterile brothers can hardly be regarded as a fortuitous association, especially when the breakpoints are almost identical to those of the sole inversion previously found in an azoospermic male. If their father was a carrier, the observed sterility may be the result of 'chromosome anticipation', a phenomenon already invoked for certain familial chromosomal rearrangements.     

Assessment of the genotoxicity of atenolol in human peripheral blood lymphocytes: Correlation between chromosomal fragility and content of micronuclei

The antihypertensive drug atenolol was found to induce chromosome loss, detected as micronuclei in the peripheral lymphocytes of treated patients. The fundamental question which chromosomes the micronuclei were derived from remains to be answered. Analysis of structural chromosomal aberrations (CAs) and expression of fragile sites (FS) were pursued in this study. They revealed a significantly higher incidence of chromosomal aberrations (chromatid and chromosome breaks) in patients compared with controls, where 10 FS emerged as specific. Also, the band 17q12–21, where known fragile sites have not been reported, was only expressed in atenolol-treated patients. Fluorescence in situ hybridization using chromosome-specific probes revealed the preferential involvement of chromosomes 7, 11, 17 and X in the micronuclei (MN) of patients. The results also suggest a correlation between chromosomal fragility and content of MN, and support the findings for a linkage between hypertension and a locus on chromosome 17.     

Oculo-auriculo-vertebral (Goldenhar) spectrum associated with pericentric inversion 9: coincidental findings or etiologic factor?

Oculo-auriculo-vertebral (OAV) spectrum or Goldenhar syndrome is a complex and heterogeneous condition characterized by hemifacial microsomia (unilateral ear abnormalities and ipsilateral mandibular hypoplasia) as well as vertebral anomalies and epibulbar dermoids or lipodermoids. Although most cases of OAV spectrum are sporadic, both autosomal recessive and autosomal dominant inheritance have been reported. Furthermore, the association of OAV spectrum with different types of chromosomal abnormalities has been described. We present a premature newborn delivered after 36 weeks of gestation, whose birth weight was 2,100 g, birth length 43 cm and head circumference 32.5 cm. OAV spectrum with associated axial skeleton anomalies, eventration of the right hemidiaphragm, accessory spleen, unlobulated right lung, agenesis of right kidney, right ovary and right uterine horn, and partial agenesis of corpus callosum were found. She was the second child of unrelated parents, who have a healthy boy. Both parents refused chromosomal analysis of their peripheral blood. Trypsin G-banding and C-banding chromosomal analysis of the patient's peripheral blood revealed a pericentric inversion of chromosome 9 with break points at p11 and q13. This may be a coincidental or causal finding.     

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation

Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.     

Multicolor FISH analysis of chromosomal breaks, duplications, deletions, and numerical abnormalities in the sperm of healthy men

Transmitted de novo structural chromosomal abnormalities, the majority of which are paternally derived, can lead to abnormal reproductive outcomes as well as genetic diseases in offspring. We developed and validated a new multicolor FISH procedure (sperm ACM, which utilizes DNA probes specific for the alpha [1cen], classical, [1q12], and midi [1p36.3] satellites of chromosome 1) which utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical abnormalities plus two categories of structural aberrations: (1) duplications and deletions of 1pter and 1cen, and (2) chromosomal breaks within the 1cen-1q12 region. In healthy men, the average frequencies of sperm with duplications and deletions were (a) 4.5 +/- 0.5 and 4.1 +/- 1.3 per 10(4) involving 1pter and (b) 0.9 +/- 0.4 and 0.8 +/- 0.3 per 10(4) involving 1cen, respectively. The frequency of sperm exhibiting breaks within the 1cen-1q12 region was 14.1 +/- 1.2 per 10(4). Structural aberrations accounted for 71% of the abnormalities detected by sperm ACM, which was significantly higher than numerical abnormalities (P=2x10-8). Our findings also suggest that, for healthy men, (a) sperm carrying postmeiotic chromosomal breaks appear to be more prevalent than those carrying products of premeiotic or meiotic breakage or rearrangements, (b) the high frequency of chromosome breaks measured after "fertilization" by the hamster-egg cytogenetic method already appear to be present and detectable within human sperm by FISH, and (c) there are nonrandom and donor-specific distributions of breakpoint locations within 1q12 in sperm. FISH facilitates the analysis of much larger numbers of sperm than was possible when the hamster-egg method was used. Therefore, FISH-based procedures for simultaneously detecting chromosomal breaks, rearrangements, and numerical abnormalities in sperm may have widespread applications in human genetics, genetic toxicology, and reproductive medicine.     

Chromosomal restructurings and the distribution of chromosome fragile sites in the peripheral blood lymphocytes in a breast cancer patient after cytostatic therapy]

Cytogenetic analysis was performed repeatedly on a breast cancer patient since the beginning of the antitumor treatment. Double minute chromosomes (DMS, 2-10 per cell) were found in less than 2% of peripheral blood lymphocytes besides other chromosomal abnormalities after radiation therapy and 8 months after chemotherapy. The level of structural chromosomal aberrations two years after the therapeutic treatment was 0.13-0.14 aberrations per cell, but DMS were not observed. Estimation of the fragile site (FS) frequency and distribution at this time revealed a significant expression of the common FS FRAGF (9q1.2) after the treatment of blood culture with 5-bromo-2-deoxyuridine at dose levels of 7 and 50 g/l and enhanced fragility in chromosome band 1p35-36.1 (FRA1A) in folate-deprived conditions. Rare FS were not found. The presented data are discussed.     

Gene order, amplification, and rearrangement of chromosome band 11q23 in hematologic malignancies

Eleven genes were found to be amplified in a patient with acute myelogenous leukemia and a homogeneous staining region 11q23qter. The gene order of such region was determined by using transverse alternating field electrophoresis of normal cell DNA and Southern blots of DNA from somatic cell hybrids, each containing a single human derivative chromosome 11 from six different chromosomal defects. This in turn allowed us to uncover a breakpoint in band 11q23.3 between the CD3 gamma and the ets-1 genes in genomic rearrangements found in acute myelogenous leukemia, acute lymphocytic leukemia, and B-cell diffuse lymphoma. The breakpoint of a constitutional deletion from a patient whose mother and brother have a heritable 11q23.3 fragile site occurs in the same region.     

A complex chromosomal rearrangement with a translocation 4;10;14 in a fertile male carrier: ascertainment through an offspring with partial trisomy 14q13-->q24.1 and partial monosomy 4q27-->q28 [corrected

Complex chromosomal rearrangements (CCRs) are usually associated with infertility or subfertility in male carriers. If fertility is maintained, there is a high risk of abnormal pregnancy outcome. Few male carriers have been identified by children presenting with mental retardation/congenital malformations (MR/CM) or by spontaneous abortions of the spouses. We report a de novo CCR with five breakpoints involving chromosomes 4, 10 and 14 in a male carrier who was ascertained through a son presenting with MR/CM due to an unbalanced karyotype with partial trisomy 14 and partial monosomy 4. The child has a healthy elder brother. In the family history no abortions were reported. No fertility treatment was necessary. Cytogenetic analysis from the affected son showed a reciprocal translocation t(4;10) with additional chromosomal material inserted between the translocation junctions in the derivative chromosome 10. The father showed the same derivative chromosome 10 but had additionally one aberrant chromosome 14. Further molecular cytogenetic analyses determined the inserted material in the aberrant chromosome 10 as derived from chromosome 14 and revealed a small translocation with material of chromosome 4 inserted into the derivative chromosome 14. Thus the phenotype of the son is supposed to be associated with a partial duplication 14q13-->q24.1 and a partial monosomy 4q27-->q28. Including our case we are aware of eleven CCR cases with fertile male carriers. In eight of these families normal offspring have been reported. We propose that exceptional CCRs in fertile male carriers might form comparatively simple pachytene configurations increasing the chance of healthy offspring.