A precise interchromosomal reciprocal exchange between hot spots for cleavable complex formation by topoisomerase II in amsacrine-treated Chinese hamster ovary cells.

Among the aprt mutations induced in confluence-arrested Chinese hamster ovary D422 cells by the topoisomerase II poison amsacrine, there was a reciprocal exchange between the aprt gene and an unrelated sequence, accompanied by a chromosomal translocation at the aprt locus. The breakpoints in both parental sequences were hot spots for amsacrine-stimulated DNA cleavage in vitro, and the novel junctions formed were precisely as expected for a mechanism involving reciprocal exchange of topoisomerase II subunits followed by resealing of the breaks and correction of mismatches in the cohesive ends. The results are consistent with a role for direct subunit exchange in the production of chromosomal translocations by topoisomerase poisons, although more complex models involving double-strand breakage and repair could produce reciprocal exchanges of similar specificity.

p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells.

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.

De novo inverted tandem duplication of the short arm of chromosome 12 in a patient with microblepharon.

We present a patient with a de novo inverted duplication of nearly the entire short arm of chromosome 12 [inv dup(12)(p13.3p12)], which was characterized using GTG-banding and spectral karyotyping. The patient was noted to have microblepharon, which has not been previously described in children with a similar chromosomal rearrangement. This patient represents one of the few examples of complete and pure trisomy 12p due to inverted duplication of the short arm of chromosome 12 and expands the clinical spectrum.

Heritability and heteromorphic distributions of AluI chromosome banding variants in twins.

The heritability and heteromorphic appearance of chromosomal banding patterns induced through in situ digestion with the restriction enzyme AluI were studied by analyzing the chromosomes of 25 monozygotic and 25 dizygotic twin pairs selected at random from a juvenile twin registry. A total of 19 AluI banding variants were found to be heteromorphic, with the pericentromeric region of chromosome 3 and the satellites of chromosome 22 being most and least heteromorphic, respectively. As expected, the correlations of the semi-quantitative scores for each of the chromosomal variants were significantly higher between MZ twin pairs (ranging from 0.48 to 0.95) than DZ twin pairs (ranging from -0.02 to 0.69), suggesting that genetic factors play an important role in their appearance. This finding was confirmed in a model fitting analysis in which the heritabilities of the AluI-induced chromosome variants were found to range from 70 to 96% for 12/13 heteromorphisms studied. These consistent findings are significant in that these variants may be useful for family studies in clinical genetics.

Parental origin determination in thirty de novo Robertsonian translocations.

Cytogenetic heteromorphisms and restriction fragment length polymorphisms were used to assign the parental origins of 30 de novo non-homologous Robertsonian translocations. The balanced and unbalanced translocations studied included 20 rob(14q21q) four rob(13q14q)four rob(15q21q) one rob(13q15q), and one rob(13q21q). Significantly more maternally (26/30) than paternally (4/30) derived de novo translocations were noted and all rob(14q21q) ascertained through unbalanced probands (20/20) were maternal in origin. Interestingly, 12/13 probands who were trisomic and informative for proximal chromosome 21q loci were homozygous for the markers tested. Segregation (2:1) of the Robertsonian translocation into one daughter cell in meiosis I and subsequent failure of the chromosome 21 chromatids to separate in meiosis II may account for our observation of homozygosity for proximal chromosome 21 loci in the majority of de novo rearrangements tested.

Further characterization of 19 cases of rea(21q21q) and delineation as isochromosomes or Robertsonian translocations in Down syndrome.

We have used 9 conventional RFLPs and 6 dinucleotide repeat polymorphisms on chromosome 21q to demonstrate that 17 of 19 cases of rea(21q21q) were consistent with isochromosomes i(21q) with the remaining 2 being true Robertsonian translocations. Eight of the 17 isochromosomes were of maternal origin and 9 cases were paternally derived. The 2 Robertsonian translocations were both maternally derived. Of the 17 isochromosomes, 7 were dicentric [idic(21q)] and 10 were monocentric [i(21q)]. Both rob(21q21q) were monocentric. Our findings agree with those made in 17 previously published cases of rea(21q21q). The parental origins of the i(21q) were equally divided between maternal (n = 17) and paternal (n = 15) origins. All 4 true rob(21q21q) reported to date are of maternal origin. Collectively, it appears that most homologous rearrangements of chromosome 21 are isochromosomes and only a small proportion are consistent with true Robertsonian translocations.

Fluorescence in situ hybridization detectable mosaicism for Angelman syndrome with biparental methylation.

We present a child with mild to moderate global developmental delay including severe speech impairment, inappropriate happy demeanor, wide-based gait, frequent ear infections with mild hearing loss, deep-set eyes, a wide mouth, widely-spaced teeth, normal head circumference, and no seizures. Results of peripheral blood lymphocyte chromosomal analysis with GTG banding were normal. However, fluorescence in situ hybridization (FISH) studies showed mosaicism for a deletion of probes (D15S10 and SNRPN) from the Angelman syndrome (AS) critical region with approximately 40% of peripheral lymphocytes having the deletion. The deleted chromosome 15 also showed centromeric duplication, which was detected with a D15Z1 probe [46,XX, dic(15)(pter-->q11.1::p11.2-->q11. 1::q13-->qter)]. The same duplication pattern was observed in 30% of the nuclei obtained from a buccal smear. Methylation studies using polymerase chain reaction with sodium bisulfite-treated DNA demonstrated a normal biparental methylation pattern. To the best of our knowledge, this is the first case with AS and a FISH detectable deletion in a mosaic pattern. We recommend FISH studies for the detection of mosaicism in the patients with AS clinical findings even if results of the methylation studies are normal.

Mental retardation and Ullrich-Turner syndrome in cases with 45,X/46X,+mar: additional support for the loss of the X-inactivation center hypothesis.

Four cases having mosaicism for a small marker or ring [45,X/46,X,+mar or 45,X/46,X,+r] chromosome were ascertained following cytogenetic studies requested because of minor anomalies (cases 1, 3, and 4) and/or short stature (cases 2 and 4). While all 4 cases had traits typical of Ullrich-Turner syndrome (UTS), cases 1, 3, and 4 had manifestations not usually present in UTS, including unusual facial appearance, mental retardation/developmental delay (MR/DD) (cases 3 and 4), and syndactylies (case 1). The facial appearances of cases 1 and 3 were similar yet distinct from that of case 4. Using fluorescence in situ hybridization (FISH), each of the markers in these 4 cases was identified as having been derived from an X chromosome. The level of mosaicism for the mar/r(X) cell line in these cases varied from 70% (case 1) to 16% (case 4) but was not apparently correlated with the presence of MR/DD. Replication studies demonstrated a probable early replication pattern for the mar/r(X) in cases 1, 3, and 4, while the marker in case 2 was apparently late replicating. To date, 41 individuals having mosaicism for a small mar/r(X) chromosome have been described. Interestingly, most of the 14 individuals having a presumedly active mar/r(X) demonstrated clinical findings atypical of UTS, including abnormal facial changes (11) and MR/DD (13). MR was noted most frequently in those cases having at least 50% mosaicism for the marker or ring. In contrast, atypical UTS facial appearance or MR/DD was not noted in 14 of the 16 cases with UTS who carried a probable late replicating marker or ring. In conclusion, although the phenotype of 45,X/46,X,mar/r(X) individuals appears to be influenced by the genetic content and degree of mosaicism for the mar/r(X), the most significant factor associated with MR/DD appears to be the activity status of the mar/r(X) chromosome. Thus, our 4 cases provide further support for the hypothesis that a lack of inactivation of a small mar/r(X) chromosome may be a factor leading to the MR and other phenotypic abnormalities seen in this subset of individuals having atypical UTS.

Cytogenetic characterization of the human prostate cancer cell line P69SV40T and its novel tumorigenic sublines M2182 and M15.

Cytogenetic studies of a SV40T antigen immortalized human prostate epithelial cell line (P69SV40T) and its increasingly tumorigenic tumor sublines, designated M2182 and M15, were done with GTG-banding and multicolor fluorescence in situ hybridization (FISH). The parental line and each of the two sublines were near-diploid and contained several consistent abnormalities. Two structural chromosome anomalies were noted in all three lines; a der(7)t(5;20;7) and a der(5)t(5;9). Abnormalities that were acquired and retained in the tumor sublines after in vivo and/or in vitro selection included a der(1)t(1;8), der(3)t(3;14), der(20)t(7;20), and der(X)t(X;11). Findings unique to subline M2182 were a der(11)t(5;11) and -14. Those unique to M15 were a der(16)t(16;19) and -Y. Chromosome imbalances resulting from numerical and/or structural abnormalities in the tumor sublines involved several chromosome regions that have previously been implicated in human prostate cancer, such as loss of Xp, Y, 3p (M2182 and M15), 16q (M15), and gains for 5q (M2182) and 8q (M2182 and M15). Collectively, the characterization of these lines should assist with the localization of chromosome regions, and possibly genes, that are important in the development and progression of human prostate cancer.

Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes.

BACKGROUND: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively simple alternative method to detect the methylation status of the PWS/AS region. METHODS AND RESULTS: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. CONCLUSION: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.

Strategies and logistical requirements for efficient testing in genetic disease.

Rapid advances in the field of human genetics have led to an increase in the availability of genetic diagnostic tests. This article reviews technical approaches and diseases for which metabolic, newborn screening, molecular, and cytogenetic diagnostic tests are available currently. Overlaps in areas of diagnostic testing that have emerged from the application of new technology in the field of genetics also are discussed, as are criteria and approaches used for identifying conditions for which diagnostic, presymptomatic, prenatal, and carrier testing should be offered. Finally, the delivery of these results and necessary genetic counseling that should accompany this information are reviewed.

The application of spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) technology to determine the chromosomal content(s) of micronuclei.

DNA loss by the process of micronucleation is associated with aging, cancer and environmental exposure. The primary aim of this study was to identify the chromosomal origin of the DNA excluded into micronuclei (MN). This was achieved using a novel application of SKY and FISH technologies. Cytochalasin B (Cyt B)-treated lymphocyte cultures from three females (aged 28, 42 and 72) were analyzed. SKY revealed that the majority of MN (89.8, 82.9, and 97.6% in the 28-, 42- and 72-year-old (y.o.), respectively) had a uniform, single color, suggesting that they were comprised of DNA from a single chromosome. Using a pancentromeric probe, most of the MN (82% in 28 y.o., 69% in 42 y.o. and 80% in 72 y.o.) had one centromere signal present. Overall, the confirmation studies (using FISH with chromosome-specific WCP) were in agreement with the SKY chromosomal assignments for 71.1% of the MN. Although the SKY analysis showed that all of the 23 chromosomes (22 autosomes and the X chromosome) could be present in the MN, overall, the X chromosome was seen most frequently. DNA from the X chromosome was seen in 50.6% of MN in the 42 y.o. individual, whereas in the 28 and 72 y.o. it was seen in 12.2 and 7.1% of MN, respectively. This difference (P<0.0001) in the frequencies of X chromosome exclusion into MN among individuals was independently confirmed using a single whole chromosome painting probe (WCP) for the X chromosome. SKY also showed variation in the frequency of autosomal exclusion into MN between chromosomes and between females. Collectively, this study supports the hypothesis that the majority of MN contain DNA from a single, monocentric chromosome. The use of SKY technology for the identification of the chromosomal content(s) of MN provides an opportunity for expansion of our knowledge of the chromosomal changes that accompany MN formation.

Partial trisomy 10 mosaicism with cutaneous manifestations: report of a case and review of the literature.

A female infant with partial trisomy 10 mosaicism and hypomelanosis of Ito is presented. Features include a prominent forehead, hypertelorism, large dysplastic ears, prominent nasal root, a cleft lip and alveolar ridge, bilateral metatarsus adductus, and streaks and whorls of hypopigmented skin. The skin findings were diagnostic for hypomelanosis of Ito. A peripheral blood karyotype was normal. Fibroblasts from a junctional skin biopsy revealed mosaicism for partial trisomy of chromosome 10 [46, XX/47, XX, +del(10) (q11.2q23.2)]. The physical findings of this patient are compared to five published cases of complete trisomy 10 mosaicism and 94 cases of isolated trisomy 10p and trisomy 10q.

Mosaic "tetrasomy" 8p: case report and review of the literature.

A male infant presenting with multiple anomalies including a midline cleft palate, anasarca, hepatomegaly, pulmonary edema, agenesis of the corpus collosum, and complex congential cardiac anomalies was found to have mosaicism for an additional chromosome that appeared (following GTG-banding and FISH) to be a monocentric isochromosome of the short arm of chromosome 8 (46,XY/47,XY, +i(8p)). Nine other cases of mosaicism for an additional i(8p) were reviewed. Considerable phenotypic variation was noted. Consistent features were identified including agenesis of the corpus callosum, cardiac malformations, and minor facial dysmorphology. The phenotype of these patients partially overlaps those of trisomy 8 and trisomy 8p. By studying additional individuals with this condition, mosaic tetrasomy 8p may emerge as a recognizable clinical phenotype.

Highly conservative reciprocal translocations formed by apparent joining of exchanged DNA double-strand break ends.

Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the aprt gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-aprt parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between aprt and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually

Suspected gonadal mosaicism for isochromosomes 18p and 18q unsubstantiated by fluorescence in situ hybridization analysis of sperm.

PURPOSE: A father had two children, one with isochromosome 18p, and another with isochromosome 18q. The father was counseled that he might have gonadal mosaicism for isochromosomes 18p and 18q, which could confer a high recurrence risk. METHODS: A sperm sample from the father was analyzed with fluorescence in situ hybridization probes for 18p and 18q. RESULTS: More than 1,000 sperm were scored and none were found with two 18p or 18q signals. There were no differences in the father's specimen compared to a control. CONCLUSIONS: There was no evidence for gonadal mosaicism. It is important to confirm clinical hypotheses whenever possible.

Nucleolar organizer region variants as a risk factor for Down syndrome.

An unusual nucleolar organizer region (NOR) heteromorphism was noted among 13 of 41 parents in whom nondisjunction leading to trisomy 21 was known to have occurred. In contrast, only one of these double NOR (dNOR) variants was found among the 41 normal spouses and none were seen among 50 control individuals. In two dNOR(+) families, a second child with trisomy 21 was conceived. In both families, the extra chromosome in each child was contributed by the parent who carried the dNOR variant and resulted from a recurrent meiosis I error. Our data suggest that the dNOR heteromorphism may play a role in meiotic nondisjunction and could be associated with as much as a 20-fold increased risk for having offspring with trisomy 21.

A molecular genetic approach to the identification of isochromosomes of chromosome 21.

The largest class of de novo chromosomal rearrangements in Down syndrome are rea(21q21q). Classically, these rearrangements have been termed Robertsonian translocations, implying an attachment of two different chromosome 21 homologues. Additionally, a Robertsonian translocation between two chromosomes 21 cannot be distinguished from an isochromosome composed of genetically identical arms by cytogenetic analyses. Therefore, we have used molecular techniques to differentiate between true Robertsonian translocations and isochromosomes. Samples were obtained from 12 probands, ascertained for de novo rearrangements between homologous chromosomes 21 [11 rea(21q21q) and 1 rea (21;21)(q22;q22)], their parents (n = 24) and available siblings (n = 7). The parental origins of the de novo rearrangements were assigned using molecular and cytogenetic analyses. Although not statistically significant, there was a two-fold increase in the number of paternally derived de novo rearrangements (n = 8) as compared with maternally derived rearrangements (n = 4). To distinguish between rob(21q21q) and i(21q), we used restriction fragment length polymorphisms (RFLPs) spanning the length of chromosome 21. Using all informative and partially informative RFLPs, we used the method of maximum likelihood to assign the most likely rearrangement definition (i or rob) and parental origin in each family. The maximum likelihood estimates indicated that all rearrangements tested (n = 8) were isochromosomes. C-banding revealed two centromeres in three cases indicating that a U-type exchange occurred between sister chromatids in these rearrangements. Our results suggest that the majority of de novo rea(21q21q) are isochromosomes derived from a single parental chromosome 21.

Unusual presentation of Ewing sarcoma with t(21;22) in a 3-year-old boy.

PURPOSE: We describe a 3-year-old boy with widespread, metastatic Ewing sarcoma and an unusual translocation, involving chromosomes 21 and 22. MATERIALS AND METHODS: Cytogenetic studies were performed on a biopsy of the primary tumor. These included GTG banding and fluorescence in situ hybridization. RESULTS: A balanced translocation between chromosomes 21 and 22 was noted with translocation breakpoints at bands 21q22 and 22q12. CONCLUSIONS: The t(21;22) translocation represents a new cytogenetic abnormality that may be associated with Ewing sarcoma. Its prognostic significance, if any, remains to be determined.