In vivo loss of heterozygosity in T-cells of B6C3F1 Aprt(+/-) mice

We have used B6C3F1 mice heterozygous at Aprt (adenine phosphoribosyltransferase) as a model to study in vivo loss of heterozygosity (LOH) in normal splenic T-lymphocytes. APRT-deficient T-cells were selected in medium containing 50 microg/ml 2, 6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP(r)) T-cell variants were recovered at an average frequency of 3 x 10(-5) from 21 B6C3F1 Aprt(+/-) mice. Allele-specific PCR of Aprt showed that about 70% of 122 DAP(r) colonies were caused by loss of the nontargeted Aprt allele (Aprt(+)). Analysis of microsatellite markers along the length of chromosome 8 suggested that mitotic recombination, or chromosome loss, with or without duplication of the remaining chromosome are the predominant mechanisms resulting in loss of Aprt(+). DNA sequencing of Aprt RT-PCR products from the DAP(r) variants that retained Aprt(+) indicated that point mutation as well as other mechanisms could cause this second class of variants. The high spontaneous frequency of in vivo Aprt LOH in mouse T-cells, mediated by LOH mechanisms that are also known to produce human cancers, suggests that the Aprt heterozygous mouse is a valid model for studying the diversity of mechanisms for in vivo somatic mutagenesis.     

Intrinsic genetic instability of normal human lymphocytes and its implication for loss of heterozygosity

A combination of flow cytometry and microsatellite analysis was used to investigate loss of expression of HLA-A and/or HLA-B alleles and concurrent LOH at polymorphic chromosome 6 loci both in freshly isolated lymphocytes (in vivo mutations) and in lymphocytes cultured ex vivo. The fraction of in vivo mutants that showed LOH at 6p appeared to vary from 0%-49% for various donors. During culturing ex vivo, HLA-A(-) cells arose at a high rate and showed simultaneous loss of expression at the linked HLA-B locus. Up to 90% of the ex vivo arisen HLA-A2(-) cell population showed LOH of multiple 6p markers, and 50% had lost heterozygosity at 6q. This ex vivo spectrum resembles that found in HLA-A2 mutants obtained from lymphoblastoid cells. The HLA-A2 mutants present in vivo may reflect only a small fraction of the mutants that can be detected ex vivo. In normal lymphocytes, in vivo only mitotic recombination appears to be sustained, indicating the importance of this mechanism for tumor initiation in normal cells. Although mutations resulting in LOH at both chromosome 6 arms were shown to result in nonviable cells in normal lymphocytes, they have been shown to result in viable mutants in lymphoblastoid cells. We hypothesize that these types of mutations also occur in vivo but only survive in cells that already harbor a mutated genetic background. In light of the high rate at which these types of mutations occur, they may contribute to cancer progression.     

Expression of recessiveAprt − mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the neardiploid mouse CAK cell line have been analyzed. The Aprt gene is located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toy^r13) from whichAprt ^? mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele, with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detcted in cell lines lacking morphologically marked chromosomes.     

Molecular genetic characterization of both components of a dedifferentiated chondrosarcoma, with implications for its histogenesis

Dedifferentiated chondrosarcoma is defined as a high-grade, anaplastic sarcoma adjacent to a low-grade malignant cartilage-forming tumour. Controversy remains as to whether the anaplastic and cartilaginous components are derived from a common precursor cell, or whether they represent separate genotypic lineages (collision tumour). Both components of a case of dedifferentiated chondrosarcoma were therefore separately investigated by loss of heterozygosity (LOH) analysis, comparative genomic hybridization (CGH), DNA flow cytometry, and p53 analysis. Both showed p53 overexpression and an identical somatic 6 bp deletion in exon 7 of p53. Combination of the CGH and LOH results revealed that both components had lost the same copy of chromosome 13. These results provide compelling evidence in this case for a common origin, instead of the 'collision tumour' theory. Certain genotypic alterations were not shared. The anaplastic component showed severe aneuploidy, LOH at additional loci, and amplification and deletion of several chromosome parts. In contrast, the cartilaginous component had lost chromosomes 5, 22, 17p and part of 16p and revealed an amplification of 17q. The LOH and CGH results further demonstrated that the two components had lost a different copy of chromosome 4. Thus, a substantial number of genetic alterations have occurred after the diversion of the two components, indicating that the separation of the two clones, derived from a single precursor, was a relatively early event in the histogenesis of this case of dedifferentiated chondrosarcoma.     

Loss of heterozygosity mapping in Wilms tumor indicates the involvement of three distinct regions and a limited role for nondisjunction or mitotic recombination.

Loss of heterozygosity (LOH) for polymorphic markers is a frequently occurring event in some tumors, reflecting the role of allele loss in the development of these tumors. We have determined LOH in 38 cases of Wilms tumor for the 2 known loci on chromosome arm 11p and for a newly detected locus on chromosome arm 16q. Only 7 of the 38 tumors studied showed reduction to homozygosity of 11p13 markers. In 4 of these tumors, reduced expression of WT1 and WIT1, genes located at 11p13 and implicated in Wilms tumorigenesis, was noted. However, this was also found in 2 of 7 tumors showing LOH exclusively of 11p15 markers and in 15 of the remaining 24 tumors in which there was no LOH for 11p markers. This suggests that events not involving mitotic recombination or chromosome nondisjunction are the most common mechanisms for mutations at the 11p Wilms tumor locus. We also noted that mitotic recombination involving 11p15 loci occurred in addition to reduced expression of the 11p13 locus genes in 2 tumors, suggesting a possible interaction between these 2 loci. In addition, LOH for 16q markers was observed in 6 tumors. In one case this was coincident with reduction of WT1 and WIT1 gene expression, and in 3 other cases it occurred in addition to 11p LOH. This indicates that an additional locus on 16q is likely to be involved in Wilms tumorigenesis.     

Genetic events in tumour initiation and progression in multiple endocrine neoplasia type 2

Multiple endocrine neoplasia type 2 (MEN 2) is a familial cancer syndrome arising from mutation at a locus or loci in chromosome region 10p11.2-q11.2. The disease is characterized by medullary thyroid carcinoma (MTC) and pheochromocytoma (Pheo). To assess the genetic events in tumour initiation and progression in this disease, we have compiled an allelotype for MTC and Pheo tumours using polymorphic marker loci from each chromosome arm. Using a panel of 58 tumours, we found frequent allele losses on chromosome arms 1p (42%), 3p (30%), 3q (38%), 11p (11%), 13q (10%), 17p (8%), and 22q (29%). Loss of heterozygosity (LOH) for loci on chromosome 10 was detected in a single tumour where one whole chromosome copy was lost. We used a panel of polymorphic markers for each of chromosomes 1, 3, 11, and 17 to define a shortest region of overlap for these regions. The most frequent allele losses were on chromosome 1, spanning the entire short arm of the chromosome but not loci on 1q. LOH on chromosome 3 encompassed a minimal common region of 3q12-qter. The regions of allelic deletion on chromosome 11(11pter-p13), 17(17pter-p11.2), and 13 (13q) encompass known tumour suppressor loci (WTI, TP53, RBI) which must therefore be candidates for genes contributing to MTC and Pheo development. Our data suggest allele loss on chromosome 11, 13, or 17 occurs predominantly in tumours with losses on chromosome 3, potentially reflecting the accumulation of genetic change in tumour progression. These events may be associated with more advanced disease in MTC. We suggest that at least 7 genes contribute to tumour development in MEN 2, including an initiating locus on chromosome 10 and loci on chromosomes 1, 3, 11, 13, 17, and 22 which have a progressional role in these tumours. © 1993 Wiley-Liss, Inc.     

Exposure of mice to arsenic and/or benzo[a]pyrene does not increase the frequency of Aprt-deficient cells recovered from explanted skin of Aprt heterozygous mice

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Aprt(+/-) hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[a]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAP(r)) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DAP(r) colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data.     

Early onset of gastric carcinoma and constitutional deletion of 18p.

We report on the association of a gastric carcinoma and a constitutional deletion of the short arm of chromosome 18 in a 14-year-old patient. The phenotype of the patient, including microcephaly, ptosis, micrognathia, tetralogy of Fallot, and mental retardation, fits well with previously reported cases of del(18p); she also showed a positive serology against Helicobacter pylori. The comparison of the alleles of polymorphic loci located on the short arm of chromosome 18 between the patient and her parents showed a maternal origin of the abnormal chromosome. Loss of heterozygosity (LOH) for loci located in the long arm of chromosome 18 is a frequent event in gastric carcinomas; it was observed in the tumoral mass of our patient and again, the alleles lost were of maternal origin. We postulate that the constitutional chromosomal abnormality may have favored the loss of the abnormal chromosome in some cells and that the loss of the deleted chromosome 18 (demonstrated by LOH for this chromosome in the tumoral mass) has been an early step in the pathogenesis of the gastric carcinoma of our patient with Helicobacter pylori infection acting as a cofactor.     

Differences in malsegregation rates obtained by scoring ana-telophases or binucleate cells

In this work we have applied in situ hybridization with alphoid centromeric probes specific to chromosomes 7 and 11 to ana-telophase cells from human primary fibroblasts. The aim was to visualize the events leading to aneuploidy directly during anaphase, analyse the induction of aneuploidy during this mitotic stage and compare the frequencies of chromosome malsegregation observed in ana-telophases with the estimated malsegregation obtained in binucleate cells after a short cytochalasin B treatment. Significantly higher frequencies of chromosome loss and chromosome non-disjunction were observed in fibroblasts undergoing ana-telophase during recovery from a nocodazole-induced mitotic arrest compared with binucleate cells obtained by a further 30 min incubation with cytochalasin B. Using the same experimental schedule, analysis of hybridization signals in mononucleate cells showed higher frequencies of polyploid nuclei in cytochalasin B-treated cultures, indicating that part of the ana-telophases observed after release from the nocodazole-induced mitotic arrest may give rise to polyploid mononucleate cells instead of binucleate ones. A reduced distance between spindle poles was also measured in cells undergoing ana-telophase in the presence of cytochalasin B. Our study suggests that in nocodazole and cytochalasin B-treated cultures the shorter pole-to-pole distance may favour the reformation of a single membrane around telophase chromosomes, especially when several lagging chromosomes lie between the two future daughter nuclei. This would give rise to polyploid mononucleate cells at the ensuing interphase.     

Region-specific loss of heterozygosity on chromosome 19 is related to the morphologic type of human glioma

Allelic mutation on chromosome 19 has previously been reported as a frequent genetic event in human glial tumors. In an effort to localize specific regions of importance on this chromosome better, 13 highly polymorphic genetic markers distributed along the length of chromosome 19 were used for evaluation of loss of heterozygosity (LOH) and microsatellite instability in a total of 100 brain tumors, including 75 astrocytomas (55 grade 4; 7 grade 3; 5 grade 2; 6 grade 1; and 2 other), 17 oligodendrogliomas (1 grade 4; 5 grade 3; 10 grade 2; and 1 grade 1), and 8 mixed oligoastrocytomas (MOA) (3 grade 4; 2 grade 3; and 3 grade 2). No microsatellite expansion was observed in these glial tumors for any of the chromosome 19 loci examined. LOH for loci on chromosome 19 was detected in 23/74 informative astrocytomas (31%), 11/17 oligodendrogliomas (65%), and 3/8 MOA (38%). Partial deletion of chromosome 19 occurred more frequently (31/37 cases) than did loss of one whole copy of the chromosome, and a morphology-specific pattern of LOH was observed. In 12/14 (86%) instances of chromosome 19 deletion in oligodendrogliomas and MOA, the 19q arm showed LOH, whereas the 19p arm showed no loss for all informative loci. Conversely, in 17/23 (74%) instances of chromosome 19 deletion in astrocytomas, the 19p arm showed LOH, whereas the 19q arm showed no loss for one or more loci. Thus, loss of 19q and retention of 19p are strongly associated with oligodendroglioma and MOA, whereas loss of 19p and retention of distal 19q is associated with astrocytoma. These data indicate that two or more tumor suppressor genes may reside on chromosome 19, one on 19p important in the development of astrocytomas, and one on 19q important in oligodendrogliomas and MOA.     

Molecular genetic alterations in carcinoma ex-pleomorphic adenoma: a putative progression model?

To determine the genetic changes associated with the development of carcinoma ex-pleomorphic adenoma (Ca Ex-PA), we analyzed 15 microsatellite loci at chromosome arms 8q, 12q, and 17p on DNA from 26 neoplasms (including 8 microdissected benign and malignant components), and 13 pleomorphic adenomas for comparison. Pleomorphic adenomas and the adenoma component of Ca Ex-PAs showed a higher incidence of loss of heterozygosity (LOH) at chromosome arms 8q (52%) and 12q (28%) than at 17p (14%) loci. In the carcinoma component, the combined LOH at chromosome arm 8q, 12q, and 17p regions was 69%, 50%, and 69%, respectively; within these chromosomal regions, 8q11.23-q12 (42%), 12q23-qter (39%), 17p13 (41%), and 17p11 (45%) loci manifested the highest incidence of LOH. Eight carcinomas (30.7%) showed loss at all three chromosomal arms tested. Of the eight microdissected Ca Ex-PAs analyzed, four adenoma and corresponding carcinoma components (50%) had the same LOH at 12q loci and additional LOH at 17p loci only in carcinomas. Chromosome arm 17p alterations correlated significantly with high disease stage and an increased proliferative rate in these tumors. Our results indicate that alterations at regions on chromosome arms 8q and/or 12q may constitute early events associated with pleomorphic adenomas; that LOH at 12q loci may identify a subset of adenoma with potential progression to carcinoma; that acquisition of additional alterations at chromosome arm 17p loci might represent an event preceding malignant transformation and progression; and that 8q, 12q, and 17p regions may harbor tumor suppressor genes involved in the genesis of PA and Ca Ex-PA. Genes Chromosomes Cancer 27:162-168, 2000.     

Loss of heterozygosity on chromosome XP22.2-p22.13 and Xq26.1-q27.1 in human breast carcinomas.

In an attempt to investigate the X chromosome harboring putative tumor suppressor genes (TSGs) in sporadic breast carcinoma, we performed loss of heterozygosity (LOH) studies on 23 breast carcinomas using 15 polymorphic markers covering the whole X chromosomes. Matched DNA extracted from tumor samples and corresponding normal tissues were analyzed by polymerase chain reactions (PCR) using microsatellite markers. In 10 cases (43.5%), LOH was detected for at least 1 of the 15 polymorphic markers of the X chromosome tested. Four cases carried a LOH at Xp, and three cases LOH on Xp and Xq. Three cases carried a LOH Xq. Percentage of LOH was relatively high in DXS987 (26.7%), DXS999(30.0%), HPRT(21.4%), DXS1062(23.1%) loci. Common regions of deletions were found on Xp22.2-p22.13 (30% of LOH) measuring about 4.5Mb and Xq26.1-q27.1 (23.1% of LOH) measuring 10 Mb. The deleted allele was an active copy of the X chromosome. The results indicate the TSGs on the X chromosome are involved in breast cancer.     

Analysis of large and small colony L5178Y tk-\- mouse lymphoma mutants by loss of heterozygosity (LOH) and by whole chromosome 11 painting: detection of recombination

Analysis of 122 spontaneous large and small colony mutants derivedfrom L5178Y tk^+/–mouse lymphoma cells at 28 heteromorphicmicrosatellite loci on chromosome 11 showed that extensive lossof heterozygosity (LOH) is common in both large colony and smallcolony mutants, eliminating most chromosome 11 loci as candidatesfor a putative growth control locus. These results, in conjunctionwith historical cytogenetic data, suggest that a putative growthcontrol locus lies distal to the thymidine kinase (Tk1) gene,near the telomere. Thirty seven mutants were hybridized witha chromosome 11-specific whole chromosome painting probe foranalysis of rearrangements. Generally, painting confirmed earlierobservations that large colony mutants are karyotypically normal,whereas small colony mutants frequently have detectable rearrangements.A point probe distal to Tk1revealed no evidence of chromosomebreakage in small colony mutants that appeared normal on whole11 painting and had no LOH. Therefore, the molecular differencebetween large and small colony mutants remains unknown. Modelsto explain large and small colony mutants consistent with ourfindings are presented, including loss of a putative growthcontrol gene, differential mechanisms of chromosome breakage/repairand second site mutations as planations for small colony mutants.Painting revealed translocations and aneuploidy and showed thatnon-disjunction was not a common explanation for complete LOH.The most common finding was that large regions of LOH do notresult from deletions, demonstrating that these cells can detectrecombination events as well as previously observed chromosomalrearrangements, deletions and point mutations. ^*To whom correspondence should be addressed. Tel: +1 407 768 2048; Fax: +1 407 727 2643; Email: Liechty{at}appliedgenetics.com     

Direct and indirect non-disjunction in the origin of trisomy in cultured human lymphocytes

The aim of the present work was to investigate the processes involved in the origin of trisomic karyotypes, i.e. co-migration of sister chromatids (mitotic non-disjunction, MND) and recovery of micronuclei (MN) originating from lagging chromosomes/chromatids at anaphase (mitotic indirect non-disjunction, MIND), and to evaluate their relative contribution to aneuploidy in human lymphocytes mitotically activated in vitro. Therefore, phytohaemagglutinin-stimulated human lymphocytes from one donor were treated with 10 and 25 nM colchicine and analysed through two cell cycles by means of both molecular (FISH with centromeric DNA probes specific for chromosomes 7 and 11) and classical cytogenetic techniques. The following events were analysed: (i) chromosome/chromatid loss (a MN-generating event) in M(1) bipolar ana-telophases; (ii) MN recovery in M(2+) prophases; (iii) non-disjunction and loss of chromosomes 7 and 11 by FISH analysis in cytochalasin B-induced binucleate cells; (iv) spontaneous frequency of trisomic cells by chromosome counting and FISH analysis in M(1) c-metaphases; (v) induced frequency of trisomic cells by chromosome counting and FISH analysis in M(2) c-metaphases. Our results indicate that MND plays a major role compared with MIND in the origin of trisomic karyotypes, being approximately 4- to 5-fold higher in colchicine-treated cells. Moreover, remarkable reductions in the observed frequencies of trisomic cells were recorded in comparison with the expected ones, with an observed/expected frequency ratio of trisomic M(2) c-metaphases ranging between 1/3 and 1/6.     

Atypical ductal hyperplasia of the breast: clonal proliferation with loss of heterozygosity on chromosomes 16q and 17p.

AIMS--To determine if allelic loss on chromosomes 16q and 17p, commonly encountered in in situ and invasive ductal carcinomas, is present in atypical ductal hyperplasia (ADH); to determine whether ADH is a neoplastic (clonal) or hyperplastic (polyclonal) proliferation. METHODS--Fourteen cases of ADH were examined for allele loss at loci on chromosome 16q and 17p using a microdissection technique, polymorphic DNA markers and the polymerase chain reaction (PCR). RESULTS--Loss of heterozygosity (LOH) was detected in five of nine informative cases on chromosome 16q at the microsatellite D16S413 and two of eight informative cases on chromosome 17p at D17S796. CONCLUSIONS--The incidence of LOH at these loci is similar to that previously observed in ductal carcinoma in situ and in invasive ductal carcinoma. Because of the nature of the technique used, our findings also demonstrate that ADH is a monoclonal, and hence, neoplastic proliferation rather than a hyperplastic (polyclonal) condition as its name suggests. There is thus a case for including ADH, as presently defined, within the spectrum of ductal carcinoma in situ.     

Folate deficiency in human peripheral blood lymphocytes induces chromosome 8 aneuploidy but this effect is not modified by riboflavin

Chromosome 8 aneuploidy is a common event in certain cancers but whether folate (F) deficiency induces chromosome 8 aneuploidy is not known. Furthermore the impact of riboflavin (R) deficiency, which may alter activity of a key enzyme in folate metabolism, on these events is unknown. Therefore, the aim of our research was to test the following hypotheses: (a) F deficiency induces chromosome 8 aneuploidy; (b) chromosome 8 aneuploidy is affected by F deficiency to a similar degree as chromosome 17 and (c) R deficiency aggravates the risk of aneuploidy caused by F deficiency. These hypotheses were tested in long‐term cultures of lymphocytes from twenty female healthy volunteers (aged 30–48 years). Lymphocytes were cultured in each of the four possible combinations of low (L) and high (H) F (LF, 20 nmol/L, HF 200 nmol/L, respectively) and L and H R (LR 1 nmol/L, HR 500 nmol/L, respectively) media (LFLR, LFHR, HFLR, HFHR) for 9 days. Chromosomes 8 and 17 aneuploidy was measured in mononucleated (MONO) and cytokinesis‐blocked binucleated (BN) cells using dual‐color fluorescence in situ hybridization (FISH) with fluorescent centromeric probes specific for chromosomes 8 and 17. Culture in LF media (LFLR or LFHR) induced significant and similar increases in frequencies of aneuploidy of chromosomes 8 and 17 (P < 0.001) relative to culture in HF media (HFLR or HFHR). There was no significant effect of R concentration on aneuploidy frequency for either chromosome. We conclude that F deficiency is a possible cause of chromosome 8 aneuploidy. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

Chromosomal fragile sites FRA3B and FRA16D show correlated expression and association with failure of apoptosis in lymphocytes from patients with thyroid cancer

It has been suggested that common fragile sites (cFSs) are related to cancer development. This appears to be the case for FRA3B and FRA16D, localized in two tumor-suppressor genes (FHIT and WWOX, respectively) that are altered by deletions or loss of heterozygosity (LOH) in many cancers. The features responsible for fragility have not yet been identified. Furthermore, it is still unclear whether instability at these regions causes chance deletions and loss of function of the associated genes, or whether the gene function itself is related to the appearance of fragility. In this study, we analyzed cFS expression in lymphocytes from 20 healthy or thyroid cancer-affected subjects exposed to radiation after the Chernobyl accident. The same cells were examined for apoptosis, a principal function of both the FHIT and WWOX genes. Exceptionally elevated chromosome fragility was observed, particularly in cancer patients, affecting FRA3B, FRA16D, and a cluster of less highly expressed cFSs; levels of chromosome fragility were found to be correlated among these cFSs. Interestingly, most expressed cFSs were sites of LOH reported for thyroid tumors; moreover, cells with the highest fragility also had a reduced ability to undergo apoptosis. These findings reveal previously unknown genetic interactions affecting fragile loci, suggestive of a shared function inside mitotic cells. Attenuation of checkpoint control and apoptosis resistance seem to be the cell phenotypes associated with unusual chromosome fragility. We propose that breakage at specific cFS could derive from early epigenetic events at loci involved in radiation carcinogenesis. This article contains supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Distribution of aneuploidy in human gametes: comparison between human sperm and oocytes

The frequency and distribution of aneuploidy was compared in 11,615 karyotyped human sperm and 772 karyotyped human oocytes to determine if all chromosomes are equally likely to be involved in aneuploid events or if some chromosomes are particularly susceptible to nondisjunction. The frequency of hypohaploidy and hyperhaploidy was compared among different chromosome groups and individual chromosomes for human sperm and oocytes. In general, hypohaploid chromosome complements were more frequent than hyperhaploid complements, in sperm and oocytes. The distribution of chromosome loss in the hypohaploid complements indicated that significantly fewer of the large chromosomes and significantly more of the small chromosomes were lost, suggesting that technical loss predominantly affects small chromosomes. A conservative estimate of aneuploidy (2 X hyperhaploidy) was approximately 3-4% in the human sperm and 18-19% in human oocytes. All chromosome groups were represented among hyperhaploid human sperm and oocytes. For human sperm, the observed frequency of hyperhaploidy equaled the expected frequency based on the assumption that the frequency of nondisjunction is equal for all chromosome groups, with two exceptions: group G and the sex chromosomes. Among individual chromosomes in human sperm, chromosomes 1 and 21 and the sex chromosomes had a significant excess of hyperhaploidy. For human oocytes, there were fewer hyperhaploid oocytes than expected for chromosome groups C and F and more than expected for chromosome groups D and G. Among individual chromosomes there was a significant excess for chromosome 21. These results indicate that all chromosomes are susceptible to nondisjunction but that chromosome 21 is particularly prone to aneuploidy in both human sperm and oocytes. They also demonstrate that sex chromosome aneuploidy is common in human sperm but not in human oocytes.     

Bladder cancer genotype stability during clinical progression

Genomic instability is manifested by the accumulation of large numbers of mutations. The rate at which mutations accumulate has been difficult to estimate because serial comparisons are required. For further insight into how quickly mutations accumulate during clinical progression, cell lines sequentially isolated 6 or 11 months apart from two patients with metastatic bladder cancer were compared for loss of heterozygosity (LOH). The genomes were scanned at approximately 200 polymorphic microsatellite loci to increase the resolution and sensitivity for losses. The cell lines from both patients had evidence of genomic instability, with aneuploidy, chromosomal instability, and fractional allelic losses of 0.15 and 0. 48, respectively. However, additional changes were relatively infrequent, with more than 90% identity between the initial and recurrent cell lines. Allelic losses were not randomly scattered, but clustered on specific chromosomes. Therefore, the numbers of loci with further LOH during the clinical progression of some bladder cancers are small relative to the total number of loci with LOH, suggesting that most allelic losses accumulate before clinical presentation.     

Clonality and loss of heterozygosity of WT genes are early events in the pathogenesis of nephroblastomas

Nephrogenic rests (NRs), putative precursor lesions of nephroblastomas (Wilms' tumors), are found in 25% to 40% of kidneys presenting with nephroblastomas. Nephroblastomas are clonal tumors that, according to a genetic multistep model, are thought to arise as subclonal proliferations from NRs by accumulating genetic alterations. Different candidate genes for the pathogenesis of nephroblastomas have been identified, including those at chromosomes 11p13 (WT1 gene), 11p15 (WT2 gene), and 16q (WT3 gene). We investigated clonality and loss of heterozygosity (LOH) at these loci in different subtypes of NR. After microdissection under microscopic control, we analyzed a highly polymorphic locus of the human androgen receptor gene (HUMARA) for nonrandom X-inactivation of genomic DNA using a methylation-sensitive restriction enzyme to investigate clonality. Out of 14 patients, we found that 1 case each of adenomatous and hyperplastic NR and 2 of 7 cases of sclerosing NR were monoclonal. Five patients were noninformative. We assessed LOH at chromosomes 11p13, 11p15, and 16q by analyzing polymorphic gene loci at these regions. One hyperplastic NR and the corresponding tumor showed LOH at 11p13 and 11p15; 1 sclerosing NR and the corresponding tumor exhibited LOH at chromosome 16q. We demonstrate for the first time that sclerosing NRs can exhibit genetic alterations found in nephroblastomas, namely monoclonality and LOH at the WT gene loci. The histological morphology is no different between NRs with these genetic alterations and NRs without them. We conclude that these genetic changes are early events in the multistep genetic pathogenesis of nephroblastomas; however, they do not seem to fully determine a malignant potential of NR.