Molecular genetic analysis of recessive mutations at a heterozygous autosomal locus in human cells

We have investigated the genotypic changes that lead to expression of a recessive allele at a heterozygous autosomal locus in a human cell line. Mutant clones lacking thymidine kinase activity were derived from a B-cell lymphoblastoid line initially heterozygous at the tk locus, and restriction mapping was performed to detect intragenic structural alterations in the tk gene. In addition, informative molecular markers located elsewhere on chromosome 17 were analysed in order to detect large-scale (multilocus) events. We report that among 325 spontaneous and induced mutants, allele loss was more common than intragenic rearrangements or point mutations; in many cases, loss of heterozygosity appears to have extended well beyond the locus under selection. Cytogenetic analysis of a subset of these mutants showed that expression of the recessive TK-deficient phenotype and the associated loss of heterozygosity for chromosome 17 markers was not typically associated with detectable chromosomal changes.     

Gene inactivation as a mechanism for the expression of recessive phenotypes.

A series of Chinese hamster ovary cell hybrids were constructed which were heterozygous at the emtB and chr loci. These loci encode two recessive drug-resistance genes (emetine resistance and chromate resistance, respectively) located on a structurally hemizygous region on the long arm of chromosome 2. These heterozygous hybrids therefore exhibit wild-type sensitivity to both emetine and chromate. Drug-resistant variants were then selected in medium containing either emetine or chromate, and the mechanism of reexpression of the recessive drug-resistant allele was determined by karyotypic analysis of the resultant colonies. In previous studies at these loci we have determined that segregation of the recessive phenotype occurs primarily by (1) the loss of the chromosome 2 carrying the wild-type, drug-sensitive, allele, (2) deletion of the long arm of chromosome 2, or (3) loss of one chromosome 2 followed by duplication of the remaining homologue. However, a small proportion of segregants have also been detected which may have arisen by the mechanisms of de novo gene inactivation or mutation. In this report, hybrids are described which were constructed to allow selection for the retention of the chromosome carrying the wild-type allele and which therefore optimize isolation of these rare segregants. We demonstrate by karyotypic analysis, mutation frequency analysis, and microcell-mediated chromosome transfer that these rare segregants occur primarily by gene inactivation. We also demonstrate a dramatic increase in the proportion of segregants occurring by gene inactivation in two of these hybrids as compared with those previously reported, indicating that this mechanism may be an important mode of phenotype segregation in diploid cells and, therefore, in the development of cancers--such as the childhood tumors retinoblastoma and Wilms tumor--resulting from recessive alleles     

Molecular genetics of human cancer predisposition and progression

The development of human cancer is generally thought to entail a series of events that cause a progressively more malignant phenotype. Such a hypothesis predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each of the events less the last one and so on. A dissection of the pathway from a normal cell to a fully malignant tumor may thus be viewed as the unraveling of a nested set of aberrations. In experiments designed to elucidate these events we have compared genotypic combinations at genomic loci defined by restriction endonuclease recognition site variation in normal and tumor tissues from patients with various forms and stages of cancer. The first step, inherited predisposition, is best described for retinoblastoma in which a recessive mutation of a locus residing in the 13q14 region of the genome is unmasked by aberrant, but specific, mitotic chromosomal segregation. Similar mechanisms involving the distal short arm of chromosome 17 are apparent in astrocytic tumors and the events are shared by cells in each malignancy state. DNA sequencing indicates that these events accomplish the homozygosis of mutant alleles of the p53 gene. Copy number amplification of the epidermal growth factor receptor gene occurs in intermediate and late-stage tumors whereas loss of heterozygosity for loci on chromosome 10 is restricted to the ultimate stage, glioblastoma multiforme. These results suggest a genetic approach to defining degrees of tumor progression and the locations of genes involved in the pathway as a prelude to their molecular isolation and characterization.     

Isochromosome 6p,a unique chromosomal abnormality in retinoblastoma: Verification by standard staining techniques,new densitometric methods,and somatic cell hybridization

Summary Study of chromosome rearrangements in retinoblastoma tumors revealed that all tumors contained either an unusual isochromosome and/or extra copies of chromosome 1q. Extra copies of chromosome 1q occur in many malignancies. The pattern of G-bands suggested that the isochromosome was derived from either the short arm of chromosome 6, i(6p), or the long arm of chromosome 17, i(17q). Standard staining techniques using G-, C-, Q-, and R-banding; high resolution G-banding; and density profile analysis were consistent with the characteristic isochromosome of retinoblastoma being i(6p), rather than i(17q). This conclusion was substantiated by the analysis of segregants derived from retinoblastoma X mouse hybrid cells which had been grown in bromodeoxyuridine to select for loss of chromosome 17. The unique isochromosome was not lost under these conditions confirming that it is an i(6p) rather than an i(17q). The i(6p) abnormality has not been observed frequently in other tumors, but occurs in 60% of retinoblastoma tumors. Thus, although the mutation predisposing to retinoblastoma is known to map at 13q14, somatic amplification of genes on 1q and 6p may play a role in the pathogenesis of this tumor.     

Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy,interchromosomal rearrangement,and the simultaneous incidence of both events during mitotic growth

The SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ helicase family, which includes the human BLM, WRN and RECQL4 genes responsible for Bloom and Werner's syndrome and Rothmund-Thomson syndrome, respectively. Cells defective in any of these genes exhibit a higher incidence of genome instability. We previously demonstrated that various genetic alterations were detectable as events leading to loss of heterozygosity (LOH) in S. cerevisiae diploid cells, utilizing a hemizygous URA3 marker placed at the center of the right arm of chromosome III. Analyses of chromosome structure in LOH clones by pulse field gel electrophoresis (PFGE) and PCR, coupled with a genetic method, allow identification of genetic alterations leading to the LOH. Such alterations include chromosome loss, chromosomal rearrangements at various locations and intragenic mutation. In this work, we have investigated the LOH events occurring in cells lacking the SGS1 gene. The frequencies of all types of LOH events, excluding intragenic mutation, were increased in sgs1 null mutants as compared to the wild-type cells. Loss of chromosome III and chromosomal rearrangements were increased 13- and 17-fold, respectively. Further classification of the chromosomal rearrangements confirmed that two kinds of events were especially increased in the sgs1 mutants: (1) ectopic recombination between chromosomes, that is, unequal crossing over and translocation (46-fold); and (2) allelic crossing over associated with chromosome loss (40-fold). These findings raise the possibility that the Sgs1 protein is involved in the processing of recombination intermediates as well as in the prevention of recombination repair during chromosome DNA replication. On the other hand, intrachromosomal deletions between MAT and HMR were increased only slightly (2.9-fold) in the sgs1 mutants. These results clearly indicate that defects in the SGS1 gene function lead to an elevated incidence of LOH in multiple ways, including chromosome loss and interchromosomal rearrangements, but not intrachromosomal deletion.     

Promiscuous genes and chromosomal rearrangements of hematopoietic malignancies

The nonrandomness of chromosomal abnormalities of hematopoietic malignancies, which has been established twenty years ago, has evidenced a more or less close relationship between some structural chromosomal abnormalities and leukemia subtypes. The same relation was, then, shown between gene and chromosome rearrangements. It becomes now obvious that genes involved in malignant proliferations may rearrange several different partner genes, as for instance the genes MLL, localised to chromosome band 11q23, and ETV6/TEL to 12p13. The study of these rearrangements is of particular importance in order to improve our knowledge of the functions of rearranged genes as well as their normal counterparts, and to analyse mechanisms favoring the occurrence of chromosomal rearrangements in malignancies.     

Loss of alleles at loci on chromosome 13 in human primary gastric cancers

Mitotic events leading to the loss of the normal allele corresponding to a mutated gene are important for tumorigenesis in rare heritable tumors such as retinoblastoma and Wilms tumor. As reported for both colorectal and breast cancers, some common tumors seem to develop because of the same mitotic events. We examined constitutional and tumor genotypes defined by polymorphic DNA clones in 36 patients with gastric cancer. In 14 cases, constitutional heterozygosity at loci on chromosome 13 had been lost. Loss of alleles was also detected at a locus on chromosome 18 in two cases and at a locus on chromosome 17 in one case. The frequent loss of alleles at loci on chromosome 13 (41%) suggests that elimination of genes on this chromosome may be of importance in the tumorigenesis of human primary gastric cancers.     

Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in<Emphasis Type="Italic"> Streptomyces ambofaciens</Emphasis> ATCC 23877

In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD ( amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.     

Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids.

Resistance to phosphonacetyl-L-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.     

Nondisjunction reduplication of chromosome 3 is not a common mechanism in the development of human renal cell tumors

Because of the recurrent loss of regions of the chromosome 3 short arm in renal cell carcinomas, a chromosomal mechanism for the expression of recessive cancer genes has been implicated in the development of this type of tumor. Nondisjunction and subsequent reduplication of a mutant chromosome is one of the presumed mitotic mechanisms leading to the expression of recessive cancer genes. Using variant fluorescence at the centromeric region of chromosome 3 and a restriction fragment length polymorphism on chromosome 3p, we found chromosome 3 heteromorphism in the constitutional cells of 14 of 15 patients with renal tumors showing two normal chromosomes 3. This heteromorphism was maintained in each tumor. Therefore, the mechanism of nondisjunction and reduplication in the development of homozygosity for a mutant chromosome 3 in renal tumors remains questionable.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Chromosome 13 homozygosity in osteosarcoma without retinoblastoma.

We provide evidence that some human osteosarcomas arise subsequent to the development of homozygosity at loci on the long arm of chromosome 13. The resulting chromosome 13q homozygosity allows the phenotypic expression of any recessive allele on that chromosome. Clinical evidence suggests that it is the retinoblastoma locus within 13q14 that is involved in the formation of these bone tumors.     

Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae

Since chromosomes of fungi are difficult to observe directly, strains have been developed in which chromosome loss can be detected by the use of genetic markers. In the diploid D61.M strain of Saccharomyces cerevisiae, the loss of a copy of chromosome VII that carries 3 dominant wild-type alleles is measured by expression of 3 recessive mutant alleles carried on the other remaining copy of chromosome VII. We have tested the hypothesis that expression of the 3 recessive alleles might be due to 3 simultaneous independent genetic events other than chromosome loss, such as mutation or recombination. We have measured, when possible, the frequencies of expression for each of these recessive alleles, independently and in combination one with another, under both selective and non-selective conditions. Our results show that simultaneous expression of these 3 recessive alleles is attributable to chromosome loss (greater than 98%). Similarly, at least 99% of the nocodazole-induced events are attributable to chromosome loss. In contrast, most if not all of the apparent chromosome loss induced by ethyl methanesulfonate is due to multiple events of mutation or recombination.     

The role of chromosomal alterations in human cancer development

Cancer cells become unstable and compromised because several cancer-predisposing mutations affect genes that are responsible for maintaining the genomic instability. Several factors influence the formation of chromosomal rearrangements and consequently of fusion genes and their role in tumorigenesis. Studies over the past decades have revealed that recurring chromosome rearrangements leading to fusion genes have a biological and clinical impact not only on leukemias and lymphomas, but also on certain epithelial tumors. With the implementation of new and powerful cytogenetic and molecular techniques the identification of fusion genes in solid tumors is being facilitated. Overall, the study of chromosomal translocations have revealed several recurring themes, and reached important insights into the process of malignant transformation. However, the mechanisms behind these translocations remain unclear. A more thorough understanding of the mechanisms that cause translocations will be aided by continuing characterization of translocation breakpoints and by developing in vitro and in vivo model systems that can generate chromosome translocation.     

Cytogenetic changes induced in human diploid fibroblasts by tsA58 SV40 at permissive and restrictive temperatures

Human diploid fibroblasts have been transformed by ts A58 SV40. At the permissive temperature, apparent chromosome and chromatic rearrangements were observed in a high percentage of cells and the frequency of SCE increased. If the transformed phenotype returned to normal at the restrictive temperature these alterations also returned to normal levels. Chromosome banding demonstrated many apparent chromosomal rearrangements in which diffuse staining material was joining intact chromosomes end-to-end and forming pseudostructural abnormalities. Homogeneously staining regions associated with gene amplification or virus-induced alterations in the coiling and stickiness of telomeric regions are possible mechanisms.     

A Mapping Method for SACCHAROMYCES CEREVISIAE Using rad52-Induced Chromosome Loss

Saccharomyces cerevisiae diploids homozygous for the rad52-1 mutation have previously been shown to lose chromosomes mitotically. Spontaneous events and events following low levels of X-ray or methyl methanesulfonate treatment result in monosomic diploids, whereas higher levels of treatment result in near haploidization. This rad52-1-dependent chromosome loss has been used to develop a new mapping method which can be used to assign a previously unmapped gene to a chromosome. Chromosome loss mapping can be done in either of two ways: if a diploid, homozygous for rad52-1 but heterozygous for a variety of other recessive markers, is constructed with an unmapped recessive mutation in coupling with known chromosomal markers, chromosome loss will result in the coordinate expression of the mutation and other recessive markers on the same chromosome; if, however, the diploid is constructed with the unmapped mutation in repulsion to chromosomal markers, then even haploidization will never result in the coordinate expression of the unmapped mutation and other markers on the same homologous chromosome pair--This mapping method and subsequent tetrad analyses have been used to locate hom6 on chromosome X, ade4 on chromosome XIII and cdc31 on chromosome XV and to demonstrate that met5, previously assigned to chromosome V, actually maps to chromosome X; the met- marker on chromosome V has been shown to be met6. GAL80 and SUP5, previously assigned to an unmapped fragment, have now been mapped to the right arm of chromosome XIII.     

Loss of heterozygosity in chondrosarcomas for markers linked to hereditary multiple exostoses loci on chromosomes 8 and 11.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant condition characterized by growth of multiple benign cartilage-capped tumors. EXT greatly increases the relative risk to develop chondrosarcoma, although most chondrosarcomas are sporadic. This observation suggests that, like the genes responsible for retinoblastoma and other dominantly inherited cancer susceptibility disorders, the genes that cause EXT may have tumor-suppressor function and may play a role in the pathogenesis of the related sporadic tumors. To investigate this hypothesis, we evaluated chondrosarcomas for loss of constitutional heterozygosity (LOH) at polymorphic loci linked to three recently identified genomic regions containing genes involved in EXT. LOH for markers linked to EXT1 on chromosome 8 was detected in a chondrosarcoma that arose in a man with EXT. Four of 17 sporadic tumors showed LOH for markers linked to EXT1, and 7 showed LOH for markers linked to EXT2 on chromosome 11. In all, LOH was observed for markers linked to EXT1 or EXT2 in 44% of the 18 tumors, whereas heterozygosity was retained for markers on 19p linked to EXT3. These findings support the hypothesis that genes on 8q and the pericentromeric region of 11 have tumor-suppressor function and play a role in the development of chondrosarcomas.     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

Complex repetitive arrangements of gene sequence in the candidate region of the spinal muscular atrophy gene in 5q13.

Childhood-onset proximal spinal muscular atrophy (SMA) is a heritable neurological disorder, which has been mapped by genetic linkage analysis to chromosome 5q13, in the interval between markers D5S435 and D5S557. Here, we present gene sequences that have been isolated from this interval, several of which show sequence homologies to exons of beta-glucuronidase. These gene sequences are repeated several times across the candidate region and are also present on chromosome 5p. The arrangement of these repetitive gene motifs is polymorphic between individuals. The high degree of variability observed may have some influence on the expression of the genes in the region. Since SMA is not inherited as a classical autosomal recessive disease, novel genomic rearrangements arising from aberrant recombination events between the complex repeats may be associated with the phenotype observed.