Common polymorphisms and somatic mutations in human base excision repair genes in ovarian and endometrial cancers.

The purpose of this study was to determine whether the human APEX and OGG1 genes, encoding proteins important in base excision repair (BER) of DNA, contain nucleotide sequence polymorphisms or are mutated somatically in tumors from women diagnosed with ovarian or endometrial cancer. Based upon the analysis of germline DNA from 83 individuals, 63 with ovarian cancer and 20 with endometrial cancer, we found two missense polymorphisms in APEX (Q51H and D 148E) and two missense (A3P and S326C) and one intronic (Exon 5-15 bp) polymorphism in OGG1. The frequencies of the various alleles (in the ovarian and endometrial cancer patients combined) were 4.8% for 51-His and 56.2% for 148-Glu in APEX, and 1.0% for 3-Pro and 20.0% for 326-Cys in OGG1. Somatic mutations in APEX (P112L, W188X and R237C) were identified in three of 20 endometrial tumors, but no mutations were identified in APEX in 43 ovarian tumors, or in OGG1 at either tumor site. Given the crucial role of the APEX and OGG1 proteins in BER of oxidative DNA damage, the identified polymorphisms are good candidates for genetic epidemiologic studies of cancer susceptibility, while the finding that three of 20 (15%) endometrial tumors have somatic mutations in APEX suggests that inactivation of the BER pathway is important for the development of endometrial cancer in at least a subset of cases.

Loss of heterozygosity in usual and special variant carcinomas of the endometrium.

Endometrial carcinoma is the most common invasive malignancy of the female genital tract, and it exists as two different clinicopathologic forms: an estrogen-dependent, "usual" type and an estrogen-independent "special variant" type. Despite the frequency of endometrial cancer, little is known about the molecular genetic events that contribute to its pathogenesis. The accumulation of genetic alterations identified through the study of loss of heterozygosity (LOH), gene mutation, and gene activation in tumor DNA has been associated with the establishment and progression of a variety of human malignancies. A relatively low incidence of LOH has been reported in usual type endometrial cancers; however, special variant tumors have rarely been included in the reported studies. To understand the molecular events that contribute to both forms of endometrial cancer, 31 tumors have been surveyed for events of LOH on all chromosomes. The study groups included 18 tumors of the usual type and 13 special variant tumors. Polymorphic loci were studied by Southern blot analysis and polymerase chain reaction (PCR) of microsatellite loci. Normal tissue in each case served as a control. Both frequency and patterns of LOH differed greatly between the two tumor types. Although LOH was frequently detected in the special variant tumors, it was rare in the usual type tumors. LOH was detected in only 8 of the 18 usual tumors, with chromosomes 17, 13, and 2 being the most frequently affected (22%, 20%, and 19%, respectively). In contrast, LOH was detected in all cases of special variant tumors, with chromosomes 17p, 14, and 12 showing the highest LOH (83%, 77%, and 40%, respectively). Two cases of microsatellite instability (MI) were detected among the usual type tumors. These findings suggest that the clinicopathologic phenotypes observed in these tumor types are likely caused by different tumorigenic pathways that reflect alterations of different cancer-controlling genes.

Evidence for a unifocal origin in familial ovarian cancer.

OBJECTIVE: The purpose of this investigation was to determine the pattern of loss of heterozygosity in multiple tumor sites from familial ovarian cancer cases. If ovarian cancer arises focally in one ovary and then metastasizes to other sites, a similar pattern should be seen in all tumor sites. However, if ovarian cancer arises multifocally throughout the peritoneal cavity, a different pattern of loss would be expected among the different sites. STUDY DESIGN: The presence or absence of loss of specific alleles for 9 loci on chromosomes 1, 6, 11, 13, 16, and 17 was determined in multiple tumor sites from 12 familial ovarian cancer cases. RESULTS: The frequency of loss of heterozygosity was as follows: chromosome 17 (100%), chromosome 13 (82%), chromosome 6 (80%), chromosome 16 (73%), chromosome 1 (57%), and chromosome 11 (22%). In every case an identical pattern was present for at least one locus. In four cases loss of the same allele was present in tumor from the ovary and all metastatic sites for all informative loci. In the remaining eight cases loss of the same allele for one to five (mean three) loci was detected. CONCLUSIONS: The pattern of loss of heterozygosity in the 12 familial ovarian cancers included in this investigation favors a unifocal origin of disease. A dual primary origin could not be absolutely excluded in 3 cases. High frequencies on chromosomes 17q and 13 suggest that loss of whole or part of these chromosomes is important in ovarian carcinogenesis.

Genetic alterations distinguish different types of ovarian tumors.

Seventy-four sporadic ovarian tumors were studied for loss of heterozygosity (LOH) and microsatellite instability (MI) with 20 polymorphic markers on chromosome 17 and at least I marker on every other chromosome. Additionally, activation of the K-ras oncogene was examined through mutation analysis of codon 12. A majority of the tumors analyzed were low grade and/or of the mucinous histologic type. A negative correlation between LOH on chromosome 17 and K-ras activation was observed, with the former alteration present in the majority of high grade serous and endometrioid tumors and the latter most commonly found in the mucinous and low malignant potential (LMP) tumors. In 60% of cases where LOH on chromosome 17 was present, it was observed at all informative markers, indicating chromosome loss. In these cases, frequent events of LOH were observed on the other chromosomes. When confined events of LOH were observed on chromosome 17, fewer events of LOH were observed on the other chromosomes. In the absence of LOH on chromosome 17, LOH on other chromosomes was rare. K-ras activation was most commonly observed in tumors with no LOH events. Two endometrioid tumors and 2 mucinous tumors demonstrated MI. Our data support the involvement of different molecular pathways in the development of different types of ovarian tumors.

The molecular basis of ovarian cancer.

BACKGROUND: Molecular genetic studies of ovarian cancer have been limited by the inaccessible location of the ovary, the advanced stage of tumors available for analysis, and the lack of a well-defined precursor lesion. However, genetic alterations important in ovarian tumorigenesis have been identified recently. METHODS: Molecular genetic evaluation of ovarian cancer primarily has utilized mutation analysis, immunohistochemical techniques, and loss of heterozygosity (LOH) studies. RESULTS: Overexpression of the HER-2/neu oncogene is present in approximately one third of ovarian cancers and is associated with poor prognosis. Mutations of the K-ras oncogene have been identified in a similar proportion of mucinous ovarian tumors, including borderline tumors. The study authors as well as others have frequently detected LOH on chromosome 17, including the p53 and BRCA1 loci, and at 17p3.3 and 1717q22-23. Genetic linkage analysis indicates that the majority of inherited ovarian cancers are caused by mutations in the BRCA1 gene. Mutations in mismatch repair genes have been identified in ovarian cancers that occur as part of the hereditary nonpolyposis colon cancer syndrome. CONCLUSIONS: Sporadic ovarian tumors are the end result of a complex pathway involving multiple oncogenes and tumor suppressor genes, including HER-2/neu, K-ras, p53, BRCA1, and additional tumor suppressor genes on chromosome 17. The majority of inherited ovarian cancers are due to mutations in the BRCA1 gene, which appears to be a tumor suppressor gene. It is hoped that an increased understanding of the molecular basis of ovarian cancer will lead to advances in prevention, diagnosis, and treatment.

Loss of heterozygosity and genomic instability in synchronous endometrioid tumors of the ovary and endometrium.

BACKGROUND: The unknown etiology of endometrioid carcinomas of the ovary and the relatively high frequency of a concomitant carcinoma of the endometrium in these patients warrants study of such tumors. The aim of this study was to identify the genetic alterations involved in endometrioid ovarian cancer development, and to determine whether primary tumors of the endometrium and synchronous primary endometrioid tumors of the ovary could be distinguished based on differing patterns of genetic alterations. The distinction of metastatic carcinoma of the ovary from other synchronous primary tumors is often difficult but has important therapeutic and prognostic implications. METHODS: This study examined the genetic alterations at 28 polymorphic DNA markers in the DNA of tumors of 17 patients with endometrioid carcinoma of the ovary, including 5 nonmetastatic ovarian tumors, 5 ovarian tumors metastatic to the uterus, and 7 endometrioid ovarian tumors with a synchronous primary endometrial tumor. RESULTS: Chromosomes 17 and 22 were found to be the most common sites of loss of heterozygosity (LOH) in the 17 patients studied. Loss of heterozygosity on chromosome 17 was associated with advanced stage ovarian tumors. In 96% of LOH events in the metastatic tumors, LOH was observed in the primary tumor and in the metastatic site. Conversely, in four of seven synchronous tumors in which LOH was observed, LOH was confined to the ovarian tumor. Genomic instability was identified in two of seven patients with synchronously occurring tumors that did not demonstrate LOH. A positive family history was noted for these two patients. CONCLUSIONS: A lack of shared genetic alterations and in synchronously occurring endometrial and endometrioid ovarian tumors indicates independent developmental pathways for these tumors. Loss of heterozygosity on chromosome 17 in endometrioid ovarian carcinoma may indicate transition to a more aggressive tumor.

Genetic alterations on chromosome 17 distinguish different types of epithelial ovarian tumors.

Epithelial tumors of the ovary are the most common ovarian tumors of adult women. They exist in several different histological patterns and exhibit varying degrees of aggressiveness. Molecular genetic studies in epithelial ovarian cancer have shown that loss of heterozygosity (LOH) for regions of chromosome 17 is a common event, probably reflecting the inactivation of one or more tumor suppressor genes present on this chromosome. We examined 87 sporadic epithelial ovarian tumors of different grade and histological type at 16 loci on this chromosome and found that 35% of them showed LOH for chromosome 17. Of these, 84% showed LOH for all informative markers, suggesting that loss of the entire chromosome 17 homologue may have occurred. Interestingly, chromosome 17 loss was observed frequently in serous tumors (49%), was less common in endometrioid tumors (15%), and was rare in mucinous tumors (4%) (P = .01 and P = .0002, respectively). Our findings support the concept that the histological subtypes of epithelial ovarian cancer may be the result of different molecular genetic events.

Hypermethylation at a chromosome 17 "hot spot" is a common event in ovarian cancer.

Alterations of normal DNA methylation patterns have been reported in various types of human tumors. These alterations are represented by genome wide hypomethylation and by region specific hypermethylation. One commonly hypermethylated region is 17p13.3 (D17S5), the putative site of a tumor suppressor gene. In this study we report that hypermethylation at this locus occurs frequently (33%) in ovarian tumors. We reported previously that loss of chromosome 17 is a common event in serous epithelial ovarian tumors. A correlation of the methylation event and chromosome 17 loss suggests that hypermethylation at D17S5 precedes chromosome 17 loss.

Molecular genetic changes in human epithelial ovarian malignancies.

The frequent finding of loss of heterozygosity (LOH) for a specific chromosomal marker in tumor DNA compared to normal DNA suggests the presence of a closely linked tumor-suppressor gene. Using Southern blot analysis, 34 primary ovarian epithelial tumors were examined for the presence of tumor-specific allelic losses, using six probes for chromosomes 6q, 11p, 13q, 16q, and 17p. A high incidence of LOH was observed on 11p, 13q, and 17p. LOH for 17p was present in 3 of 4 (75%) informative benign ovarian tumors, 1 of 5 (20%) borderline tumors, and 16 of 24 (67%) invasive ovarian cancers. Allelic loss with the H-ras1 probe on 11p was present in 10 of 19 (53%) invasive tumors but was not identified in 6 benign or borderline tumors. LOH on 13q was present in 18 of 31 (58%) informative cases including 8 of 10 (80%) Stage 1 tumors. This preliminary study suggests that loss of tumor-suppressor genes on chromosomes 13q and 17p may be early events in ovarian tumorigenesis and that changes on chromosome 11p are later events.

DNA methylation represses FMR-1 transcription in fragile X syndrome.

Fragile X syndrome is the most frequent form of inherited mental retardation and segregates as an X-linked dominant with reduced penetrance. Recently, we have identified the FMR-1 gene at the fragile X locus. Two molecular differences of the FMR-1 gene have been found in fragile X patients: a size increase of an FMR-1 exon containing a CGG repeat and abnormal methylation of a CpG island 250 bp proximal to this repeat. Penetrant fragile X males who exhibit these changes typically show repression of FMR-1 transcription and the presumptive absence of FMR-1 protein is believed to contribute to the fragile X phenotype. It is unclear, however, if either or both molecular differences in FMR-1 gene is responsible for transcriptional silencing. We report here the prenatal diagnosis of a male fetus with fragile X syndrome by utilizing these molecular differences and show that while the expanded CGG-repeat mutation is observed in both the chorionic villi and fetus, the methylation of the CpG island is limited to the fetal DNA (as assessed by BssHII digestion). We further demonstrate that FMR-1 gene expression is repressed in the fetal tissue, as is characteristic of penetrant males, while the undermethylated chorionic villi expressed FMR-1. Since the genetic background of the tissues studied is identical, including the fragile X chromosome, these data indicate that the abnormal methylation of the FMR-1 CpG-island is responsible for the absence of FMR-1 transcription and suggests that the methylation may be acquired early in embryogenesis.

A transposon-like element in the deletion-prone region of the dystrophin gene.

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.

The sulfatase gene family: cross-species PCR cloning using the MOPAC technique.

Several human sulfatase cDNAs have recently been cloned, revealing highly conserved domains of protein similarity. We have used this information for the isolation of sulfatase genes in different species using the polymerase chain reaction (PCR). Degenerate oligonucleotide primers corresponding to these regions of identity among human arylsulfatases A, B, and steroid sulfatase (ARSA, ARSB, and STS) were designed. The primers were used in the PCR amplification of reverse transcribed RNA (RT-PCR) from multiple tissues in human and mouse. Amplification products were obtained from mouse liver and from human liver, lymphoblasts, kidney, intestine, heart, muscle, and brain cDNA samples. Each of the PCR products was subcloned into a plasmid vector, and several subclones were characterized by colony hybridization and DNA sequencing. All the previously identified human ARSA, ARSB, and STS were found among our clones, indicating the power of the technique. Sequence analysis of two mouse clones showed high degrees of homology with the human ARSA and ARSB sequences, respectively, and likely represent the murine homologues of these enzymes. These are the first sulfatase genes isolated in the mouse. A murine equivalent for STS could not be identified, suggesting its strong diversity from the human homologue.

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.

A gene deleted in Kallmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules.

Kallmann's syndrome (clinically characterized by hypogonadotropic hypogonadism and inability to smell) is caused by a defect in the migration of olfactory neurons, and neurons producing hypothalamic gonadotropin-releasing hormone. A gene has now been isolated from the critical region on Xp22.3 to which the syndrome locus has been assigned: this gene escapes X inactivation, has a homologue on the Y chromosome, and shows an unusual pattern of conservation across species. The predicted protein has significant similarities with proteins involved in neural cell adhesion and axonal pathfinding, as well as with protein kinases and phosphatases, which suggests that this gene could have a specific role in neuronal migration.

Absence of expression of the FMR-1 gene in fragile X syndrome.

We previously reported the isolation of a gene (FMR-1) expressed in brain at the fragile X locus. One exon of this gene lies within an EcoRI fragment that exhibits length variation in fragile X patients. This exon also contains the CGG repeat within the CpG island hypermethylated in fragile X patients. To study the involvement of the FMR-1 gene in the fragile X syndrome, its expression was studied in lymphoblastoid cell lines and leukocytes derived from patients and normal controls. FMR-1 mRNA was absent in the majority of male fragile X patients, suggesting a close involvement of this gene in development of the syndrome. Normal individuals and carriers all show expression. The methylation status of the BssHII site at the CpG island was also studied by Southern blot analysis of DNA from patients, carriers, and controls. The minority of fragile X affected males that show expression of FMR-1 demonstrated an associated incomplete methylation of the BssHII site.

Alu-primed polymerase chain reaction for regional assignment of 110 yeast artificial chromosome clones from the human X chromosome: identification of clones associated with a disease locus.

Over 400 yeast artificial chromosome (YAC) clones were isolated from the human X chromosome, and 110 of these were assigned to regions defined by chromosome translocation and deletion breakpoints. Polymerase chain reaction using Alu primers was applied to YAC clones in order to generate probes, to identify overlapping clones, and to derive "fingerprints" and sequence data directly from total yeast DNA. Several clones were identified in regions of medical interest. One set of three overlapping clones was found to cross a chromosomal translocation implicated in Lowe syndrome. The regional assignment of groups of YAC clones provides initiation points for further attempts to develop large cloned contiguous sequences, as well as material for investigation of regions involved in genetic diseases.

Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.