Affiliations of authors: R. E. Buller (Division of Gynecologic Oncology and Department of Pharmacology), A. K. Sood, T. Lallas, T. Buekers, J. S. Skilling (Division of Gynecologic Oncology), the Departments of Obstetrics and Gynecology, The University of Iowa Hospitals and Clinics, Iowa City.
Correspondence to: Richard E. Buller, M.D., Ph.D., Division of Gynecologic Oncology, Departments of Obstetrics and Gynecology, 200 Hawkins Dr., #4630 JCP, Iowa City, IA 52242-1009 (e-mail: richard-buller{at}uiowa.edu).
Present address: J. S. Skilling, Department of Obstetrics & Gynecology, Division of Gynecologic Oncology, University of California, Davis Medical Center, Sacramento, CA.
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ABSTRACT |
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INTRODUCTION |
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Due to its highly informative nature, study of the CAG repeat of exon 1 of the androgen receptor (AR) has facilitated tumor clonality studies (6-10). Four distinct potential methylation sites located upstream of this repeat control expression of the AR locus. When these sites are methylated, as on the inactive X chromosome, the gene is not transcribed. When these sites are unmethylated, as on the active X chromosome or in males, the AR gene is transcribed. The methylation sites are sequences recognized and cut by the restriction endonucleases HhaI (GCGC) and HpaII (CCGG) only when they are unmethylated. Resistance to digestion by these enzymes thus identifies the inactive X chromosome (11). At least two laboratories studying tumor clonality with markers less informative than this CAG repeat have noted nonrandom X-chromosome inactivation in germline DNA of both healthy and cancer-affected individuals (12,13). Two large AR analyses of X-chromosome inactivation patterns in more than 400 healthy females have confirmed that while patterns generally follow a normal distribution, sizable segments of the population are skewed in their pattern of X-chromosome inactivation. (14,15)
Tumor suppressor genes exert their effects according to the Knudson hypotheses in one of two ways (16,17). For most sporadic cancers, loss of one allele (loss of heterozygosity = LOH) is followed by mutation within the remaining allele. This mechanism explains the role of classic tumor suppressor genes, such as p53 (also known as TP53) and retinoblastoma (also known as Rb) (18). Additionally, germline carriers of a tumor suppressor gene mutation are predisposed to cancer on the basis of the need only to lose the wild-type allele because the remaining allele already carries a mutation. This mechanism explains the predisposition to a variety of cancers seen in individuals with Li-Fraumeni syndrome carrying germline p53 mutations (19). It also explains early onset, bilateral retinoblastomas in individuals carrying germline Rb mutations. Hereditary, early onset, breast, and ovarian cancers have been attributed to germline mutations of the BRCA1 and BRCA2 tumor suppressor genes (20-24). All four of these genes are autosomal in location so that normal human cells carry two copies of each gene. In contrast, for putative X-linked tumor suppressor genes, nonrandom X-chromosome inactivation is equivalent to a functional LOH in all affected cells. This equivalency is because each cell carries only a single active X chromosome. Therefore, either a mutation or a loss of the active allele from the preferentially active X chromosome would render the cell without a functional copy of putative X-linked tumor suppressor genes in a single step.
Several authors have demonstrated that in ovarian cancer, frequent LOH at several regions of the X chromosome suggests that this chromosome may indeed harbor one or more tumor suppressor genes (25-28). Nonrandom X-chromosome inactivation has been shown to play a role in the development of X-linked recessive disorders such as Wiskott-Aldrich syndrome (29). Thus, Mendelian inheritance of nonrandom X-chromosome inactivation associated with a germline putative X-chromosome-linked tumor suppressor gene mutation could contribute to some cases of familial cancers by short-circuiting the traditional Knudson model (16,17). To test this hypothesis as a mechanism of hereditary ovarian cancer, we evaluated germline DNA from a cohort of ovarian cancer patients and, where possible, from their affected first- and second-degree relatives. The frequency of nonrandom X-chromosome inactivation was also determined for healthy, unrelated control female subjects without a family history of breast, ovarian, or colon cancer.
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MATERIALS AND METHODS |
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X-Chromosome inactivation studies. The highly polymorphic
trinucleotide repeat (CAG) in the human AR gene was studied to
determine X-chromosome inactivation (10). Germline DNA was
subjected to restriction enzyme digestion at 37 °C by mixing
50-200 ng of DNA and 20 U of HhaI restriction endonuclease
(New England Biolabs, Inc., Beverly, MA) in New England buffer #4 (New
England Biolabs, Inc.) in a total volume of 10 µL for the times
indicated, usually 12 hours. In parallel, each sample served as its own
control by omitting the restriction enzyme and normalizing the volume
by the addition of distilled water. Reactions were terminated by heat
inactivation at 95 °C for 10 minutes. Alternatively,
substitution of the restriction enzyme HpaII for HhaIwas carried out to confirm results. Each digest sample (2 µL)
was amplified in a 10-µL polymerase chain reaction (PCR) reaction
containing 1 µM of 32[P]end-labeled AR sense
primer, 2 µM of unlabeled AR antisense primer, 100
mM deoxyadenosine triphosphate (dATP), 100 mM
deoxycytidine triphosphate (dCTP), 100 mM deoxyguanosine
triphosphate (dGTP), 100 mM deoxythymidine triphosphate
(dTTP), 0.1 U Taq polymerase (Promega Corp., Madison, WI), 2.0
mM MgCl2, 50 mM KCl, 10 mM
Tris-HCl buffer, pH 9.0, 0.1 mg/µL of gelatin. PCR amplification
was performed with an initial denaturation step of 95 °C for
5 minutes followed by 29 cycles as follows: 95 °C for 45
seconds, 61.5 °C for 30 seconds, and 72 °C for 30
seconds. The PCR products were analyzed on 8% polyacrylamide
sequencing gels. Labeling only one of the primers enhanced band
resolution. Primers used in this reaction were: AR-sense: 5'-TCC
AGA ATC TGT TCC AGA GCG TGC-3' and AR-antisense: 5'-GCT GTG AAG
GTT GCT GTT CCT CAT-3' (10). In an attempt to quantitate
differences in methylation for informative DNA samples, we evaluated
the ratio of AR amplimer band intensity on autoradiograms. The lower
band (shorter CAG repeat) was taken as the reference band. For
informative samples, calculation of a modified allelic cleavage ratio
was performed:
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The expected value of the modified allelic cleavage ratio is 1.0 because germline tissues are predicted to contain equal populations of active maternal and paternal AR alleles, according to the Lyon hypothesis. Several investigators have noted some degree of asymmetry or skewing in the distribution of this parameter and have defined nonrandom X-chromosome inactivation variously as twofold (32), threefold (12), or fourfold (13) differences in the band intensities of the X-linked gene following appropriate restriction digest. Initially, we quantitated this ratio for all DNA samples from control subjects, both digested and undigested (see the "Results" section), by optical scans on a laser densitometer (Protein & DNA Image Wave System, Huntington Station, NY). This analysis allowed us to confirm the published observation of Mutter et al. (32) that differences of this magnitude are readily detectable visually. We found significant variability in the calculated modified allelic cleavage ratio for a given sample. Parameters influencing this variability included the observer, band separation on a given gel, whether 35S or 32P-adenosine triphosphate (ATP) was used, and the scan technique such as adjustment of baseline and whether peak height or area under the curve was used. Accordingly, we arbitrarily chose to utilize a visual difference of greater than 3 : 1 between band intensities to denote nonrandom X-chromosome inactivation for our analysis of the germline DNA samples from patients with ovarian cancer. This corresponds to a modified allelic cleavage ratio of less than 0.33 or greater than 3.0 and resulted in a minimum difference between band intensities of at least twofold, independent of observer, and scan technique determined by repetitive scans made by several observers. Furthermore, as shown below in the "Results" section, this difference exceeds any potential systematic error. Substitution of 7-deaza-2'-dGTP for dGTP was performed on selected samples exhibiting nonrandom X-chromosome inactivation to rule out PCR bias, which may occur when amplifying GC-rich targets (33).
Determination of loss of heterozygosity. LOH at various loci in available paired ovarian cancer (somatic) and germline DNA samples was determined using standardized PCR methodology. p53 LOH was determined by allelotyping the tumor on the basis of the codon 72 polymorphism, Alu repeats, and mutational analysis as we have described (31). BRCA1 LOH was determined using the microsatellite repeats D17S855, D17S1322, and D17S1323 (34). The D17S1322 and D17S1323 markers can be multiplexed. BRCA2 LOH was based on allelic loss of either D13S1700, D13S1701, or D13SBR2. In each case, approximately 20 ng of DNA was added to a reaction mix that contained 32[P] end-labeled primer, 100mM dATP, 100mM dCTP, 100mM dGTP, 100mM dTTP, and 1 U of Taq polymerase for amplification as we have described (31,35-36). For this analysis, we selected 102 probands from our 213 case patient cohort for which paired germline and ovarian cancer (somatic) DNA samples were available and required the tumor DNA samples to be informative at a minimum of two of the three study loci (BRCA1, BRCA2, or p53). The samples were also selected to render as many informative (100 of 102) at the AR locus as possible to maximize the power of the LOH analysis.
Family history, pedigree, pathologic confirmation of cancers, and follow-up. A complete pedigree was obtained on each individual studied with pathologic follow-up of reported breast or ovarian cancers among family members when possible. Family history of breast or ovarian cancer was determined by reviewing the proband's pedigree to determine the number of relatives affected by these cancers. For this analysis, we counted only first-, second-, and third-degree relatives. A positive family history was noted if one additional ovarian cancer or two additional breast cancers were documented by pathologic review or by death certificate.
Statistical analysis. Statistical analyses included population sample statistics, skewness, kurtosis, Pearson chi-squared testing of categorical or t tests of continuous variables. The binomial distribution was used to calculate the probability of a specific combination of BRCA1 mutations in association with nonrandom X-chromosome inactivation (14). These calculations were performed by the BMDP Statistical Software (Biomathematical Data Package Statistical Software, Inc., Los Angeles, CA) package on a desktop computer. P values less than .05 were considered significant. All statistical tests were two-sided.
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RESULTS |
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The pedigrees of many ovarian cancer probands with nonrandom
X-chromosome inactivation were striking in that a family history of
breast and/or ovarian cancer was commonly reported. Pedigrees of four
such individuals are shown in Fig. 4. Fig.
5
represents a gel analysis of available germline DNA
samples from three of the families from Fig. 4
. In these figures, a
Roman numeral represents the generation and an Arabic subscript serves
to differentiate between individuals within each generation. When two
bands appear on PCR amplification of the control (undigested) DNA
sample, but only one from the PCR amplified restriction nuclease
digested germline DNA sample, one is able to determine if the same X
chromosome is active (disappears with digest) in the germline of
multiple family members and the association of this relationship to
cancers in the families. For a given family, the presence of a band at
the same position in the gel for two different family members indicates
a shared AR allelotype. Several important observations emerge from a
careful study of these two figures. Individuals in different
generations of the same family can demonstrate germline nonrandom
X-chromosome inactivation associated with either ovarian or breast
cancer or both. This association did not hold for all individuals in a
given family who developed one of these cancers. The potential for
mother-daughter (families 8, 23, and 26) as well as father-daughter
transmission (Family 15) exists. Finally, not all affected individuals
share the same active or inactive AR allelotypes. Thus nonrandom
X-chromosome inactivation may play a complex role in some hereditary
breast and ovarian cancer families.
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DISCUSSION |
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The original reports of skewed patterns of X-chromosome inactivation in normal tissue emanated from tumor clonality studies (12,13). There are no previous reports of this phenomenon related to ovarian cancer. A 33% incidence of nonrandom X-chromosome inactivation among healthy control subjects in the present study is higher than the 23% found in the hematologic normal individuals studied by Gale et al. (12) in England and much higher than the 3/81 (4%) reported by Vogelstein et al. (13). However, if we apply the same 3 : 1 difference in relative band intensities to the data from 365 normal females reported by Naumova et al. (14), we calculate a nonrandom X-chromosome inactivation rate of approximately 32%. This rate is precisely in agreement with the rate among the control individuals in our study. In marked contrast, nonrandom X-chromosome inactivation was found in 53% of germline DNA from probands we have studied with invasive ovarian cancer. This frequency is a remarkable finding that is statistically significantly different from: 1) our healthy control subjects without a family history of breast or ovarian cancer, 2) individuals with borderline ovarian cancers, and 3) the frequency noted among a large series of healthy females previously reported in the literature (14). Clearly, the use of the AR polymorphism to determine X-chromosome inactivation in tumor clonality studies should be very carefully controlled.
Occasionally, we have drawn blood samples during chemotherapy. Because profound neutropenia and lymphocytopenia can occur after chemotherapy, it is theoretically possible that we sampled clonal expansions following pancytopenic nadirs for a small subset of patients (12). This explanation is unlikely because we have repeated blood draws more than a year following completion of chemotherapy and the same X-chromosome was inactivated in the follow-up specimens (data not shown). In addition, we have retrieved several normal tissue samples such as large and small bowel removed as part of the cytoreductive ovarian cancer surgery for three individuals who demonstrated skewed patterns of X-chromosome inactivation. In each case, the same skewed pattern of X-chromosome inactivation was present in the non-hematologic sample as well. These studies require expansion. However, on a preliminary basis it appears that nonrandom X-chromosome inactivation is not tissue specific, but rather can be a characteristic of a given individual. This finding argues in favor of nonrandom X-chromosome inactivation occurring due to selection for a gene that offers proliferative advantage not only to lymphoid cells, but to precursor cells for any organ of the body as well. The association of nonrandom X-chromosome inactivation with germline BRCA1 mutation could in part explain why there are increases in prostate cancer, and colon cancer in addition to breast and ovarian cancer in hereditary breast or ovarian cancer families (42).
Gale et al. (43) have observed both similar and dissimilar patterns of skewed X-chromosome inactivation between normal hematologic and nonhematologic tissues. These authors offer an alternative explanation for skewed X-chromosome inactivation arguing that this phenomenon is still random. Specifically, they support the hypothesis of McLaren (44) that the number of cells present in the embryo at the time of inactivation or, the number of cells that generate a given organ, modifies the pattern of X-chromosome inactivation.
The pedigrees and AR allelotype analyses we have presented suggest that nonrandom X-chromosome inactivation can be inherited. By studying additional family members, the development of more cancers in the study families, and the use of other highly informative X-chromosome markers to render some of our noninformative AR samples informative, we hope to determine in future studies whether or not the inheritance pattern is Mendelian.
We have shown that a common AR allelotype and the same active X chromosome can be shared among individuals with early onset breast and ovarian cancers. The two probands from Family 15, however, relate the independence of nonrandom X-chromosome inactivation and the AR allelotype. Thus, a factor other than the AR itself is responsible for this process. Naumova et al. (14) have proposed a model of nonrandom X-chromosome inactivation that results from an X-chromosome associated factor, X-inactive specific transcript (XIST), distinct from the X-chromosome inactivation center. These authors used their Family K1362 to demonstrate that nonrandom X-chromosome inactivation was unlikely to be due to an autosomal factor since there is a low probability that such a factor could be transferred from the grandmother to her son, and to all seven granddaughters. None the less, we have observed two distant relatives in our Family 15 with autosomal germline 17q21 BRCA1 germline mutation who both demonstrate nonrandom X-chromosome inactivation. Overall, 9 of 11 informative ovarian cancer probands carrying a germline BRCA1 mutation also demonstrated a skewed pattern of X-chromosome inactivation. We estimated that the probability of this association happening by chance alone ranged between .0002<P<.008. In addition, down-regulation of BRCA1 independent of BRCA1 mutation is a common occurrence in ovarian cancer (45). Therefore, it seems likely that some X-chromosome factor may modify the expression or action of the BRCA1 gene product. With only one functionally active X chromosome, inheritance of a germline mutation in such a factor on the active X chromosome, but associated with a skewed X-chromosome inactivation pattern could provide an explanation for some cases of hereditary breast and ovarian cancer independent of BRCA1 and BRCA2 mutation.
Female expression of at least two X-linked recessive genetic disorders has now been reported to result from nonrandom X-chromosome inactivation. These include Snyder-Robinson syndrome, a form of X-linked mental retardation, linked to Xp21.3-22.12 (15), and Wiskott-Aldrich syndrome linked to mutation of the WASP protein encoded at Xp11.22-23 (29). In the former, a mutation in the minimal promoter of the XIST gene at Xq13.2 was implicated as causal of nonrandom X-chromosome inactivation, while in the latter, a germline mutation in exon 4 of WASP coupled with nonrandom X-chromosome inactivation was responsible for disease. Since, the XIST promoter mutation was found to be sufficiently rare in the general population (less than 1 in 1166 independent chromosomes), we must hypothesize that other factors contribute to nonrandom X-chromosome inactivation associated with ovarian cancer. Indeed, nonrandom X-chromosome inactivation in general is much more prevalent than are XIST promoter mutations. Several authors have proposed that a candidate lies in the vicinity of Xq27 (46-49).
Another mechanism by which nonrandom X-chromosome inactivation could occur is via disruption of the normal methylation process. Methylation abnormalities, including both hypomethyation and hypermethylation, are frequently seen in cancer (50-52). Indeed, hypermethylation appears to be an important mechanism of gene suppression in cancer. The degree to which germline nonrandom X-chromosome inactivation was found in ovarian cancer probands tempts one to speculate that these individuals might carry a genetic predisposition to methylation abnormalities in general. If this is the case, nonrandom X-chromosome inactivation could represent an independent risk factor for the development of many cancers.
Cheng et al. (27) performed an analysis of borderline ovarian cancer tumor DNA samples that supports the presence of at least one tumor suppressor gene on the X chromosome. Other tumor suppressor genes are certain to be found based upon existing LOH analyses (25-26). Recognition of the importance of the X chromosome in cancer is emerging (53). X-chromosome tumor suppressor genes found in concert with nonrandom X-chromosome inactivation could provide a powerful carcinogenic stimulus. This combination is equivalent to a functional LOH. This combination could contribute to a one-step mechanism of carcinogenesis that contrasts with the classical Knudson two-step model (16,17).
In conclusion, we have begun to characterize an unusual and unpredicted phenomenon that departs from the classic Lyon hypotheses of random X-chromosome inactivation. We found that nonrandom germline X-chromosome inactivation is prevalent among individuals who develop invasive ovarian cancer, but not borderline ovarian cancer. Failure of this phenomenon to associate with borderline ovarian cancer provides additional evidence along with genomic patterns of LOH (27,54), microsatellite instability (55), and K-ras mutation (56,57) that borderline cancers do not evolve into invasive ovarian cancer. It may have important molecular epidemiological ramifications as an alternative mechanism for the development of hereditary breast and ovarian cancer independent of BRCA1 mutation.
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NOTES |
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We thank Colleen Fullenkamp, Krista Larson, and Sara McClain for their technical assistance.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Gartler SM, Ziprowski L, Krakowski A, Ezra R, Szeinberg A, Adam A. Glucose 6 phosphate dehydrogenase mosaicism as a tracer in the study of hereditary multiple trichoepithelioma. Am J Human Genetics 1966;18:282-7.
2 Beutler E, Collins Z, Irwin LE. Value of genetic variants of glucose-6-phosphate dehydrogenase in tracing the origin of malignant tumors. N Engl J Med 1967;276:389-91.[Medline]
3 Smith JW, Townsend DE, Sparks RS. Genetic variants of glucose-6-phosphate dehydrogenase in the study of carcinoma of the cervix. Cancer 1971;28:529-32.[Medline]
4 Lyon M. X-chromosome inactivation as a system of gene dosage compensation to regulate gene therapy. Prog Nucleic Acid Res Mol Biol 1989;36:119-30.[Medline]
5 Gartler SM, Riggs AD. Mammalian X-chromosome inactivation. Ann Rev Genet 1983;17:155-90.[Medline]
6 Jacobs IJ, Kohler MF, Wiseman RW, Marks JR, Whitaker R, Kerns BA, et al. Clonal origin of epithelial ovarian carcinoma: analysis by loss of heterozygosity, p53 mutation, and X-chromosome inactivation. J Natl Cancer Inst 1992;84:1793-8.[Abstract]
7 Tsao SW, Mok CH, Knapp RC, Oike K, Muto MG, Welch WR, et al. Molecular genetic evidence of a unifocal origin for human serous ovarian carcinomas. Gynecol Oncol 1993;48:5-10.[Medline]
8 Enomoto T, Fujita M, Inoue M, Tanizawa O, Nomora T, Shroyer KR. Analysis of clonality by amplification of short tandem repeats. Carcinomas of the female reproductive tract. Diagn Mol Pathol 1994;3:292-7.[Medline]
9 Pejovic T, Heim S, Mendahl N, Elmfors B, Furgyik S, Floderus UM, et al. Bilateral ovarian carcinoma: cytogenetic evidence of unicentric origin. Int J Cancer 1991;47:358-61.[Medline]
10 Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen receptor gene correlates with X chromosome inactivation. Am J Hum Genet 1992;51:1229-39.[Medline]
11 Razin A, Riggs AD. DNA methylation and gene function. Science 1980;210:604-10.[Medline]
12 Gale RE, Wheadon H, Linch DC. X-chromosome inactivation patterns using HPRT and PGK polymorphisms in haematologically normal and post-chemotherapy females. Br J Haematol 1991;79:193-7.[Medline]
13 Vogelstein B, Fearon ER, Hamilton SR, Preisinger AC, Willard HF, Michelson AM, et al. Clonal analysis using recombinant DNA probes from the X-chromosome. Cancer Res 1987;47:4806-13.[Abstract]
14 Naumova AK, Plenge RM, Bird LM, Leppert M, Morgan K, Willard HF, et al. Heritability of X chromosomeinactivation phenotype in a large family. Am J Hum Genet 1996;58:1111-9.[Medline]
15 Plenge RM, Hendrich BD, Schwartz C, Arena JF, Naumova A, Sapienza C, et al. A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Nat Genet 1997;17:353-6.[Medline]
16 Knudson AG Jr. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A 1971;68:820-3.[Abstract]
17 Knudson AG Jr. Antioncogenes and human cancer. Proc Natl Acad Sci U S A 1993;90:10914-21.[Abstract]
18 Hollingsworth RE, Lee WH. Tumor suppressor genes: new prospects for cancer research. J Natl Cancer Inst 1991;83:91-6.[Abstract]
19 Li FP, Garber JE, Friend SH, Strong LH, Patenaude AF, Juengst ET, et al. Recommendations on predictive testing for germ line p53 mutations along cancer-prone individuals. J Natl Cancer Inst 1992;84: 1156-60.[Medline]
20 Boyd J, Rubin SC. Hereditary ovarian cancer: molecular genetics and clinical implications. Gynecol Oncol 1997;64:196-206.[Medline]
21 Couch FJ, Weber BL. Mutations and polymorphisms in the familial early-onset breast cancer (BRCA1) gene. Breast Cancer Information Core. Hum Mutat 1996;8:8-18.[Medline]
22 Tavtigian SV, Simard J, Rommens J, Couch F, Shattuck-Eidens D, Neuhausen S, et al. The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat Genet 1996;12:333-7.[Medline]
23 Takahashi H, Chiu HC, Bandera CA, Behbakht K, Liu PC, Couch FJ, et al. Mutations of the BRCA2 gene in ovarian carcinomas. Cancer Res 1996;56:2738-41.[Abstract]
24 Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994;266:66-71.[Medline]
25 Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC, Kenney G, et al. Human epithelial ovarian cancer allelotype. Cancer Res 1993;53: 2393-8.[Abstract]
26 Yang-Feng TL, Li S, Han H, Schwartz PE. Frequent loss of heterozygosity on chromosomes Xp and 13q in human ovarian cancer. Int J Cancer1992 ;52:575-80.[Medline]
27
Cheng PC, Gosewehr JA, Kim TM, Velicescu M, Wan M,
Zheng J, et al. Potential role of the inactivated X chromosome in ovarian epithelial tumor
development. J Natl Cancer Inst 1996;88:510-18.
28 Osborne RJ, Leech V. Polymerase chain reaction allelotyping of human ovarian cancer. Br J Cancer 1994;69:429-38.[Medline]
29
Parolini O, Ressmann G, Haas OA, Pawlowsky J, Gadner H,
Knapp W, et al. X-linked Wiskott-Aldrich syndrome in a girl. N Engl J Med 1998;338:291-5.
30 Buller RE, Skilling JS, Kaliszewski S, Niemann T, Anderson B. Absence of significant germ line p53 mutations in ovarian cancer patients. Gynecol Oncol 1995;58:368-74.[Medline]
31 Skilling JS, Powills K, Lager DJ, Anderson B, Sorosky J, Buller RE. p53 allelotypes and enhanced detection of allelic loss in ovarian cancer: lack of correlation with familial and clinical factors. Gynecol Oncol 1996;61:180-8.[Medline]
32 Mutter GL, Chaponot ML, Fletcher JA. A polymerase chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers. Am J Pathol 1995;146:501-8.[Abstract]
33 Mutter GL, Boynton KA. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies. Nucleic Acids Res 1995;23:1411-8.[Abstract]
34 Buller RE, Skilling JS, Sood AK, Plaxe S, Baergen RN, Lager DJ. Field cancerization: why late recurrent ovarian cancer is not recurrent. Am J Obstet Gynecol 1998;178:641-9.[Medline]
35 Lallas TA, Buekers TE, Buller RE. BRCA1 mutations in familial ovarian cancer. Submitted.
36 International Federation of Gynecology and Obstetrics (FIGO). Changes in definitions of clinical staging for carcinoma of the cervix and ovary. Am J Obstet Gynecol 1987;156:263-4.[Medline]
37 Lallas TA, Buller RE. Optimization of PCR and electrophoresis conditions enhances mutation analysis of the BRCA1 gene. Mol Genet Metab 1998;64:173-6.[Medline]
38
Belmont JW. Clinical interpretation of skewed X inactivation. Blood 1994;84:2375-6.
39
Gale RE, Linch DC. Interpretation of X-chromosome
inactivation patterns. Blood 1994;84:2376-8.
40 Fey MF, von Rohr A. Response. Blood 1994;84:2377-8.
41
Fey MF, Liechti-Gallati S, von Rohr A, Borisch B, Theilkas L,
Schneider V, et al. Clonality and X-inactivation patterns in hematopoietic cell populations
detected by the highly informative M27 beta DNA probe. Blood 1994;83:931-8.
42 Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE. Risks of cancer in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. Lancet 1994;343:692-5.[Medline]
43
Gale RE, Wheadon H, Boulos P, Linch DC. Tissue specificity
of X-chromosome inactivation patterns. Blood 1994;83:2899-905.
44 McLaren A. Numerology of development. Nature 1972;239:274-6.[Medline]
45 Holt JT, Thompson ME, Szabo C, Robinson-Benion C, Arteaga CL, King MC, et al. Growth retardation and tumour inhibition by BRCA1. Nat Genet 1996;12:298-302.[Medline]
46 Schmidt M, Certoma A, Du Sart D, Kalitsis P, Leversha M, Foster K, et al. Unusual X chromosome inactivation in a mentally retarded girl with an interstitial deletion Xq27: implications for the fragile X syndrome. Hum Genet 1990;84:347-52.[Medline]
47 Clarke JT, Greer WL, Strasberg PM, Pearce RD, Skomorowski MA, Ray PN. Hunter disease (mucopolysaccharidosis type II) associated with unbalanced inactivation of the X chromosomes in a karyotypically normal girl. Am J Hum Genet 1991;49:289-97.[Medline]
48 Clarke JT, Wilson PJ, Morris CP, Hopwood JJ, Richards RI, Sutherland GR, et al. Characterization of a deletion at Xq27-q28 associated with unbalanced inactivation of the nonmutant X chromosome. Am J Hum Genet 1992;51:316-22.[Medline]
49 Dahl N, Hu LJ, Chery M, Fardeau M, Gilgenkrantz S, Nivelon-Chevallier A, et al. Myotubular myopathy in a girl with a deletion at Xq27-q28 and unbalanced X inactivation assigns the MTM1 gene to a 600-kb region. Am J Hum Genet 1995;56:1108-15.[Medline]
50 Rainier S, Feinberg AP. Genomic imprinting, DNA methylation, and cancer. J Natl Cancer Inst 1994;86:753-9.[Medline]
51 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 1983;301:89-92.[Medline]
52 Jones PA. DNA methylation errors and cancer. Cancer Res 1996;56:2463-7.[Medline]
53
Brown CJ. Role of the X chromosome in cancer. J Natl
Cancer Inst 1996;88:480-2.
54 Chenevix-Trench G, Kerr J, Hurst T, Shih YC, Purdie D, Bergman L, et al. Analysis of loss of heterozygosity and KRAS2 mutations in ovarian neoplasms: clinicopathological correlations. Genes Chromosomes Cancer 1997;18:75-83.[Medline]
55 Shih YC, Kerr J, Hurst TG, Khoo SK, Ward BG, Chenevix-Trench G. No evidence for microsatellite instability from allelotype analysis of benign and low malignant potential ovarian neoplasms. Gynecol Oncol 1998;69:210-3.[Medline]
56 Teneriello MG, Ebina M, Linnoila RI, Henry M, Nash JD, Park RC, et al. p53 and Ki-ras gene mutations in epithelial ovarian neoplasms. Cancer Res 1993;53:3103-8.[Abstract]
57 Mok SC, Bell DA, Knapp RC, Fishbaugh PM, Welch WR, Muto MG, et al. Mutations of K-ras protooncogene in human ovarian epithelial tumors of borderline malignancy. Cancer Res 1993;53:1489-92.[Abstract]
Manuscript received March 10, 1998; revised December 11, 1998; accepted December 17, 1998.
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