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A Genome-wide Map Showing Common Regions of Loss of Heterozygosity/Allelic Imbalance in Breast Cancer¹

Institute of Genetics, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH [R. J. O.], and School of Life and Environmental Sciences, University Park, University of Nottingham, Nottingham NG7 2RD [M. G. H.], United Kingdom

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
We report here a new framework map that integrates data from 143 studies on the loss of heterozygosity/allelic imbalance in breast cancer. Full details of these loss of heterozygosity maps can be found at the web site (http://www.nottingham.ac.uk/pdzmgh/loh/) that accompanies this report. By combining results from these data, we have also been able to highlight and identify minimum commonly deleted regions on each chromosome. In addition to finding commonly deleted regions at both the BRCA1 and BRCA2 loci, which confirmed the power of the technique, 24 other regions were identified on 16 different chromosomes.

	Introduction

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Nearly 30 years ago, Knudson deduced that development of childhood retinoblastoma may require two rate-limiting genomic events: the "two-hit hypothesis" (1) . According to this model, the first event in tumor formation is a mutation in a specific cancer gene, which in hereditary cancers is present in the germ-line. The second "hit" in both hereditary and nonhereditary cases would be a somatic mutation leading to functional inactivation of the second copy of the gene (1, 2) . This second "hit" usually results from a large deletion of chromosomal material as a result of aberrant mitotic recombination or nondisjunction, although several other mechanisms have been suggested (2–5) . Knudson’s model provides the rationale that frequent or above background rates of loss of DNA at a specific chromosomal locus in a tumor may signify the presence of a tumor suppressor gene in the region of DNA that is lost (6) . To identify and map genes (including tumor supressor genes) that are inactivated in this manner, numerous studies have exploited the presence of mapped polymorphisms at the DNA level. If an individual is constitutively heterozygous at a given locus, the LOH³ in a tumor sample can be used to implicate that locus in the formation/prevention of cancer (2) . LOH is detected by measuring the amount of DNA from each allele from tumor cells and comparing this with the amounts found from normal tissue in the same individual. A skewed distribution in the tumor cells, compared with those found in the normal tissue, indicates an allelic imbalance. This imbalance can result from either loss of an allele (true LOH) or allelic amplification. Although some earlier articles reporting LOH may, for technical reasons, have measured allelic amplification rather than loss of an allele (e.g., reported LOH at 8q24 more likely reflects the site of MYC amplification), we have included these studies because they indicate a contribution for genes in these regions in tumor progression. This report combines the data of a large number of studies into reported LOH in breast cancer to identify the minimal regions of deletion that may be lost during tumor formation.

	Materials and Methods

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Integrated chromosome-specific maps of polymorphic markers were produced using a technique similar to that used by the UDB (see Table 1 for the URLs). This involves the cross-referencing of common markers between different maps. For the LOH map presented here, the CHCL recombination minimized female genetic map was used as a base map onto which other markers were assigned using maps from several different sources (see Table 1 ). Once these integrated maps were produced, it was possible to compare and combine the LOH results obtained in the individual studies.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

To produce an integrated map onto which the regions of LOH or allelic imbalance could be mapped, this report contains details of a new LOH framework map. Using the CHLC recombination map as a base map, the Genethon Microsatellite map was then used to integrate a number of markers from the different LOH studies [for example, on chromosome 13, the location of D13S290 is assigned by reference to two flanking markers, D13S289 and D13S260 (see Fig. 1 )]. Markers that could not be positioned using the Genethon Microsatellite maps were assigned by a stepwise process initially using NIH/CEPH Baseline maps, the Human Physical Mapping Project at the Whitehead Institute, and then the radiation hybrid mapping resource at Stanford University. Where differences were found in the marker order between the different maps, the order from the physical maps took precedence. The maps produced by GENATLAS were then used to position some remaining markers onto the consensus, and the UDB map at the Weismann Institute and the Genome Location Database were then used to check the order of some markers and, in some circumstances, to position them. GeneMap’98 at the National Center for Bioinformatics was then used to position genes in relation to their surrounding markers. Fig. 1 illustrates this stepwise process for chromosome 13. Markers from the final consensus map were then positioned onto the comprehensive physical map framework available at the GDB to produce an integrated map containing all of the markers that had been used in the different studies. We used the same rationale to produce the other consensus maps in this study (see Fig. 2 ) . More detailed maps, which include information about the nature of the imbalance (i.e., true LOH or allelic imbalance), and references to the individual studies used are available at the web site that accompanies this article.⁴ Because the majority of LOH events in breast cancer extend across an entire chromosome arm or chromosome (7) , the bars on each of these maps do not precisely delimit the area of LOH. However, by combining the results of several studies that have themselves combined the data from numerous patients, a CDR can be identified. Because the definition of a minimally CDR is highly subjective, the regions that we identified were not intended to be exhaustive, although we generally selected regions in which deletion was noted in at least four different studies. There are probably many regions other than those listed that are recurrently abnormal. However, using the data for chromosome 13 as an illustration, it is clear that by combining the data from the 22 different studies that contained information about LOH on this chromosome, two CDRs are apparent, at 13q12.3 and 13q12.2–14.3. These regions correspond to regions that contain genes that have already been implicated in breast tumorigenesis [BRCA2 (13q12.3) and RB1 (13q14.2)]. This example has been chosen to indicate the potential power of this technique. Applying the same approach to the other chromosomes, many other CDRs can be identified. In this report, we have highlighted 24 other CDRs on 16 chromosomes (see Table 2 ). This is only a sample of some of the regions, to highlight the potential application of this approach. Many of the regions we selected do not contain genes known to be involved in breast tumorigenesis, although candidate genes can be derived for them, some of which are also shown in Table 2 . The list of candidate genes within each common region of deletion is not intended to be exhaustive; the aim of this report was not to highlight individual candidate genes but to bring together a large amount of LOH and allelic imbalance data. In doing this, those regions that may not have been identified in the individual studies can be highlighted for further investigation. The identification of genes within these regions may provide insights into the pathogenesis of this complex multifactorial disease.

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Integrated maps that allow comparison of several LOH studies were produced in a stepwise manner to produce final consensus order maps. This stepwise process is illustrated here for chromosome 13. A similar approach was used to produce the integrated map for all of the other chromosomes.

View larger version (293K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. The integrated maps for each of the chromosomes indicating the regions of LOH/allelic imbalance. The position of markers on the central, integrated map are linked to the CHLC genetic map (left) and the GDB comprehensive map (right). The vertical bars correspond to the central column of markers and delimit the areas of LOH/allelic imbalance found for each specific study. Boxed numbers indicate the reference for each study; full details regarding these references and detailed images that include information about the nature of the allelic imbalance are given on the web site that accompanies this report.4

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the University of Nottingham (Nottingham, United Kingdom).

² To whom requests for reprints should be addressed. E-mail: marion.hamshere{at}nottingham.ac.uk

³ The abbreviations used are: LOH, loss of heterozygosity; CDR, commonly deleted region; CHLC, Cooperative Human Linkage Center; URL, uniform resource locator; UDB, Unified Genome Database; GDB, Genome Database; CEPH, Centre d’Etude du Polymorphisme Humain.

⁴ http://www.nottingham.ac.uk/pdzmgh/loh/.

Received 11/16/99. Accepted 6/ 2/00.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Knudson A. G. Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad. Sci. USA, 68: 820-823, 1971.[Abstract/Free Full Text]
2. Cavenee W. K., Dryja T. P., Phillips R. A., Benedict W. F., Godbout R., Gallie B. L., Murphree A. L., Strong L. C., White R. L. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature (Lond.), 305: 779-784, 1983.[Medline]
3. Tomlinson I. P., Bodmer W. F. Chromosome 11q in sporadic colorectal carcinoma: patterns of allele loss and their significance for tumorigenesis. J. Clin. Pathol, 49: 386-390, 1996.[Abstract/Free Full Text]
4. Cappione A. J., French B. L., Skuse G. R. A potential role for NF1 mRNA editing in the pathogenesis of NF1 tumors. Am. J. Hum. Genet, 60: 305-312, 1997.[Medline]
5. Huynh H., Alpert L., Pollak M. Silencing of the mammary-derived growth inhibitor (MDGI) gene in breast neoplasms is associated with epigenetic changes. Cancer Res, 56: 4865-4870, 1996.[Abstract/Free Full Text]
6. Chen L. C., Kurisu W., Ljung B. M., Goldman E. S., Moore D., Smith H. S. Heterogeneity for allelic loss in human breast cancer. J. Natl. Cancer Inst, 84: 506-510, 1992.[Abstract/Free Full Text]
7. Devilee P., Cornelisse C. J. Somatic genetic changes in human breast cancer. Biochim. Biophys. Acta, 1198: 113-130, 1994.[Medline]

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