Closing in on a breast cancer gene on chromosome 17q.

Linkage of early-onset familial breast and ovarian cancer to 11 markers on chromosome 17q12-q21 defines an 8-cM region which is very likely to include the disease gene BRCA 1. The most closely linked marker is D17S579, a highly informative CA repeat polymorphism. D17S579 has no recombinants with inherited breast or ovarian cancer in 79 informative meioses in the seven families with early-onset disease (lod score 9.12 at zero recombination). There is no evidence for linkage heterogeneity in the families with early-onset disease. The proportion of older-onset breast cancer attributable to BRCA 1 is not yet determinable, because both inherited and sporadic cases occur in older-onset families.     

Linkage to markers for the chromosome region 17q12-q21 in 13 Dutch breast cancer kindreds

We have performed linkage analysis with five markers for the chromosome region 17q12-q21 in 13 Dutch breast cancer kindreds in order to find support for the claim by Hall et al. that a gene in this region, termed “BRCA1,” is associated with predisposition to early-onset familial breast cancer. This work is part of a collaborative study, the results of which are published elsewhere in this issue. Best evidence for linkage was observed with the marker CMM86 (D17S74) in pedigrees with an average age at onset of ≤47 years (LOD score = 1.77 at 1% recombination). In one breast-ovarian cancer family with a high probability of being linked to 17q, we observed one putative recombinant between D17S250 and D17S579, which suggests that BRCA1 is proximal to D17S579.     

BRCA1 maps proximal to D17S579 on chromosome 17q21 by genetic analysis

Previous studies have demonstrated linkage between early-onset breast cancer and ovarian cancer and genetic markers on chromosome 17q21. These markers define the location of a gene (BRCA1) which appears to be inherited as an autosomal dominant susceptibility allele. We analyzed five families with multiple affected individuals for evidence of linkage to the BRCA1 region. Two of the five families appear to be linked to BRCA1. One apparently linked family contains critical recombinants, suggesting that the gene is proximal to the marker D17S579 (Mfd188). These findings are consistent with the maximum-likelihood position estimated by the Breast Cancer Linkage Consortium and with recombination events detected in other linked families. Linkage analysis was greatly aided by PCR-based analysis of paraffin-embedded normal breast tissue from deceased family members, demonstrating the feasibility and importance of this approach. One of the two families with evidence of linkage between breast cancer and genetic markers flanking BRCA1 represents the first such family of African-American descent to be reported in detail.     

A Radiation Hybrid Map of the BRCA1 Region

A locus on chromosome 17q, designated “BRCA1,” has been identified as a predisposition gene for breast cancer. A panel of chromosome 17–specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17. A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays. Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region. In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region.     

Multicolor FISH Mapping with Alu-PCR-Amplified YAC Clone DNA Determines the Order of Markers in the BRCA1 Region on Chromosome 17q12-q21

A gene designated BRCA1, implicated in the susceptibility to early-onset familial breast cancer, has recently been localized to chromosome 17q12-q21. To date, the order of DNA markers mapped within this region has been based on genetic linkage analysis. We report the use of multicolor fluorescence in situ hybridization to establish a physically based map of five polymorphic DNA markers and 10 cloned genes spanning this region. Three cosmid clones and Alu-PCR-generated products derived from 12 yeast artificial chromosome clones representing each of these markers were used in two-color mapping experiments to determine an initial proximity of markers relative to each other on metaphase chromosomes. Interphase mapping was then employed to determine the order and orientation of closely spaced loci by direct visualization of fluorescent signals following hybridization of three probes, each detected in a different color. Statistical analysis of the combined data suggests that the order of markers in the BRCA1 region is cen-THRA1-TOP2-GAS-OF2-17HSI)-248yg9-RNU2-OF3-PPY/p131-EPB3-Mfd188-WNT3-HOX2-GP3A-tel. This map is consistent with that determined by radiation-reduced hybrid mapping and will facilitate positional cloning strategies in efforts to isolate and characterize the BRCA1 gene.     

THRA1 and D17S183 flank an interval of < 4 cM for the breast-ovarian cancer gene (BRCA1) on chromosome 17q21.

In order to pinpoint the locale of the gene for early-onset familial breast and ovarian cancer (BRCA1), polymorphisms were developed within the locus for thyroid hormone receptor alpha (THRA1) and for several anonymous sequences at chromosome 17q12-q21. The THRA1 polymorphism is a dinucleotide repeat with 10 alleles and heterozygosity.79. Gene mapping in extended families with inherited, early-onset breast and ovarian cancer indicates that BRCA1 is distal to THRA1 and proximal to D17S183 (SCG43), an interval of < 4 cM. This locale excludes HER2, THRA1, WNT3, HOX2, NGFR, PHB, COLIA1, NME1, and NME2 as candidates for BRCA1 but does not exclude RARA or EDH17B. Resolving the remaining recombination events in these families by new polymorphisms in the THRA1-D17S183 interval will facilitate positional cloning of the breast-ovarian cancer gene on chromosome 17q12-q21.     

A Common Region of Deletion on Chromosome 17q in Both Sporadic and Familial Epithelial Ovarian Tumors Distal to BRCA1

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17pl3.1 and 17pl3.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17ql2-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans −25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.     

Linkage of familial breast cancer to chromosome 17q21 may not be restricted to early-onset disease.

Lod scores for linkage between familial breast and ovarian cancer and markers on chromosome 17q21 are more frequently positive among families with disease diagnosed at younger ages than they are among older-onset families, suggesting that linkage is restricted to early-onset disease. However, for late-onset cases, the relative probability of sporadic rather than inherited disease is higher than previously suggested. If this correction is made, then later-onset families are much less informative; linkage heterogeneity based on age at onset is no longer significant; and for the sample of families as a whole, linkage is significant at a recombination fraction since demonstrated to be close to the correct local. There is probably more than one gene for inherited breast cancer, but heterogeneity may not be due to age at disease onset.     

The role of MMAC1 mutations in early-onset breast cancer: causative in association with Cowden syndrome and excluded in BRCA1-negative cases.

Cowden syndrome (CS) is an autosomal dominant disorder associated with the development of hamartomas and benign tumors in a variety of tissues, including the skin, thyroid, breast, endometrium, and brain. It has been suggested that women with CS are at increased risk for breast cancer. A locus for CS was recently defined on chromosome 10 in 12 families, resulting in the identification of the CS critical interval, between the markers D10S215 and D10S541. More recently, affected individuals in four families with CS have been shown to have germ-line mutations in a gene known as "PTEN," or "MMAC1," which is located in the CS critical interval on chromosome 10. In this study, we report three novel MMAC1 mutations in CS and demonstrate that MMAC1 mutations are associated with CS and breast cancer. Furthermore, we also show that certain families and individuals with CS do not have mutations in the coding sequence of MMAC1. Finally, we did not detect MMAC1 mutations in a subpopulation of individuals with early-onset breast cancer, suggesting that germ-line mutations in this gene do not appear to be common in this group.     

Frequent loss of the BLID gene in early-onset breast cancer

The BH3-like motif-containing inducer of cell death (BLID) is an intronless gene localized on 11q24.1. Loss of that region has frequently been reported in early-onset breast cancer and is significantly associated with poor prognosis and reduced survival. Downregulation of BLID is associated with younger age, triple-negative phenotype, and reduced disease-free and overall survival of breast cancer patients. In this study, we investigated allelic loss of BLID in breast tumor specimens from 78 women with invasive breast cancer using 2 dinucleotide polymorphic markers closely linked to the BLID gene (no intragenic marker for BLID is available). Seventy-three cases were informative. Overall, loss of heterozygosity (LOH) at the BLID locus was detected in 32% of the informative cases (23/73). However, in patients 40 years old and younger, LOH was detected in 50% of the cases (9/18). Patients aged 40 years and younger were significantly more likely to experience LOH than those aged 41-55 years (p = 0.04). Specifically, the odds of BLID loss for patients aged 40 years and younger were 3.7 times the odds of loss for patients aged 41-55 years (95% CI, 1.1-13). Our findings suggest a tumor suppressor role of the BLID gene in early-onset breast cancer.     

PARK7, a Novel Locus for Autosomal Recessive Early-Onset Parkinsonism, on Chromosome 1p36

Although the role of genetic factors in the origin of Parkinson disease has long been disputed, several genes involved in autosomal dominant and recessive forms of the disease have been localized. Mutations associated with early-onset autosomal recessive parkinsonism have been identified in the Parkin gene, and recently a second gene, PARK6, involved in early-onset recessive parkinsonism was localized on chromosome 1p35-36. We identified a family segregating early-onset parkinsonism with multiple consanguinity loops in a genetically isolated population. Homozygosity mapping resulted in significant evidence for linkage on chromosome 1p36. Multipoint linkage analysis using MAPMAKER-HOMOZ generated a maximum LOD-score of 4.3, with nine markers spanning a disease haplotype of 16 cM. On the basis of several recombination events, the region defining the disease haplotype can be clearly separated, by > or =25 cM, from the more centromeric PARK6 locus on chromosome 1p35-36. Therefore, we conclude that we have identified on chromosome 1 a second locus, PARK7, involved in autosomal recessive, early-onset parkinsonism.     

Fine-mapping the putative chromosome 17q21–22 prostate cancer susceptibility gene to a 10 cM region based on linkage analysis

Prostate cancer linkage studies have suggested the existence of a prostate cancer susceptibility gene on chromosome 17q21–22. We now report the results of an extended linkage analysis including 95 new multiplex prostate cancer families and 9 additional microsatellite markers resulting in a maximum LOD score of 2.99 at approximately 81–82 cM for all 453 pedigrees. Results from these 95 new pedigrees provide additional support for a chromosome 17q21–22 prostate cancer susceptibility gene. Inclusion of the 9 additional markers significantly reduced the size of the candidate region, as defined using a 1-LOD support interval, especially when focusing analyses on subsets of pedigrees with four or more confirmed affecteds or average age of diagnosis less than or equal to 65 years. A novel subset analysis of only those families (n = 147) that had four or more prostate cancer cases and an average age of prostate cancer diagnosis ≤ 65 years results in a maximum LOD score of 5.49 at 78 cM with a 1-LOD support interval of 10 cM. This large set of pedigrees with four more prostate cancer cases characterized by early-onset disease will serve as a useful resource for identifying the putative 17q21–22 prostate cancer susceptibility gene.     

An Evaluation of Genetic Heterogeneity in 145 Breast-Ovarian Cancer Families

The breast-ovary cancer–family syndrome is a dominant predisposition to cancer of the breast and ovaries which has been mapped to chromosome region 17ql2-q21. The majority, but not all, of breast-ovary cancer families show linkage to this susceptibility locus, designated BRCA1. We report here the results of a linkage analysis of 145 families with both breast and ovarian cancer. These families contain either a total of three or more cases of early-onset (before age 60 years) breast cancer or ovarian cancer. All families contained at least one case of ovarian cancer. Overall, an estimated 76% of the 145 families are linked to the BRCA1 locus. None of 13 families with cases of male breast cancer appear to be linked, but it is estimated that 92% (95% confidence interval 76%–100%) of families with no male breast cancer and with two or more ovarian cancers are linked to BRCA1. These data suggest that the breast-ovarian cancer–family syndrome is genetically heterogeneous. However, the large majority of families with early-onset breast cancer and with two or more cases of ovarian cancer are likely to be due to BRCA1 mutations.     

Linkage analysis of familial Alzheimer disease, using chromosome 21 markers

Chromosome 21 markers were tested for linkage to familial Alzheimer disease (FAD) in 48 kindreds. These families had multiple cases of Alzheimer disease (AD) in 2 or more generations with family age-at-onset means (M) ranging from 41 to 83 years. Included in this group are seven Volga German families which are thought to be genetically homogeneous with respect to FAD. Autopsy documentation of AD was available for 32 families. Linkage to the 21 q11-q21 region was tested using D21S16, D21S13, D21S110, D21S1/S11, and the APP gene as genetic markers. When linkage results for all the families were summed, the LOD scores for these markers were consistently negative and the entire region was formally excluded. Linkage results were also summed for the following family groups; late-onset (M greater than 60), early-onset (M less than or equal to 60), Volga Germans (M = 56), and early-onset non-Volga Germans (M less than or equal to 60). For the first three groups, LOD scores were negative for this region. For the early-onset non-Volga German group (six families), small positive LOD scores of Zmax = 0.78 (recombination fraction theta = .15), Zmax = 0.27 (theta = .15), and Zmax = 0.64 (theta = .0), were observed for D21S13, D21S16, and D21S110, respectively. The remainder of the long arm of chromosome 21 was tested for linkage to FAD using seven markers spanning the q22 region. Results for these markers were also predominantly negative. Thus it is highly unlikely that a chromosome 21 gene is responsible for late-onset FAD and at least some forms of early-onset FAD represented by the Volga German kindreds.     

Absence of linkage to the ataxia telangiectasia locus in familial breast cancer

Heterozygotes for ataxia-telangiectasia (AT) are known to have an increased risk of breast cancer. The gene (or genes) responsible for almost all cases of AT has been localised to chromosome 11q by genetic linkage analysis. To examine the possibility that AT heterozygosity may account for a substantial proportion of familial breast cancer, we have typed five markers on chromosome 11q in 16 breast cancer families. We have found no evidence for linkage between breast cancer and chromosome 11q markers and conclude that the contribution of AT to familial breast cancer is likely to be minimal.     

Linkage analysis of 26 Canadian breast and breast-ovarian cancer families

We have examined 26 Canadian families with hereditary breast or ovarian cancer for linkage to markers flanking the BRCA1 gene on chromosome 17q12–q21. Of the 15 families that contain cases of ovarian cancer, 94% were estimated to be linked to BRCA1. In contrast, there was no overall evidence of linkage in the group of 10 families with breast cancer without ovarian cancer. A genetic recombinant in a breast-ovarian cancer family indicates a placement of BRCA1 telomeric to D17S776, and helps to define the region of assignment of the cancer susceptibility gene. Other cancers of interest that appeared in the BRCA1-linked families included primary peritoneal cancer, cancer of the fallopian tube, and malignant melanoma.     

Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families. The Breast Cancer Linkage Consortium.

Breast cancer is known to have an inherited component, consistent in some families with autosomal dominant inheritance; in such families the disease often occurs in association with ovarian cancer. Previous genetic linkage studies have established that in some such families disease occurrence is linked to markers on chromosome 17q. This paper reports the results of a collaborative linkage study involving 214 breast cancer families, including 57 breast-ovarian cancer families; this represents almost all the known families with 17q linkage data. Six markers on 17q, spanning approximately 30 cM, were typed in the families. The aims of the study were to define more precisely the localization of the disease gene, the extent of genetic heterogeneity and the characteristics of linked families and to estimate the penetrance of the 17q gene. Under the assumption of no genetic heterogeneity, the strongest linkage evidence was obtained with D17S588 (maximum LOD score [Zmax] = 21.68 at female recombination fraction [theta f] = .13) and D17S579 (Zmax = 13.02 at theta f = .16). Multipoint linkage analysis allowing for genetic heterogeneity provided evidence that the predisposing gene lies between the markers D17S588 and D17S250, an interval whose genetic length is estimated to be 8.3 cM in males and 18.0 cM in females. This position was supported over other intervals by odds of 66:1. The location of the gene with respect to D17S579 could not be determined unequivocally. Under the genetic model used in the analysis, the best estimate of the proportion of linked breast-ovarian cancer families was 1.0 (lower LOD-1 limit 0.79). In contrast, there was significant evidence of genetic heterogeneity among the families without ovarian cancer, with an estimated 45% being linked. These results suggest that a gene(s) on chromosome 17q accounts for the majority of families in which both early-onset breast cancer and ovarian cancer occur but that other genes predisposing to breast cancer exist. By examining the fit of the linkage data to different penetrance functions, the cumulative risk associated with the 17q gene was estimated to be 59% by age 50 years and 82% by age 70 years. The corresponding estimates for the breast-ovary families were 67% and 76%, and those for the families without ovarian cancer were 49% and 90%; these penetrance functions did not differ significantly from one another.     

Frequent occurrence of BRCA2 linkage in Icelandic breast cancer families and segregation of a common BRCA2 haplotype.

Cloning of a breast cancer-predisposing gene (BRCA2) on chromosome 13Q12-14 has been reported recently. We analyzed seven large Icelandic breast cancer families with markers from the BRCA2 region. Five families showed strong evidence of linkage. The maximum two-point LOD scores for the five BRCA2-linked families ranged from 1.06 to 3.19. Haplotype analyses revealed a region with identical allele sizes between the families, suggesting that they have inherited the mutation from a common ancestor. Cancer types other than breast cancer occur in individuals, segregating the affected haplotype within these families. This suggests that mutations in the gene may also confer some risk of other malignancies in both males and females.     

Linkage of a major breast cancer gene to chromosome 17q12-21: results from 15 Edinburgh families.

DNA from members of 15 pedigrees each containing between three and eight cases of breast cancer have been collected from southeastern Scotland. Polymorphic markers on chromosome 17q were screened to locate a putative breast cancer gene by using DNA from relevant individuals within these families. Pairwise LOD scores were calculated for markers D17S74, NM23, D17S588, and D17S579. The maximal summated LOD for the 15 families was 5.44 at theta = .034, when D17S588 (42D6) was used. In these breast cancer families, a subset which did not give evidence for linkage to this region could be identified.