The analyses of 149 families reported here represented by far the largest genome-wide linkage screen for breast cancer susceptibility loci. The only other report since the identification of BRCA1
was that by Huusko et al., (2004)
, who studied 14 BRCA1
/2 negative breast cancer families from Finland. Other reports have examined specific loci on chromosome arms 6q, 8p, and 13q (Zuppan et al., 1991
; Kerangueven et al., 1995
; Seitz et al., 1997
; Kainu et al., 2000
; Rahman et al., 2000
; Thompson et al., 2002
The rationale for the genome-wide linkage searches is that there exist further breast cancer genes in which alleles confer high risks. The pattern of familial risks indicates that such alleles are likely to be dominant, and we therefore considered the parametric analysis assuming a dominant model to be the primary analysis. To provide some protection against model misspecification, we also conducted analyses under a recessive model and using an allele sharing approach. These approaches, however, identified no further strong linkage signals.
Under the dominant model, we found three regions with HLODs in excess of 1, but none with HLODs over 2. Of these linkage peaks, one on chromosome 22 is explained entirely by a single family (EUR60). This family is the most informative in the study, containing 18 breast cancer cases. Seven cases of breast cancer have been shown to carry the CHEK2
variant 1100delC (Miejers-Heijboer et al., 2002
). Since CHEK2
is located on chromosome 22, one might hypothesize that the linkage signal is a reflection of the segregation of this variant. However, the breast cancer risk conferred by CHEK2
1100delC is only twofold, and this would not be expected to generate strong linkage evidence. Furthermore, the LOD score in the larger branch of EUR60 at CHEK2
itself is only 0.3. Thus, it remains unclear whether the linkage signal on chromosome 22 reflects the effect of CHEK2
1100delC together with chance segregation, or whether there is an additional susceptibility allele segregating in this family. If the latter is true, given the lack of any linkage evidence from other families, susceptibility alleles at this other locus must be rare.
The strongest linkage signal in our set was found on the short arm of chromosome 4. This score was also, in part, due to EUR60 (LOD score 1.91 in the larger branch), although some evidence of linkage remained when EUR60 was excluded. The third linkage peak was on 2p (HLOD 1.2). This evidence increased (HLOD 2.4) when analyses were restricted to families with at least four cases of breast cancer diagnosed below age 50 years.
Huusko et al. (2004)
reported evidence for linkage to markers on 2q32 in 14 Finnish breast cancer families, with a maximum LOD score of 3.20 close to D2S2262
. We found no evidence of linkage in this region (maximum HLOD under the dominant model 0.0, α = 0.0; nonparametric LOD = 0.05). Huusko et al. (2004)
found one other LOD score over 1 under a dominant model, at D9S283
(1.12). Again, we found no evidence for linkage in this region. Similarly, Zuppan et al. (1991)
found evidence of linkage to the estrogen receptor gene on 6q in two families. In our study, we found no evidence of linkage to this region (HLOD = 0 for both the dominant and recessive models).
Theoretical calculations indicate that, for a fully informative marker map, the expected number of regions with LOD scores of greater than 1 and 1.5 will be ~5 and 2, respectively (Lander and Kruglyak, 1995
). These predictions are not strictly comparable to our analyses, since our marker sets are not fully informative. Nevertheless, they indicate that the number of linkage peaks is not clearly in excess of the number that might be expected by chance and, therefore, that the observed peaks may reflect the play of chance rather than true susceptibility loci.
Under the admixture model, the estimated proportion of families linked to the loci are 0.18, 0.18, and 0.06 for chromosomes 2, 4, and 22, respectively. Such estimates can be misleading, since they are highly dependent on the genetic model that is assumed, and the true model is unknown. However, they indicate that, even if one or more of these linkage peaks is ultimately shown to harbor a true susceptibility locus, its contribution to the familial aggregation of breast cancer is likely to be modest. Moreover, under the assumed parametric dominant model, 87% of the genome achieved an HLOD of -1 or lower if the proportion of linked families was assumed to be 0.3, and 66% of the genome achieved an HLOD < -2, indicating that such a locus was unlikely to have been missed elsewhere in the genome.
The failure to detect strong linkage signals might reflect extensive locus heterogeneity, whereby the disease is only linked to a particular locus in a small proportion of families. Under this scenario, greater power might be achievable by considering subsets of families from more homogeneous populations where genetic heterogeneity might be reduced. We were able to examine this to a limited extent by performing separate analyses of the families in each of the four study sets. Since the Australian families were largely of British and Irish origin, these two groups might be considered comparable. The Dutch population exhibits distinct founder mutations for many diseases and this group is, to an extent, genetically distinct, while the IARC families originated from many sources and are genetically heterogeneous. In the event, no strong linkage signals were observed either in the Dutch set or in the combined United Kingdom/Australian set. In particular, the linkage peaks identified in family EUR60 were not supported by linkage evidence in other Dutch families. The linkage peak on chromosome 2 did, however, become somewhat stronger when the Dutch families were excluded.
The failure to detect strong evidence for linkage may also reflect disease heterogeneity. Recent studies have demonstrated that breast tumors can be categorized into groups on the basis of CGH profiles and expression patterns, and that these patterns differ between BRCA1
, and non-BRCA1
/2 familial breast cancer (Hedenfalk et al., 2001
; Gronwald et al., 2005
; Macguire et al., 2005
). These observations raise the possibility that mutations in other breast cancer susceptibility genes are associated with distinct tumor profiles. If so, incorporating tumor characteristics into the analyses could identify linkage signals that are not evident using breast cancer as a whole as the disease end point.
The positive signals found in this study indicate the most promising locations for further high-risk susceptibility genes, and would be worth following up in further families. Our results also indicate, however, that many genes are likely to be involved in breast cancer predisposition, with no gene accounting for a large fraction of the familial aggregation, and that alternative strategies will probably be necessary to identify them.