Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs.

A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants (UVs) include variants that are located in the intronic sequences of BRCA1 and BRCA2. The purpose of this study was to assess the use of splice-site prediction programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81-6T>A and c.4986+5G>T; BRCA2, c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68-7T>A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e., the wild-type splice-site was recognized). No false-negative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis.

Characterization of familial non-BRCA1/2 breast tumors by loss of heterozygosity and immunophenotyping.

PURPOSE: Since the identification of BRCA1 and BRCA2, there has been no major breast cancer susceptibility gene discovered by linkage analysis in breast cancer families. This has been attributed to the heterogeneous genetic basis for the families under study. Recent studies have indicated that breast tumors arising in women carrying a BRCA1 mutation have distinct histopathologic, immunophenotypic, and genetic features. To a lesser extent, this is also true for breast tumors from BRCA2 carriers. This indicates that it might be possible to decrease the genetic heterogeneity among families in which BRCA1 and BRCA2 have been excluded with high certainty (BRCAx families) if distinct subgroups of BRCAx-related breast tumors could be identified. EXPERIMENTAL DESIGN: Loss of heterozygosity (LOH) analysis with at least one marker per chromosomal arm (65 markers) was used to characterize 100 breast tumors derived from 92 patients from 42 selected BRCAx families. In addition, the immunophenotype of 10 markers was compared with that of 31 BRCA1- and 21 BRCA2-related breast tumors. RESULTS AND CONCLUSIONS: The BRCAx-related tumors were characterized by more frequent LOH at 22q relative to sporadic breast cancer (P < 0.02), and differed significantly from BRCA1- and BRCA2-related tumors in their positivity for Bcl2. However, cluster analyses of the combined data (LOH and immunohistochemistry) did not result in subgroups that would allow meaningful subclassification of the families. On chromosomes 2, 3, 6, 12, 13, 21, and 22, we found markers at which LOH occurred significantly more frequent among the tumors from patients belonging to a single family than expected on the basis of overall LOH frequencies. Nonetheless, linkage analysis with markers for the corresponding regions on chromosomes 12, 21, and 22 did not reveal significant logarithm of the odds.

The CHEK2*1100delC variant acts as a breast cancer risk modifier in non-BRCA1/BRCA2 multiple-case families.

The frame-shifting mutation 1100delC in the cell-cycle-checkpoint kinase 2 gene (CHEK2) has been reported to be associated with familial breast cancer in families in which mutations in BRCA1 and BRCA2 were excluded. To investigate the role of this variant as a candidate breast cancer susceptibility allele, we determined its prevalence in 237 breast cancer patients and 331 healthy relatives derived from 71 non-BRCA1/BRCA2 multiple-case early onset breast cancer families. Twenty-seven patients (11.4%) were carrying the CHEK2*1100delC variant. At least one carrier was found in 15 of the 71 families (21.1%). There was no evidence of cosegregation between the variant and breast cancer, but carrier patients developed breast cancer earlier than did noncarriers. We studied CHEK2 protein expression in 111, and loss of heterozygosity at CHEK2 in 88 breast tumors from these patients. Twelve of 15 tumors from carriers showed absent protein expression as opposed to 3 of 76 tumors from noncarriers (P < 0.001). CHEK2 loss of heterozygosity was associated with absence of protein expression but not with 1100delC carrier status. Thus, selecting for breast cancer cases with a strong familial background not accounted for by BRCA1 or BRCA2 strongly enriches for carriers of CHEK2*1100delC. Our results support a model in which CHEK2*1100delC interacts with an as yet unknown gene (or genes) to increase breast cancer risk.

Are ATM mutations 7271T-->G and IVS10-6T-->G really high-risk breast cancer-susceptibility alleles?

Two mutations of the ATM gene were recently suggested to confer breast cancer risks similar to mutations of BRCA1 or BRCA2. Here, we set out to confirm these findings in 961 families with non-BRCA1/BRCA2 breast cancer from diverse geographical regions. We did not detect the ATM 7271T-->G mutation in any family. The ATM IVS10-6T-->G mutation was detected in eight families, which was similar to its frequency among population-matched control individuals (pooled Mantel-Haenszel odds ratio = 1.60; 95% confidence interval = 0.48 to 5.35; P = 0.44). Bayesian analysis of linkage in the ATM IVS10-6T-->G-positive families showed an overall posterior probability of causality for this mutation of 0.008. We conclude that the ATM IVS10-6T-->G mutation does not confer a significantly elevated breast cancer risk and that ATM 7271T-->G is a rare event in familial breast cancer.

A genome wide linkage search for breast cancer susceptibility genes.

Mutations in known breast cancer susceptibility genes account for a minority of the familial aggregation of the disease. To search for further breast cancer susceptibility genes, we performed a combined analysis of four genome-wide linkage screens, which included a total of 149 multiple case breast cancer families. All families included at least three cases of breast cancer diagnosed below age 60 years, at least one of whom had been tested and found not to carry a BRCA1 or BRCA2 mutation. Evidence for linkage was assessed using parametric linkage analysis, assuming both a dominant and a recessive mode of inheritance, and using nonparametric methods. The highest LOD score obtained in any analysis of the combined data was 1.80 under the dominant model, in a region on chromosome 4 close to marker D4S392. Three further LOD scores over 1 were identified in the parametric analyses and two in the nonparametric analyses. A maximum LOD score of 2.40 was found on chromosome arm 2p in families with four or more cases of breast cancer diagnosed below age 50 years. The number of linkage peaks did not differ from the number expected by chance. These results suggest regions that may harbor novel breast cancer susceptibility genes. They also indicate that no single gene is likely to account for a large fraction of the familial aggregation of breast cancer that is not due to mutations in BRCA1 or BRCA2.