Results

In 42 of the first 100 families that consulted for familial cancer, the BRCA1 and BRCA2 genes were screened for mutations because the criteria (see Methods) for initiating the BRCA screen were met and a blood sample from a breast or ovarian cancer patient was available. As the clinic is open to all familial cancer syndromes, we consider this to be a high proportion, which can probably be explained by the prevalence of familial breast cancer and the attention the media paid to the discovery of the BRCA genes.

In a first step, the 42 families were screened for BRCA1/2 mutations at the genomic level with a PTT analysis on the large exons of BRCA1 and BRCA2, and by SSCP/HD on exons 2, 5, and 20 of BRCA1, as described in the Methods section. Mutations were found and confirmed by sequencing in 14 families (fig 1, table 1). Seven of these mutations were included in the series reported by Peelen et al.21

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Diagram of distribution of BRCA1/2 gene mutations.

We also analysed 11 families by RT-PTT to identify possible truncating mutations in the remaining smaller exons of BRCA1 and BRCA2. This method was applied for the screening of two RT-PCR fragments generated from BRCA1, while four fragments were analysed in the case of BRCA2 (see Methods). In nine out of the 66 PCR fragments investigated, the PTT generated truncated proteins not observed in control experiments. Sequence analysis of the corresponding complementary DNA fragment indicated that, in each case, the observed protein truncation resulted from abnormal splicing events since whole exons were missing. However, genomic sequence analysis of the intron/exon boundaries did not result in the detection of splice mutations.

The presence of ovarian cancer in the pedigree was a strong and significant (p=0.005) predictor for the probability of finding a BRCA1/2 gene mutation (table 2). The likelihood of finding a mutation increased with a lower average age at diagnosis of breast cancer in a family (p=0.015, table 3). There was a trend for a correlation between finding a mutation and a lower average age at diagnosis of ovarian cancer (p=0.093), the occurrence of breast and ovarian cancer in a single family member (p=0.49), or bilateral breast cancer (p=0.19). The lack of statistical significance could be related to our sample size.

There were fewer mutations found in the families that just met the lowest criteria for selection of two symptomatic first degree relatives, but the chance of finding a BRCA gene mutation did not correlate with the number of breast cancer cases in the family; there were on average 2.1 breast cancers in the families with an identified BRCA mutation versus 2.9 breast cancers in the families where no mutation was found. Even when corrected for family size by calculating the number of breast cancer cases per 100 women, no correlation was found: 17 breast cancers per 100 women in the families with an identified mutation versus 23 breast cancers in the families where no mutation was found. Four of the families that only just met the lowest criteria had ovarian cancer versus 10 of the families with a more extensive history of cancer in the family.

All mutations were found in breast/ovarian cancer families with few other cancers except for one BRCA1 mutation identified in a female rectal cancer patient belonging to a family with two (dead) breast cancer patients. Testicular cancer, diagnosed at the age of 25, also occurred in this family.

What is the interest in presymptomatic testing once a BRCA1 or 2 mutation is identified in the family? Family members who had shown an interest in testing initially, before identification of the mutation, consequently opted for testing. In contrast, of the other relatives who were eligible for testing, only a minority requested it. Overall, women tended to be somewhat more interested in the test than men: 14 women out of 52 eligible presymptomatic female relatives opted for testing (27%), whereas only eight men out of 46 eligible did so (17%). All presymptomatic women who tested positive took a conservative approach to their breast cancer risk, while two of the seven women with a previous history of breast cancer requested a prophylactic contralateral mastectomy after being notified of a positive BRCA test result.

Discussion

Previous reports and statistics on BRCA gene testing have mostly been generated based on large pedigrees with a large number of affected subjects. The stringent criteria for BRCA testing derived from these large families are not readily applicable to the commonly encountered situation of a small family in which dominant inheritance of cancer predisposition is apparent. To meet a demand for oncogenetic counselling from members of small families with only two or more affected members, less stringent rules have to be applied. The proposed minimum selection criteria for searching for a BRCA1 or BRCA2 gene mutation in small families with a limited number of cases were two or more first degree relatives with breast cancer if one of these was diagnosed before the age of 50 or if one of them had ovarian cancer at any age. The purpose of this analysis was first to verify whether the application of these selection criteria made testing still worthwhile, and what factors influence the likelihood of finding a mutation. A secondary purpose was to examine the consequences of finding a mutation in these families for the presymptomatic relatives. Forty-two families of the first 100 families that consulted for familial cancer met the criteria put forward for initiating a BRCA1/2 mutation screen and had material available for analysis. We searched for mutations in the large exons of the BRCA1 and BRCA2 genes, and in exons 2, 5, and 20 of BRCA1 that contain frequently recurring mutations described world wide. The analyses can be performed on genomic DNA and are easily incorporated into a routine diagnostic test. In 14 of the 42 families we were able to find a BRCA1 or BRCA2 mutation. The initial mutation screen covered only about 60% of the coding regions of the BRCA1 and BRCA2 genes. However, expanding the PTT analysis to all the remaining smaller exons, for which RNA material is needed, did not lead to the identification of additional truncating mutations. Recently, several authors described large deletions in the BRCA1 gene of several patients belonging to breast cancer families.22 23 For instance, in the Dutch population two mutations of this type were found to be present in more than 30% of the BRCA1 families.20 Southern blot analysis of BRCA1 in our series did not show any genomic alteration.

The success rate of 14 mutations identified in 42 families (33%) compares favourably with other data obtained in similar families.24-26 Various factors could explain this relatively high mutation rate. Mutations in the BRCA1 and BRCA2 genes have been found world wide, but the type of mutations identified can differ considerably from country to country. Only a few specific mutations are dispersed world wide (for example, 5382 ins C). Some mutations recur frequently but only in a particular country (for example, 2312 del 5 only in Belgium and The Netherlands) and many mutations have been found in only one or a few families.21The rather high proportion of ovarian cancers in the families reported here probably increased the yield of mutation detection. However, the BRCA2 gene mutations we could isolate in our breast and ovarian cancer families were not preferentially located in the ovarian cancer cluster region.27 A mutation was detected in all five families in which the average age at diagnosis of ovarian cancer among family members was less than 50 years. When analysing our site specific breast cancer families, the success rate for detecting a mutation decreases to 18%. This indicates that the mutation responsible for breast cancer “specific” predisposition in our small families cannot be picked up efficiently with the screening methods used. These mutations could be regulatory mutations or missense mutations of BRCA1 or BRCA2, or they may even be localised in a third BRCA gene, which would be responsible for the cancer predisposition in most of the small breast cancer specific families. Sequencing of the two genes to identify point mutations would be especially difficult in these families, as linkage analysis showing the pathogenicity of these point mutations would be difficult if not impossible.

Once a BRCA1 or 2 mutation was found, testing was offered to the family members. Surprisingly, only a minority of the presymptomatic men and women in the families with a BRCA gene mutation subsequently came forward to request testing. Since most of them were not interviewed, the reasons for this abstention are not known. One might speculate that some do not want testing, while others may not have been fully informed by their family members. This should be elucidated in the future as the relative responsibilities of health care workers versus family members for diffusing this knowledge within these families are better defined. The current lack of data on the cost/benefit of preventive surgery versus close observation justifies a neutral counselling position with regard to this matter and we are satisfied that presymptomatic female carriers opt for careful follow up for the moment. It is, however, noteworthy that some of the women with previous cancer and treatment experience chose a more invasive preventive strategy. We expect that the prospective follow up of mutants will help redefine the preventive options in the future.

So far, our experience has shown BRCA gene mutation analysis based genetic counselling to be feasible in small families when there are two or more symptomatic first degree relatives, if one of them had ovarian cancer, or if one breast cancer was diagnosed before the age of 50. It seems appropriate to continue screening small families in view of the fact that a BRCA gene mutation was identified in 24% of the families that only just met the minimal criteria for selection. Technical advances may allow the initial mutation analysis to be expanded. Commercial full mutation analysis may be appropriate in a limited number of families.