Rapid detection of BRCA1 mutations by the protein truncation test.

More than 75% of the reported mutations in the hereditary breast and ovarian cancer gene, BRCA1, result in truncated proteins. We have used the protein truncation test (PTT) to screen for mutations in exon 11, which encodes 61% of BRCA1. In 45 patients from breast and/or ovarian cancer families we found six novel mutations: two single nucleotide insertions, three small deletions (1-5 bp) and a nonsense mutation identified two unrelated families. Furthermore, we were able to amplify the remaining coding region by RT-PCR using lymphocyte RNA. Combined with PTT, we detected aberrantly spliced products affecting exons 5 and 6 in one of two BRCA1-linked families examined. The protein truncation test promises to become a valuable technique in detecting BRCA1 mutations.

Instability of short tandem repeats (microsatellites) in human cancers.

The allele sizes of polymorphic microsatellite repeats in DNA from human cancers were compared to normal DNA from the same patients. In 16 out of 196 paired samples (8%), we found evidence of an extra allele of a different size in the tumour which was not present in the normal DNA. Sequence analysis confirmed that the extra allele originates from the appropriate locus and that the size change is attributable to alteration in the number of repeat units. This form of instability was more common in tri- and tetranucleotide repeats than in dinucleotide repeats. In any single tumour sample only one repeat in the set examined was abnormal, the remainder showing identical patterns in normal and tumour DNA or evidence of allele loss. The pattern of instability in diverse types of cancer differs from that reported in colorectal neoplasms.

Evidence for a gene on 17p13.3, distal to TP53, as a target for allele loss in breast tumors without p53 mutations.

In breast cancer, loss of heterozygosity (LOH) on 17p is a frequent event and a likely target is the p53 gene on 17p13.1. However, several LOH mapping studies have indicated that, in some breast tumors, LOH affects only the most distal 17p markers, suggestive of a second tumor suppressor locus in 17p13.3. In order to distinguish which gene has most probably served as the target for LOH on 17p, we have screened 141 breast tumors for somatic mutations in the p53 gene in conjunction with detailed LOH mapping on the short arm of chromosome 17. A total of 32 mutations were detected in 31 tumors, 15 of which have never been reported in breast cancer before. The majority are point mutations leading to an amino acid change in the protein. In addition, we have stained a subset of 87 tumors for the p53 protein by immunohistochemistry. In 21 of these tumors (24%), nuclear staining was detected in over 25% of the tumor cells with the anti-p53 antibody DO7. A positive correlation was found between p53-positive staining and p53 mutation (P < 0.001). A strong association was observed between p53 mutation and LOH at the TP53 locus but not between p53 expression and LOH on 17p. In breast tumors without a detectable p53 mutation but with LOH on 17p, the 17p13.3 region is always involved and, in some cases, even exclusively involved. These results suggest that a second tumor suppressor gene, located distal to TP53, is targeted by LOH on 17p in some breast tumors and that a substantial number of breast tumors stabilize p53 through mechanisms other than mutation.

Allele loss patterns on chromosome 17q in 109 breast carcinomas indicate at least two distinct target regions.

Loss of heterozygosity (LOH) of markers for chromosome 17 is the most frequent genetic change observed in breast cancer to date. To assess whether the location of several candidate target genes is compatible with patterns of allele losses in the individual tumors, we examined the LOH status of chromosome 17 in 109 primary breast tumors with 15 polymorphic DNA markers (three for 17p and 12 for 17q). Allelic imbalance (AI) at 17q was observed in 44 of the 97 informative cases. A significant correlation was found between AI at the long arm and AI at the short arm of chromosome 17. The patterns of AI on 17q in the tumors differed and were highly complex in some cases. A number of tumors showed AI distal to the growth hormone locus, whereas others showed AI exclusively proximal of this marker. These results indicate that there are at least two different regions of allele loss on 17q.

The majority of 22 Dutch high-risk breast cancer families are due to either BRCA1 or BRCA2.

We have analyzed, by a combination of mutation and linkage analysis, the genetic basis of 22 breast cancer families in which at least 4 cases of either breast cancer diagnosed under the age of 60 or ovarian cancer had occurred. Chain-terminating mutations in BRCA1 were evidenced in 6 families, and posterior probabilities of > 0.90 of being linked to BRCA1 in 3. The breast versus ovarian cancer ratio in these 9 families was approximately 2:1. Among the remaining 13 families, significant linkage to markers flanking BRCA2 was established in the admixture test with a maximum multipoint lod score of 3.38, but there was no statistical evidence for genetic heterogeneity. The breast:ovarian cancer ratio in these families was 7:1, suggesting BRCA2 confers a much lower risk for ovarian cancer than does BRCA1. These results suggest that BRCA2 will explain a significant proportion of hereditary breast cancer in the Netherlands, and, together with BRCA1, account for the majority of all high-risk families.

Genes responsible for familial breast cancer.

A family history for breast cancer appears to be a major risk factor for breast cancer. It has been estimated that 5% of all breast cancers are hereditary. In the last five years much progress has been made in the identification of genes responsible for breast cancer. Much interest is focused on the BRCA-1 gene, which is associated with early onset breast and ovarian cancers. Heterogeneity within and across families in the pattern of cancer susceptibility has suggested that different susceptibility alleles may exist. The BRCA-1 gene has been cloned but the function of its product has not been determined. BRCA-1 mutations seem not to be involved in sporadic breast cancer. A second breast cancer susceptibility gene, BRCA-2, has been localized to chromosome 13q12-q13 but has not been identified as yet. Loss of heterozygosity of 13q is observed in 25% of sporadic breast tumors, which indicates that BRCA-2 might be a tumor suppressor gene. BRCA-2 confers only a low ovarian cancer risk. The TP53 gene has also been associated with breast cancer but to a much more limited extent than BRCA-1. Germline TP53 mutations have been found in patients with familial breast cancer. Other genes that have been associated with breast cancer risk are the androgen receptor (AR) gene and the ataxia teleangiectasia (AT) gene. The importance of the AR gene appears to be limited but the AT gene might be of considerable importance. It is to be expected that additional breast cancer susceptibility genes will be identified in the near future.

An evaluation of immune competence in different swine breeds.

The purpose of this investigation was to establish parameters for the evaluation of immune competence in different swine breeds. Cellular and humoral immune reactivity against keyhole limpet haemocyanin (KLH) was analysed using the lymphocyte stimulation test (LST), a skin allergy test and the IgG response. Various characteristics of the KLH-specific immune response were studied in 988 sows of four breeds. KLH-specific immune responses showed considerable variability. The applied statistical model explained 63 to 73 per cent of this variation. Genetic influences, expressed as the heritability estimate, were rather high for the IgG response (0.33), as well as for the skin reaction (0.26) and the LST (0.41-0.45). A positive correlation between the various immune parameters was found. Selective breeding for immune responsiveness seems to be feasible, but selection for cellular as well as humoral immune reactivity also seems to be possible with KLH as the antigen.

TP53 mutations and breast cancer prognosis: particularly poor survival rates for cases with mutations in the zinc-binding domains.

Acquired mutations in TP53 as well as immunohistochemically detectable protein expression have been implicated as prognostic factors for breast cancer. We have evaluated the relationship between mutations detected in 119 breast tumours and various clinicohistopathological indices, stratifying the mutations according to the functional domains as defined by the recent elucidation of the crystal structure of the protein. Patients with missense mutations located in regions encoding parts of the protein involved in zinc-binding had significantly decreased disease-free and overall survival relative to patients whose tumours had mutations in other domains. These results indicate that these biochemically defined domains also have biological relevance in terms of breast cancer disease course, and suggest that some mutations in TP53, more than others, can contribute to the development of clinically more aggressive and perhaps treatment resistant breast tumours. When confirmed, this will be of potential importance in predicting the clinical behaviour of breast cancer and its responsiveness to therapy.

High allele loss rates at 17q12-q21 in breast and ovarian tumors from BRCAl-linked families. The Breast Cancer Linkage Consortium.

Loss of heterozygosity (LOH) was evaluated in 174 breast and ovarian tumors derived from 94 families with at least 3 first-degree relatives affected with either of these cancers. By linkage analysis 26 families were identified as having a high posterior probability of being due to BRCAl (the breast/ovarian cancer susceptibility locus on 17q12-21) with lod scores varying from 0.51 to 9.49. Tumor genotypes were determined at at least 2 constitutionally heterozygous markers flanking BRCAl in a total of 58 tumors from these families. These tumors were derived from 52 patients, the BRCAl mutation carrier status of which was evidenced by DNA sequencing in 20, and inferred by reconstructing haplotypes in the remainder. LOH was detected in 50 (86%) tumors, and invariably involved the wild-type allele. Where informative, this allele was of paternal origin in 33 cases and of maternal origin in 10 cases. These results strongly suggest that BRCAl is a tumor suppressor gene and that LOH is greatly favored to fully inactivate it.

Age at diagnosis as an indicator of eligibility for BRCA1 DNA testing in familial breast cancer.

We searched for criteria that could indicate breast cancer families with a high prior probability of being caused by the breast/ovarian cancer susceptibility locus BRCA1 on chromosome 17. To this end, we performed a linkage study with 59 consecutively collected Dutch breast cancer families, including 16 with at least one case of ovarian cancer. We used an intake cut-off of at least three first-degree relatives with breast and/or ovarian cancer at any age. Significant evidence for linkage was found only among the 13 breast cancer families with a mean age at diagnosis of less than 45 years. An unexpectedly low proportion of the breast-ovarian cancer families were estimated to be linked to BRCA1, which could be due to a founder effect in the Dutch population. Given the expected logistical problems in clinical management now that BRCA1 has been identified, we propose an interim period in which only families with a strong positive family history for early onset breast and/or ovarian cancer will be offered BRCA1 mutation testing.

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 < or = 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.

Three germline mutations in the TP53 gene.

Three germline mutations in the TP53 tumor-suppressor gene are reported, two of which are not reported previously. A missense mutation at codon 265 of TP53 was found in three patients of a family that complied with the definition of the Li-Fraumeni syndrome. A nonsense mutation in codon 306 was found in a woman who had had a rhabdomyosarcoma at age 4 and a subsequent breast cancer at age 22. She was part of a Li-Fraumeni-like family, but the parental origin of the mutation could not be traced. Finally, while screening for somatic alterations in TP53 in a series of 141 sporadic breast tumors, we detected a constitutional missense mutation in codon 235 in a woman diagnosed with breast cancer at age 26 and a recurrence 4 years later. The recurrence, but not the primary tumor, showed an additional missense mutation at codon 245 as well as loss of the wild-type allele. This suggests that the 245 mutation was particularly important for tumor progression and that there might exist heterogeneity in terms of cancer predisposition potential among the various germline TP53 mutations.