Identifying the BRCA1 c.-107A > T variant in Dutch patients with a tumor BRCA1 promoter hypermethylation.

An inherited single nucleotide variant (SNV) in the 5'UTR of the BRCA1 gene c.-107A > T was identified to be related to BRCA1 promoter hypermethylation and a hereditary breast and ovarian cancer phenotype in two UK families. We investigated whether this BRCA1 variant was also present in a Dutch cohort of breast and ovarian cancer patients with tumor BRCA1 promoter hypermethylation. We selected all breast and ovarian cancer cases that tested positive for tumor BRCA1 promoter hypermethylation at the Netherlands Cancer Institute and Sanger sequenced the specific mutation in the tumor DNA. In total, we identified 193 tumors with BRCA1 promoter hypermethylation in 178 unique patients. The wild-type allele was identified in 100% (193/193) of sequenced tumor samples. In a large cohort of 178 patients, none had tumors harboring the previously identified c.-107A > T SNV in BRCA1. We therefore can conclude that the germline SNV is not pervasive in patients with tumor BRCA1 promoter hypermethylation.

TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes.

BACKGROUND Li-Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome. Most families fulfilling the classical diagnostic criteria harbour TP53 germline mutations. However, TP53 germline mutations may also occur in less obvious phenotypes. As a result, different criteria are in use to decide which patients qualify for TP53 mutation analysis, including the LFS, Li-Fraumeni-like (LFL) and Chompret criteria. We investigated which criteria for TP53 mutation analysis resulted in the highest mutation detection rate and sensitivity in Dutch families. We describe the tumour spectrum in TP53-positive families and calculated tumour type specific relative risks. METHOD A total of 180 Dutch families referred for TP53 mutation analysis were evaluated. Tumour phenotypes were verified by pathology reports or clinical records. RESULTS A TP53 germline mutation was identified in 24 families. When the Chompret criteria were used 22/24 mutations were detected (sensitivity 92%, mutation detection rate 21%). In LFS and LFL families 18/24 mutations were found (sensitivity 75%). The two mutations detected outside the 'Chompret group' were found in a child with rhabdomyosarcoma and a young woman with breast cancer. In the mutation carriers, in addition to the classical LFS tumour types, colon and pancreatic cancer were also found significantly more often than in the general population. CONCLUSION We suggest TP53 mutation testing for all families fulfilling the Chompret criteria. In addition, TP53 mutation testing can be considered in the event of childhood sarcoma and breast cancer before 30 years. In addition to the risk for established LFS tumour types, TP53-positive individuals may also have an elevated risk for pancreatic and colon cancer.

The single-nucleotide polymorphism 309 in the MDM2 gene contributes to the Li-Fraumeni syndrome and related phenotypes.

Li-Fraumeni syndrome (LFS) is an autosomal-dominant cancer predisposition syndrome of which the majority is caused by TP53 germline mutations and is characterised by different tumour types occurring at relatively young age. Recently, it was shown that a single-nucleotide polymorphism (SNP) in the MDM2 gene, SNP309 (T>G variation), was associated with accelerated tumour formation in LFS patients who carry a TP53 germline mutation. To confirm this finding in different populations, we screened 25 Dutch and 11 Finnish TP53 mutation carriers for the presence of the SNP309 G allele in the MDM2 gene. Additionally, we investigated whether the SNP309 G allele plays a role in 72 Dutch TP53-negative LFS and LFS-related patients. In the TP53 germline mutation carriers, a significant difference was seen in the mean age of tumour onset for the SNP309 G allele group, that is, 29.7 years as compared to the SNP309 homozygous T group 45.5 years (P=0.005). In patients of LFS and LFS-related TP53-negative families, no difference was seen in the mean age of tumour onset. However, this TP53-negative group did show a significantly higher percentage of SNP309 homozygotes (G/G) compared to the general population (P=0.02). In conclusion, TP53 germline mutation carriers who have an SNP309 G allele have an earlier onset of tumour formation. The higher prevalence of MDM2 SNP309 homozygous G/G carriers in the TP53-negative group suggests that this allele contributes to cancer susceptibility in LFS and LFS-related families.

Late-onset common cancers in a kindred with an Arg213Gln TP53 germline mutation.

Li-Fraumeni syndrome (LFS) is an autosomal-dominant condition characterized by early-onset sarcoma, breast cancer and other specific tumour types. In most LFS kindreds germline TP53 mutations have been identified. In general, TP53 germline mutations are not associated with late-onset common cancers. We encountered a large kindred in which a wide spectrum of tumour types occurred, including melanoma, breast, ovarian, colorectal, stomach and renal cell cancer, without clear-cut early ages at onset of disease. An Arg213Gln TP53 germline mutation was detected in 12 out of 15 affected family members whereas testing for other cancer susceptibility genes in selected patients was negative. In vitro testing indicated that the specific TP53 mutation inactivates the protein transcriptionally. Our findings suggest that this TP53 germline mutation is a causative factor in this family and that specific TP53 germline mutations can be associated with relatively late-onset common cancers.

Screening for polymorphisms in the PXR gene in a Dutch population.

OBJECTIVE: Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of over 50% of all drugs currently in use. However, CYP3A4 expression shows a large inter-individual variation that cannot only be explained by genetic polymorphisms identified in this gene. The pregnane X receptor (PXR) has been identified as a transcriptional regulator of CYP3A4. Single nucleotide polymorphisms (SNPs) in the PXR gene could influence PXR activity and thereby CYP3A4 expression. This study was therefore aimed at determining the frequencies of known SNPs and detecting yet unknown SNPs in the PXR gene in a Dutch population. METHODS: Genomic DNA was isolated from blood samples obtained from 100 healthy volunteers and subjected to PCR amplification, followed by DNA sequencing. The population, of which the ethnicity was 93% Caucasian, consisted of 79 female individuals and 21 males. RESULTS: A total of 24 SNPs were found in the PXR gene, eight of which are previously unknown. The allelic frequencies found in this population varied from 0.5 to 73%. Most of the previously detected SNPs were located in introns. One new SNP, T8555G in exon 8, causes an amino acid change of C379G and is located in the Ligand Binding Domain of PXR. CONCLUSION: Several SNPs were detected in the PXR gene, one of which is located in the ligand binding domain (LBD). These SNPs may influence PXR-mediated CYP3A4 induction.

Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method.

We applied a novel method to detect single or multiple exon deletions and amplifications in the BRCA1 gene. The test, called multiplex ligation-dependent probe amplification (MLPA), uses probes designed to hybridize adjacently to the target sequence. After ligation, the joined probes are amplified and quantified. Our two diagnostic laboratories have tested in the recent years 805 families by conventional PCR-based techniques, and found 116 BRCA1 and 28 BRCA2 mutation-positive families. Using MLPA, we have tested the remaining 661 noninformative breast cancer families and identified five distinct BRCA1 germ-line mutations in five families: a deletion of exon 8, a deletion of exons 20-22, a duplication of exon 13 and exons 21-23, respectively, and a triplication, encompassing exons 17-19. Genomic deletions of BRCA1 constitute a substantial fraction of mutations in Dutch breast cancer families. If MLPA had been included in our initial BRCA1 testing, 33 families with a deletion or duplication would have been identified, representing 27% of the total 121 BRCA1 mutation-positive families. The MLPA test for BRCA1 ensures a sensitive and comprehensive high-throughput screening test for genomic rearrangement and can easily be implemented in the molecular analysis of BRCA1.

Detection of circulating breast tumor cells by differential expression of marker genes.

PURPOSE: We undertook a systematic approach to identify breast cancer (BC) marker genes with molecular assays and evaluated these marker genes for the detection of minimal residual disease in peripheral blood mononuclear cells (PBMCs). EXPERIMENTAL DESIGN: We used serial analysis of gene expression to identify a range of genes that were expressed in BC but absent in the expression profiles of blood and bone marrow cells. Next, we evaluated a panel of four marker genes (p1B, PS2, CK19, and EGP2) by real-time quantitative PCR in 103 PBMC samples from patients with metastatic BC (stage III/IV) and in 96 PBMC samples from healthy females. RESULTS: Increased marker gene expression of at least one marker was seen in 33 of 103 patients. Using quadratic discriminant analysis including all four marker genes, we determined a discriminant value with 29% positivity in the BC patient group that did not yield false positive results among the healthy females. CONCLUSIONS: Real-time PCR for the simultaneous expression of multiple cancer-specific genes may ensure the specificity required for the clinical application of mRNA expression-based assays for occult tumor cells.