Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families.

The hereditary breast cancer gene BRCA2 was recently cloned and is believed to account for almost half of site-specific breast cancer families and the majority of male breast cancer families. We screened 49 site-specific breast cancer families for mutations in the BRCA2 gene using single strand conformation analysis (SSCA) followed by direct sequencing. We found mutations in eight families, including all four families with male breast cancer. The eight mutations were small deletions with the exception of a single nonsense mutation, an all were predicted to interrupt the BRCA2 coding sequence and to lead to a truncated protein product. Other factors which predicted the presence of a BRCA2 mutation included a case of breast cancer diagnosed at age 35 or below (P = 0.01) and a family history of pancreatic cancer (P = 0.03). Two mutations were seen twice, including a 8535delAG, which was detected in two French Canadian families. Our results suggest the possibility that the proportion of site-specific breast cancer families attributable to BRCA2 may be overestimated.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Mutation of the PTEN tumor suppressor gene in endometrial hyperplasias.

Mutation and deletion of the PTEN tumor suppressor gene occurs in about 40% of endometrial carcinomas. The purpose of this study was to determine whether PTEN mutations also are present in endometrial hyperplasias, which are premalignant precursors of invasive endometrial adenocarcinomas. Genomic DNA from 51 endometrial hyperplasias was extracted from paraffin blocks, and PCR was used to amplify the nine exons of the PTEN gene. These products were screened using single-strand conformation analysis, and variant bands were sequenced. Somatic mutations in the PTEN gene were seen in 10 of 51 cases (20%), and two mutations were found in one case. An identical 4-bp deletion in exon 8 was seen in three cases, and 8 of 11 PTEN mutations predicted truncated protein products. There was no higher frequency of PTEN mutations in endometrial hyperplasias with atypia (6 of 32; 19%) relative to those without atypia (4 of 19; 21%). These data suggest that inactivation of the PTEN tumor suppressor gene is an early event in the development of some endometrial cancers.

Aberrant splicing of the TSG101 tumor suppressor gene in human breast and ovarian cancers.

OBJECTIVE: To determine whether large deletions or other alterations in the putative tumor suppressor gene TSG101 play a role in the molecular pathogenesis of breast and ovarian cancers. METHODS: Expression of TSG101 transcripts was examined in breast and ovarian cancers using the reverse transcriptase-polymerase chain reaction (RT-PCR), and selected transcripts were sequenced. Southern blot analysis was performed to determine whether there were genomic deletions in the TSG101 gene, and Northern blot analysis was used to examine the relative abundance of various transcripts. RESULTS: All the cancerous and normal breast tissue examined expressed full length 1145 base pair (bp) TSG101 transcripts. Additional truncated transcripts were seen using the RT-PCR in 57 (64%) of 89 primary breast cancers, 1 (20%) of 5 breast cancer cell lines, 3 (50%) of 6 normal breast tissues, 16 (64%) of 25 primary ovarian cancers and 1 (33%) of 3 ovarian cancer cell lines. Only the primary breast (21%) and ovarian (24%) cancers had three or more truncated transcripts. None of the normal tissues or cell lines examined had more than two aberrant transcripts. DNA sequencing revealed that the most commonly expressed truncated transcript arises because of loss of 902 bp between codons 153 and 1055. Only full length TSG101 transcripts were seen on Northern blot analysis of breast cancer cell lines, however. There was no evidence of genomic deletions in the TSG101 gene on Southern blot analysis. CONCLUSION: Truncated TSG101 transcripts that probably represent splice variants are present in some breast and ovarian cancers, but there is no evidence to suggest that loss of this putative tumor suppressor gene plays a role in the molecular pathogenesis of these cancers.

Increased baseline levels of platelet P-selectin, and platelet-endothelial cell adhesion molecule-1 in patients with acute myocardial infarction as predictors of unsuccessful thrombolysis.

BACKGROUND: Platelets play a pivotal role in the pathogenesis of acute myocardial infarction and their activation can cause thrombolysis to fail. METHODS: Baseline aggregation of platelets and expression of major surface receptors measured by flow cytometry were compared with clinical outcome after thrombolysis for 23 patients enrolled in the GUSTO-III trial. RESULTS: Failure to reperfuse (n = 3) and recurrent ischemia (n = 2) were observed in five patients who subsequently underwent emergency angioplasty. These patients were treated later in their course of disease than were the 18 patients who were successfully reperfused and remained free of recurrent ischemia. Greater than normal expression of platelet P-selectin (33.7 +/- 1.1 versus 28.1 +/- 1.9, P = 0.01) and expression of platelet--endothelial cell adhesion molecule-1 (PECAM-1; (57.1 +/- 2.3 versus 50.2 +/- 2.8, P = 0.02) were observed in members of the group with recurrent ischemia or failed reperfusion. CONCLUSION: Greater than normal baseline expression of P-selectin and PECAM-1 platelet receptors was correlated to delayed and unsuccessful coronary thrombolysis among patients presenting with acute myocardial infarction.

Bcl10 is not a target for frequent mutation in human carcinomas.

The recently described Bcl10 gene has been suggested to be a major target gene for inactivation in a variety of human cancers. In order to further evaluate the role of this gene in human adult malignancies, we have analysed a series of carcinomas for mutations in the Bcl10 gene. We have screened a panel of 174 carcinoma samples in total, comprised of 47 breast, 36 epithelial ovarian, 36 endometrial, 12 cervical, 23 colorectal and 20 head/neck carcinomas, all unselected for grade or stage. This panel reflects, in part, tumours reported to have involvement of the 1p22 region of chromosome 1, the region harbouring the Bcl10 gene. No deleterious mutations were detected in any of the samples analysed, strongly suggesting that Bcl10 is not a common target for inactivation in adult malignancies and that BCL10 is not the gene targeted for frequent inactivation at 1p22.

K-ras mutations in Müllerian inclusion cysts associated with serous borderline tumors of the ovary.

OBJECTIVE: Müllerian inclusion cysts (MIC) are small benign appearing glands that are occasionally noted in lymph nodes and peritoneal biopsies. They occur most frequently in women with serous ovarian tumors, with borderline tumors (SBOT) having a higher incidence than invasive cancers. The aim of this study was to examine whether MIC and SBOT have identical K-ras mutations, which would suggest that they are related. Methods. Six patients in whom adequate tissue was available from SBOT, MIC, and normal tissue were identified from a consecutive series of patients with SBOT who underwent lymph node sampling from 1992 to 1997 at Duke University Medical Center. DNA extraction was performed using laser capture microdissection. Exon 1 of the K-ras gene was amplified using PCR and subjected to single-strand conformation analysis to screen for mutations. Shifted bands were sequenced to confirm the presence of mutations. RESULTS: Mutations in codon 12 of K-ras were found in three of six (50%) SBOT. In two of these three cases, the identical mutation was found in the SBOT and the MIC (gly to val in both cases), but not in the corresponding normal DNA. In one case, a mutation was seen in the ovarian tumor (gly to asp), but not in the corresponding MIC. CONCLUSIONS: Mutations in codon 12 of the K-ras gene are a hallmark of serous borderline tumors. The presence of identical K-ras mutations in some SBOT and their associated MIC suggests that they are related processes. Both may arise due to a field effect, or alternatively some MIC may represent metastases from the primary ovarian tumor.

Confirmation of a gene locus for medullary cystic kidney disease (MCKD2) on chromosome 16p12.

BACKGROUND: Autosomal-dominant medullary cystic kidney disease (MCKD) is an interstitial nephropathy characterized by structural renal tubular defects that result in salt wasting and a reduction in urinary concentration. The condition has clinical and morphological similarities to autosomal-recessive juvenile nephronophthisis. Two genes predisposing to MCKD have been localized. MCKD1 on chromosome 1q21 was localized in two Cypriot families, and MCKD2 on chromosome 16p12 was localized in a single Italian family. We have evaluated a large Welsh MCKD family for linkage at these two loci. METHODS: Clinical data and DNA samples were collected from affected family members. Polymorphic microsatellite markers spanning the critical regions on chromosome 1 and chromosome 16 that encompass MCKD1 and MCKD2 were analyzed. Two-point and multipoint LOD scores were calculated. RESULTS: The family fulfilled previously published criteria for the diagnosis of MCKD, but hyperuricemia and gout were not prominent features. Twenty-one affected individuals were identified. Mean age at death or end-stage renal disease was 47 years (37 to 60). Linkage and haplotype analysis generated strongly negative results at MCKD1 on chromosome 1q21 (two-point LOD score = -5.32). Strong evidence of linkage to MCKD2 was generated with a maximum multi-point LOD score of 3.75. CONCLUSION: These results provide the first independent confirmation of a gene predisposing to MCKD on chromosome 16p12 and indicate that mutation of this gene is not restricted to a single family or population. The absence of hyperuricemia and gout in our family indicates that these are not obligatory features of MCKD2 mutations.

Allele loss on chromosome 1p36 in epithelial ovarian cancers.

OBJECTIVES: Prior studies have shown that allelic loss on chromosome 1p36 occurs frequently in ovarian as well as several other types of cancer. This suggests that inactivation of gene(s) in this region may play a role in the pathogenesis of these cancers. The aim of this study was to further delineate the region of loss on chromosome 1p36 in ovarian cancers and to identify associated patient or tumor characteristics. METHODS: Paired normal/cancer DNA samples from 75 ovarian cancers (21 early stage I/II and 54 advanced stage III/IV) were analyzed using microsatellite markers. RESULTS: Forty-nine of 75 (65%) ovarian cancers had loss of at least one marker. The marker demonstrating the most frequent loss was D1S1597, which was lost in 29/57 (51%) informative cases. Allele loss on 1p36 was significantly more common in poorly differentiated ovarian cancers (73%) relative to well or moderately differentiated cases (48%) (P = 0.03). Evidence was obtained for two common regions of deletion: one flanked by D1S1646/D1S244 and another more proximally by D1S244/D1S228. CONCLUSION: These findings further delineate regions on chromosome 1p36 proposed to contain tumor suppressor gene(s) that may play a role in the development and/or progression of epithelial ovarian carcinoma. Allele loss on 1p36 is associated with poor histologic grade.

Identification of the breast cancer susceptibility gene BRCA2.

In Western Europe and the United States approximately 1 in 12 women develop breast cancer. A small proportion of breast cancer cases, in particular those arising at a young age, are attributable to a highly penetrant, autosomal dominant predisposition to the disease. The breast cancer susceptibility gene, BRCA2, was recently localized to chromosome 13q12-q13. Here we report the identification of a gene in which we have detected six different germline mutations in breast cancer families that are likely to be due to BRCA2. Each mutation causes serious disruption to the open reading frame of the transcriptional unit. The results indicate that this is the BRCA2 gene.

BRCA1 and BRCA2 in breast cancer families from Wales: moderate mutation frequency and two recurrent mutations in BRCA1.

Mutations in the BRCA1/BRCA2 genes account for varying proportions of breast cancer families studied, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations in 17 families from Wales with two or more cases of breast cancer under age 50 and/or ovarian cancer. Eight out of 17 (47%) families had demonstrable mutations. Six out of 17 (35%) carried BRCA1 mutations and 2 out of 17 (12%) carried BRCA2 mutations. Two recurrent mutations in BRCA1 were identified, which appear to represent founder mutations in this population. These data support the existence of additional breast and ovarian cancer susceptibility genes.

Depressed platelet status in an elderly patient with hemorrhagic stroke after thrombolysis for acute myocardial infarction. GUSTO-III Investigators.

BACKGROUND: Impaired platelet function has been reported in acute myocardial infarction (AMI) and stroke. However, prospective data on the changes of platelet status in patients before the occurrence of hemorrhagic stroke after thrombolytic therapy are unavailable. CASE DESCRIPTION: An 86-year-old male patient was among the 23 AMI patients enrolled in the platelet study for the GUSTO-III trial. He received 325 mg of aspirin daily for at least 6 years, suffered an AMI, and was successfully reperfused with alteplase, but after 44 hours developed a large hemorrhagic stroke resulting in paraplegia. Platelet aggregation and receptor expression were measured by flow cytometry and ELISA before thrombolysis and at 3, 6, 12, and 24 hours thereafter. The percentage of platelet aggregation was lower in the stroke patient at every time point when induced by 5 micromol/L of ADP, by 10 micromol/L of ADP, and by thrombin than in the rest of the AMI group. Ristocetin and collagen-induced aggregability were within the group range. Decreased platelet glycoprotein Ib, IIb, IIIa, and IIb/IIIa and vitronectin receptor expression were observed in the stroke patient. No other differences in p24 (CD9), very late antigen-2, P-selectin, and platelet/endothelial cell adhesion molecule-1 expression were determined. CONCLUSIONS: Profound depression of platelet status preceded the occurrence of hemorrhagic stroke in an elderly long-term aspirin user treated with thrombolytic therapy. Initial "exhausted" platelets may be responsible for the increased risk for hemorrhagic stroke after coronary thrombolysis.

Effect of cyclooxygenase-2 inhibition on renal function in elderly persons receiving a low-salt diet. A randomized, controlled trial.

BACKGROUND: Most nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both cyclooxygenase-1 (COX-1), whose inhibition is associated with gastrointestinal ulceration, and COX-2, whose inhibition is associated with therapeutic benefits. Although agents that do not produce COX-1 activity may have fewer adverse effects, targeted disruption of the COX-2 allele in mice has resulted in severe renal problems, suggesting that COX-2 inhibition may also produce adverse effects. OBJECTIVE: To determine the effect of rofecoxib, a member of the coxib class of drugs and a specific inhibitor of the COX-2 enzyme, on renal function in elderly patients. DESIGN: A randomized, three-period, single-dose crossover study and a randomized, parallel-group, multiple-dose study. SETTING: Clinical research units. PATIENTS: 75 patients 60 to 80 years of age. INTERVENTION: In the first study, single doses of rofecoxib, 250 mg (about 5-fold to 20-fold the recommended dose); indomethacin, 75 mg; and placebo were administered to 15 patients. In the second study, multiple doses of rofecoxib, 12.5 or 25 mg/d; indomethacin, 50 mg three times daily; or placebo were administered to 60 patients. Patients in both studies received a low-sodium diet MEASUREMENTS: Glomerular filtration rate, creatinine clearance, and urinary and serum sodium and potassium values. RESULTS: Compared with placebo, single doses of rofecoxib and indomethacin decreased the glomerular filtration rate by 0.23 m/s (P < 0.001) and 0.18 mL/s (P = 0.003), respectively. In contrast, respective decreases of 0.14, 0.13, and 0.10 mL/s were observed after multiple doses of rofecoxib, 12.5 mg/d (P = 0.019); rofecoxib, 25 mg (P = 0.029), and indomethacin (P = 0.086) were administered. Changes in creatinine clearance and serum and urinary sodium and potassium were less pronounced. CONCLUSIONS: The effects of COX-2 inhibition on renal function are similar to those observed with nonselective NSAIDs. Thus, COX-2 seems to play an important role in human renal function.