Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.

The aim of this study was to evaluate the prevalence of simple sequence variation in the BRCA2 gene. To this end, 71 breast and breast-ovarian cancer (HBC/HBOC) families along with 95 control individuals from a wide range of ethnicities were analyzed by means of denaturing high-performance liquid chromatography (DHPLC) and direct sequence analysis. In the coding (10 257 bp) and non-coding (2799 bp) sequences of BRCA2, 82 sequence variants were identified. Three different, apparently disease-associated BRCA2 mutations were found in six HBC/HBOC families (8%): two splice site mutations in introns 5 and 21, and one frameshift mutation in exon 11. In the coding region, 53 simple sequence variants were found: 35 missense mutations, one 2 bp deletion (CT) resulting in a stop at codon 3364, one nonsense mutation with a stop at codon 3326, one deletion of a complete codon (AAA) resulting in the loss of leucine, and 15 silent mutations. In the non-coding region, 26 polymorphisms were detected. Of the 79 sequence variants that were not obviously disease-associated, eight were detected only in HBC/HBOC families. The remaining 71 variants were identified in both HBC/HBOC families and control individuals. Sixty three sequence variants (80%) were specific for a continent. Forty two percent (33 out of 79) of the sequence variants were detected exclusively in Africa, though only 13% of the 332 chromosomes screened were of African origin. Our data indicate that, in BRCA2, simple sequence variation is frequent [in the coding region 1 in 194 bp (straight theta = 2.2 x 10(-4)), and in the non-coding region 1 in 108 bp (straight theta = 4.4 x 10(-4)), respectively].

We identified 17 BRCA1 mutations in 86 Austrian breast and ovarian cancer families (20%) that were screened for mutations by denaturing high-performance liquid chromatography (DHPLC) and the protein truncation test (PTT). Eleven distinct mutations were detected, 4 of them (962del4, 2795del4, 3135del4 and L3376stop) not previously reported in families of non-Austrian origin. In addition, 6 rare missense mutations (allele frequency F 1%) with unknown biological effects were identified. Four mutations occurred more than once in the Austrian population: 2795del4 (3 times), Cys61Gly (3 times) 5382insC (2 times) and Q1806stop (2 times). Haplotype analysis of the 4 recurrent mutations suggested a common ancestor for each of these. Thirty-four breast cancer cases from 17 families with BRCA1 mutations were further analyzed. We observed a low median age of onset (39.5 years). Sixty-eight percent of all BRCA1 breast cancer cases had negative axillary lymph nodes. This group showed a significant prevalence of a negative estrogen and progesterone receptor status and stage I tumors compared with an age-related, node-negative control group. The prevalence of grade III tumors was marginally significant . Survival analysis either with a control group matched for age (within 5 years), grade, histologic subtype and estrogen receptor status, or with an age-related, node-negative comparison group, showed no statistical difference. Int.

Mutation analysis of the BRCA1 and BRCA2 genes has become widely available to hereditary breast and ovarian cancer families (HBOC) or breast-cancer-only families (HBC) in North America and Europe. To date, hundreds of these families have been identified with BRCA mutations and the majority has received counselling regarding the test results. Follow-up care of mutation carriers has focused on cancer surveillance and surgical options (Burke et al, 1997). However, little is known about the impact carrier status has on the development of depression, or the resulting changes in the individual's body image (Lerman et al, 1996Croyle et al, 1997;Lynch et al, 1997). In the Lerman et al (1996) study population, 18% stated their intent to undergo prophylactic mastectomy (PM) and 33% stated their intent to under prophylactic oophorectomy (PO). The motivational issues involved in deciding for or against prophylactic surgery, however, still await detailed study. Similarly, hardly anything is known about the feelings associated with this decision or their effect on the individual's quality of life in general.The major aim of this study was to study the attitude of mutation carriers in regard to PM and PO. Furthermore, it assessed the short-term effect of knowledge about carrier status on the occurrence of depression. PATIENTS AND METHODS Study populationA total of 138 individuals, from 35 families, that had been selected and counselled through the genetic counselling service of the Division of Senology, University of Vienna, were enrolled on the basis of previously detected BRCA1 or BRCA2 mutations. None of the family members had received the results of the BRCA testing. All individuals who requested their results were informed about this study. Family members who elected to participate gave their written consent. Prior to mutation result disclosure and counselling study participants received the first questionnaire of this study. The sequence in which the various elements of the survey were conducted is depicted in Figure 1.All female mutation carriers are examined every 6 months for the purposes of cancer surveillance and psychosocial follow-up. The observation period for this study began in January 1996 and is currently still being continued. This study was approved by the University of Vienna Institutional Review Board. Attitude of mutation carriers towards the option of PM and/or POFemale mutation carriers received a questionnaire comprising 12 items concerning PM and 11 items concerning PO to be rated on a five-point scale. Since the relevant literature does not provide a standardized questionnaire on this subject, two special questionnaires were developed on the basis of past conversations with SummaryThe intent of this study was to evaluate the effect that an awareness of being a BRCA1 or BRCA2 mutation carrier has on the attitude towards prophylactic surgery and on developing depression symptoms. Thirty-five families were selected on the basis of previously detected BRCA1 or 2 mutations and 90 family members were giv...