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.

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In long-term IDDM, the role of CF-SG autoantibodies, which tend to be more frequent in patients with ECG-based CAN, requires further investigations. The persistence of GAD and IA-2 autoantibodies is not related to ECG-based CAN.

BackgroundThe birth year‐dependent onset of breast cancer (BC) in BRCA1/2 mutation carriers suggests a risk‐modifying role for reproductive and life style factors. We therefore examined possible associations between these factors and age at diagnosis.MethodsCox regression analysis and log‐Rank testing were used to estimate the effect of potential life style factors on the onset of BC in 197 BRCA mutation carriers.ResultsNulliparous BRCA mutation carriers developed BC earlier than those who had delivered (36.4 vs. 40.9; P = 0.001). Similarly, smokers and women who had used oral contraceptives experienced an earlier cancer onset (39.0 vs. 41.4; P = 0.05 and 39.3 vs. 44.9; P = 0.0001, respectively). In multivariate analysis, oral contraceptive use (HR: 1.7; P = 0.006) and birth cohort (< vs. ≥1965 HR: 4.5; P = 0.001) were associated with an earlier BC onset, while previous pregnancies led to a delay (HR: 0.2; P = 0.04). Mutation carriers born ≥1965 were less likely to have experienced pregnancies and more likely to have used oral contraceptives, and consequently developed BC at an earlier age (median age: 42 vs. 58; P < 0.0001 log‐Rank test).ConclusionWe here demonstrate that in BRCA1/2 mutation carriers the birth cohort‐associated differences in the onset of BC are profound and influenced by reproductive factors.

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We investigated the prevalence of germline BRCA mutations in a population‐based cohort of Austrian women diagnosed with ovarian cancer and its association with family history of cancer. We prospectively collected family pedigrees of 443 Austrian ovarian cancer patients who had been tested for the presence of a germline BRCA or 2 mutations and correlated the familial breast and ovarian cancer burden with the prevalence of BRCA mutations and disease onset. The probability of carrying a gBRCA mutation in patients without family history of cancer is 14% (95% CI 9%‐22%), as opposed to 45% (95% CI 31%‐59%) of patients with at least one family member with ovarian cancer, and 47% (95% CI 40%‐54%) if other relatives have developed breast cancer. If both breast and ovarian cancer are diagnosed in the family, the probability of carrying a germline BRCA1 or 2 mutations is 60% (95% CI 50%‐68%). germline BRCA1 or mutations in families with ovarian cancer only are commonly located in the Ovarian Cancer Cluster Regions when compared to families with both breast and ovarian cancer (P = 0.001, and P = 0.020, respectively). While gBRCA mutation carriers with ovarian cancer do not have a significantly different age at onset than patients with a family history of cancer, gBRCA1 carriers in general have an earlier onset than gBRCA2 carriers (P = 0.002) and patients without a mutation (P = 0.006). The rate of germline BRCA1 or 2 mutations in ovarian cancer patients without a family history or breast or ovarian cancer is low. However, in women with additional family members affected, the prevalence is considerably higher than previously reported.