It appears that all types of genomic nucleotide variations can be deleterious by affecting normal pre-mRNA splicing via disruption/creation of splice site consensus sequences. As it is neither pertinent nor realistic to perform functional testing for all of these variants, it is important to identify those that could lead to a splice defect in order to restrict transcript analyses to the most appropriate cases. Web-based tools designed to provide such predictions are available. We evaluated the performance of six of these tools (Splice Site Prediction by Neural Network [NNSplice], Splice-Site Finder [SSF], MaxEntScan [MES], Automated Splice-Site Analyses [ASSA], Exonic Splicing Enhancer [ESE] Finder, and Relative Enhancer and Silencer Classification by Unanimous Enrichment [RESCUE]-ESE) using 39 unrelated retinoblastoma patients carrying different RB1 variants (31 intronic and eight exonic). These 39 patients were screened for abnormal splicing using puromycin-treated cell lines and the results were compared to the predictions. As expected, 17 variants impacting canonical AG/GT splice sites were correctly predicted as deleterious. A total of 22 variations occurring at loosely defined positions (+/-60 nucleotides from an AG/GT site) led to a splice defect in 19 cases and 16 of them were classified as deleterious by at least one tool (84% sensitivity). In other words, three variants escaped in silico detection and the remaining three were correctly predicted as neutral. Overall our results suggest that a combination of complementary in silico tools is necessary to guide molecular geneticists (balance between the time and cost required by RNA analysis and the risk of missing a deleterious mutation) because the weaknesses of one in silico tool may be overcome by the results of another tool.

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Constitutional mutations of the RB1 gene are associated with a predisposition to retinoblastoma. It is essential to identify these mutations to provide appropriate genetic counseling in retinoblastoma patients, but this represents an extremely challenging task, as the vast majority of mutations are unique and spread over the entire coding sequence. Since 2001, we have implemented RB1 testing on a routine basis as part of the clinical management of retinoblastoma. As most screening techniques do not meet the requirements for efficient RB1 testing, we have devised a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a quantitative multiplex PCR of short fluorescent fragments (QMPSF) approach to screen for gene rearrangements. We report the results of this comprehensive screening of all exons and promoter of RB1 in 192 unrelated patients, mostly of French origin. Among 102 bilateral and/or familial cases and 90 unilateral sporadic probands, mutations were identified in 83 (81.5%) and 5 (5.5%) cases, respectively. A total of 43 mutations have not been previously reported. The mutational spectrum was found to be significantly different from previous published series, displaying a surprising amount of splice mutations and large deletions. This study demonstrates the reliability of DHPLC for RB1 analysis, but also illustrates the need for a deletion scanning approach. Finally, considering the benefits to retinoblastoma patients, RB1 testing should be widely implemented in routine healthcare because our study clearly illustrates its feasibility.

PURPOSE To provide precise age-specific risk estimates of cancers other than female breast and ovarian cancers associated with pathogenic variants (PVs) in BRCA1 and BRCA2 for effective cancer risk management. METHODS We used data from 3,184 BRCA1 and 2,157 BRCA2 families in the Consortium of Investigators of Modifiers of BRCA1/2 to estimate age-specific relative (RR) and absolute risks for 22 first primary cancer types adjusting for family ascertainment. RESULTS BRCA1 PVs were associated with risks of male breast (RR = 4.30; 95% CI, 1.09 to 16.96), pancreatic (RR = 2.36; 95% CI, 1.51 to 3.68), and stomach (RR = 2.17; 95% CI, 1.25 to 3.77) cancers. Associations with colorectal and gallbladder cancers were also suggested. BRCA2 PVs were associated with risks of male breast (RR = 44.0; 95% CI, 21.3 to 90.9), stomach (RR = 3.69; 95% CI, 2.40 to 5.67), pancreatic (RR = 3.34; 95% CI, 2.21 to 5.06), and prostate (RR = 2.22; 95% CI, 1.63 to 3.03) cancers. The stomach cancer RR was higher for females than males (6.89 v 2.76; P = .04). The absolute risks to age 80 years ranged from 0.4% for male breast cancer to approximately 2.5% for pancreatic cancer for BRCA1 carriers and from approximately 2.5% for pancreatic cancer to 27% for prostate cancer for BRCA2 carriers. CONCLUSION In addition to female breast and ovarian cancers, BRCA1 and BRCA2 PVs are associated with increased risks of male breast, pancreatic, stomach, and prostate (only BRCA2 PVs) cancers, but not with the risks of other previously suggested cancers. The estimated age-specific risks will refine cancer risk management in men and women with BRCA1/2 PVs.

Epidemiological studies have suggested that ataxia-telangiectasia (AT) heterozygotes have a predisposition to cancer, especially breast cancer in women. Now, haplotyping can identify heterozygotes for AT mutation (ATM) in AT families, allowing the risk of cancer associated with ATM heterozygosity status to be better assessed. We report a family study of AT patients, in which we estimated the risk of cancer according to ATM heterozygosity status. We analyzed demographic characteristics and occurrence of cancer in 1,423 relatives of AT patients. Haplotyping was performed in living relatives. The probability of being heterozygotes for ATM was calculated for deceased relatives. The risk of developing cancer was estimated in the cohort of relatives, and expected numbers of cancer cases were calculated from French age period-specific incidence rates. The number of cancers at all sites in the total population of relatives was not higher than expected. However, significant heterogeneity was found according to ATM heterozygosity status. This is mainly due to the increased risk of breast cancer previously observed in obligate heterozygotes. In obligate heterozygotes, relative risk (RR) was non-significantly increased for thyroid cancer, leukemia and liver cancer. Risks of ovarian, lung, pancreatic, kidney, stomach and colorectal cancers were non-significantly increased in the group with 0.5 probability of being heterozygotes. The RR was not significantly increased for any site of cancer, except for breast. Therefore, there is no evidence that specific screening of relatives of AT patients would be justified at particular sites other than the breast. However, the amplitude of the risk of breast cancer estimated in heterozygous women does not appear to justify a separate screening program from that already available to women with a first-degree relative affected by breast cancer. © 2001 Wiley-Liss, Inc. Key words: ataxia-telangiectasia; heterozygosity; cancer risk; family study; breast cancerAtaxia-telangiectasia (AT), which is caused by mutations in the ATM (AT-mutated) gene, is a rare autosomal recessive disorder, characterized by progressive neuronal degeneration, immunological deficiency, radiosensitivity and an increased risk of cancer. Most cancers in patients with AT are lymphoid-type. 1,2 Epidemiological studies in AT families have suggested that AT heterozygotes may have increased cancer risk, especially for breast cancer, 3-11 and possibly increased risk of ischemic heart disease. 12 Combined analysis of the available data in 1994 led to an estimate of 3.9 for the increased breast-cancer risk in women. 13 Although an increased risk of developing all cancers has often been found, little is known about increased risk at particular sites, except for breast cancer. 14 Despite suggestions, there is no evidence that the incidence of ovarian, lung, gastric, pancreatic, basal-cell, colon, cervical or biliary system cancers; leukemias; or lymphomas is increased in AT families. 4,11,15 Although homozygotes are rare, 1 in 40...