Original article Age-dependent penetrance of different germline J Clin Pathol: first published as 10.1136/jcp.2008.062646 on 27 March 2009. Downloaded from mutations in the BRCA1 gene F Al-Mulla,1 J M Bland,2 D Serratt,3 J Miller,3 C Chu,3 G T Taylor4 c Additional data are published ABSTRACT carriers of the germline BRCA1 and BRCA2 remains online only at http://jcp.bmj. Aims: BRCA1 gene mutations have been extensively controversial2 and the controversy could partly com/content/vol62/issue4 studied in relation to breast and ovarian cancer have stemmed from the use of different popula- 1 Department of Pathology, susceptibility. Various genotype–phenotype correlation tions and study designs, and the lack of specific Molecular Pathology Unit, attempts have yielded important data pertaining to the mutation stratification.8 Faculty of Medicine, Safat, Kuwait; 2 Department of Health consequences of BRCA1 mutations. However, little is The Breast Cancer Information Core Database Sciences, University of York, known about the effects of recurrent BRCA mutations on (BIC) lists more than 1560 and 1880 mutations and York, UK; 3 Yorkshire Regional expressivity and the age of onset of cancer in a polymorphisms in the BRCA1 and BRCA2 genes Genetics Service, St James’s 4 population. This study addresses whether different exon respectively. The influences of each mutation type Hospital, Leeds, UK; St (genotype) on expressivity, cancer type and age- James’s Hospital Leeds, mutations have variable expressivity especially in relation Genomic Services, Institute for to the age of onset of breast cancer. dependent penetrance (phenotype) remain either Cancer Research, Cancer Methods: Using a step-wise systematic approach, controversial or unexplored. In the last decade, Research UK culminating in the sequencing of all BRCA1 and BRCA2 several reports have been published supporting the exons with the addition of multiplex ligation-dependent hypothesis that different mutations in the BRCA1 Correspondence to: Dr Fahd Al-Mulla, Department of probe amplification, the relationship between disease or BRCA2 genes confer different cancer-related Pathology, Molecular Pathology phenotypes and gene mutations in 219 individuals and risks. For example, Gayther et al (1995) reported Unit, Faculty of Medicine, PO their family members was examined. that the risk of ovarian cancer relative to the risk of Box 24923, Safat, Kuwait breast cancer was higher in families with muta- 13110; [email protected] Results: It is shown that different BRCA1 gene mutations have distinct effects that influence the age of onset of tions located 59 to exon 13 of the BRCA1 gene 9 Accepted 11 November 2008 breast or ovarian cancer. Mutations in exon 2 of the compared with families with mutations located 3 9 BRCA1 gene had significantly lower penetrance compared to exon 13. However, this finding was not 10–12 with mutations of exons 11, 13 and 20. The median age confirmed in later reports. Also, an ovarian of affliction with breast cancer was 55 years for cancer cluster region (OCCR) in the BRCA2 gene 13 185delAG in exon 2 (95% confidence interval (CI) 46.7 to was described by Gayther et al (1997). A later 59.5), 47 years for the 4184delTCAA mutation in exon 11 international collaborative study provided no (95% CI 39 to 55.4), and 41 years for exon 13 duplication statistically significant support for the existence of such a region,14 rather it was suggested that a (95% CI 32.9 to 49.7) of the BRCA1 gene. Moreover, 14 http://jcp.bmj.com/ novel mutations in BRCA1 and BRCA2 genes in the reduced absolute risk of breast cancer for OCCR Yorkshire/Humberside population were identified. mutations contributed substantially to the effect 15 Conclusions: The 185delAG mutation of the BRCA1 gene described. All the above data pooled different but is a low penetrance mutation that is age dependent neighbouring BRCA1 or BRCA2 mutations and especially when compared with the exon 13 duplication associated them with phenotypes. However, does a mutation. The data have important ramifications on specific mutation in the BRCA1 or BRCA2 gene screening, genetic counselling and prophylactic treatment confer different phenotypes compared with other on September 23, 2021 by guest. Protected copyright. ? of BRCA1 gene mutation carriers. common mutations in the same gene This is a question that remains unanswered especially per- taining to the recurrent BRCA1 exon 2, 11 and 13 mutations. The answer to this question could have Breast cancer is the most prevalent malignancy in important implications for screening, genetic women.1 In the western hemisphere the lifetime counselling and prophylactic treatment of carriers. risk of developing breast cancer is more than 10%. Therefore, there is currently a need to study the Ovarian cancer, on the other hand accounts for a genotype–phenotype correlation among common lifetime risk of 1.8%.2 The majority of breast and mutations in BRCA1 and BRCA2 genes. Our paper ovarian cancers are sporadic or not inherited. addresses this need. Germline mutations in the breast and ovarian cancer susceptibility genes BRCA1 (MIM 113705)3 and BRCA2 (MIM 600185)45 confer a high risk of METHODS developing breast and ovarian cancers. Women Patients with a mutation in the BRCA1 gene have a lifetime A total of 241 patients and their family members risk of 80–90% of developing breast cancer, and 40– from Yorkshire and Humberside, UK, were chosen 65% chance of developing ovarian cancer.6 For for BRCA1 and BRCA2 mutation testing. They BRCA2 gene mutation carriers, the estimated belonged to 131 families classified as moderate to cumulative risk of breast cancer has been shown high risk of carrying a BRCA1 or BRCA2 mutation. to be 28% by age 50 years, and 84% by age High-risk individuals were members of families This paper is freely available 7 online under the BMJ Journals 70 years. The risk of ovarian cancer has been with four confirmed cases of breast and/or ovarian unlocked scheme, see http:// shown to be 0.4% by age 50 years and 27% by age cancers, with breast cancer occurring under the jcp.bmj.com/info/unlocked.dtl 70 years.7 Nevertheless, the magnitude of risk to age of 60 years or ovarian cancer at any age. 350 J Clin Pathol 2009;62:350–356. doi:10.1136/jcp.2008.062646 Original article J Clin Pathol: first published as 10.1136/jcp.2008.062646 on 27 March 2009. Downloaded from Moderate-risk groups were defined as families with three cases Mutation testing of cancer. Patients were followed up for a mean of High-quality DNA was extracted from 5 ml EDTA-treated 81.87 months (range 0.33–343.6 months), and age at diagnosis venous blood samples using a standard phenol–chloroform and overall survival were recorded. Mutational and correspond- procedure. All DNA samples were screened for mutations at ing clinical data were available for 219 cases. For calculating age exon 2 (185delAG), exon 20 (5382insC) of BRCA1, and exon 11 at diagnosis, the age and date at which cancer of the breast or (6147delT) of BRCA2, using amplification refractory mutation ovary occurred was noted. For calculating overall survival, the system PCR and duplication (Exon13dup6kb) of exon 13 by date of death was recorded. In addition, for other subjects with multiplex ligation-dependent probe amplification (P002; MRC- a follow-up date, but no date of death, this was coded as Holland, Amsterdam, The Netherlands) as instructed by the kit censored. For one subject with no follow-up time recorded but manufacturer (level 1 testing). DNA negative for these muta- dates of diagnosis and follow-up, the follow-up time was tions was subjected to level 2 testing, which involved direct calculated from the dates. sequencing of all exon 11 in eight large PCR fragments. Negative All patients and family members were counselled and signed a samples were then subjected to level 3 testing that called for written informed consent before testing. The procedures single-strand conformation polymorphism (SSCP) analysis and followed were in accordance with the ethical standards sequencing of all BRCA1 coding exons. Therefore, all samples implemented at the University of Kuwait and with the that were negative for levels 1, 2 and SSCP were sequenced. In Declaration of Helsinki 1975, as revised in 1983. all reactions, positive (DNA with known mutations) and negative controls (wild-type DNA from normal individuals) were included. Primer sequences and reaction conditions are Table 1 Breakdown of families with the most frequent BRCA1 available online.16 mutations No of Statistical analysis Gene/ No of mutation No Family Mutation exon individuals carriers affected Two outcomes were considered: age at diagnosis and time from diagnosis to death. As both of these are time to event data, Cox 761 185delAG BRCA1/2 410 proportional hazards regression was used. Four groups of 2802 185delAG BRCA1/2 26 11 3 subjects were compared, defined by the exon where the 5358 185delAG BRCA1/2 211 mutation took place: exon 2, exon 11, exon 13 and all other 9603 185delAG BRCA1/2 110 9877 185delAG BRCA1/2 441 exons. The hazard ratio for each group, exon 2, 11 and 13, 12654 185delAG BRCA1/2 111 compared to the other exons group were calculated, with an 13376 185delAG BRCA1/2 211 overall significance test of the effect of exon and confidence 17240 185delAG BRCA1/2 222 intervals for each hazard ratio. Patients were grouped into 17924 185delAG BRCA1/2 211 pedigrees, where more than one patient may belong to the same 21347 185delAG BRCA1/2 222 pedigree. Clustering within pedigrees was be adjusted for using 17 27388 185delAG BRCA1/2 421 robust standard errors by the method of Lin and Wei. For the http://jcp.bmj.com/ 30231 185delAG BRCA1/2 211 time to death, we also controlled for age at diagnosis, to allow 2592 4184delTCAA BRCA1/11 321 for potentially greater mortality among older women.