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Keywords:

  • Bahamas;
  • BRCA1;
  • breast cancer;
  • hereditary

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We sought to identify the full range of founder mutations in BRCA1 and BRCA2 in the Bahamas and to estimate the proportion of all BRCA1 and BRCA2 mutations that are accounted for by founder mutations. We studied 214 Bahamian women with invasive breast cancer, unselected for age or family history. A founder mutation had previously been identified in 49 patients. We conducted full sequencing of the BRCA1 and BRCA2 genes and multiplex ligation-dependent probe amplification (MLPA) for 156 patients. A novel founder mutation in BRCA2 (exon 17 818delA) was seen in four different patients and five other unique mutations in BRCA1 and BRCA2, including a large deletion (exons 8–9) in BRCA1. In total, a mutation was seen in 58 of the 214 patients (27%); 92% of carriers carried one of the seven founder mutations. Approximately 27% of unselected cases of breast cancer in the Bahamian population are attributable to a mutation in BRCA1 or BRCA2, a prevalence which far exceeds that of any other country. The majority of women who carry a mutation in the Bahamas, carry one of the seven founder mutations, making it possible to offer genetic testing to all women at risk for breast cancer in the Bahamas.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Bahamas is a Carribean island country with a population of approximately 300,000. There are approximately 100 new cases of breast cancer per year in the Bahamas. The average age of onset of breast cancer in the Bahamas is 42 years and one half are diagnosed in pre-menopausal women. In 2011, we reported six founder mutations in BRCA1, one of which was present in 49 of 214 unselected breast cancer patients (23%) [1]. The mutation prevalence was particularly high (45%) for women diagnosed with breast cancer before age 40. In the context of developing a national cancer genetics program, we asked if there were founder mutations beyond these six (particularly in BRCA2) and we sought to estimate what proportion of all BRCA mutations in the Bahamian population are accounted for by founder mutations. To do so, we performed full gene sequencing of both BRCA1 and BRCA2 and multiplex ligation-dependent probe amplification (MLPA) analysis on 156 unselected breast cancer patients for whom a BRCA mutation had not been found.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Breast cancer patients were recruited from public and private clinics in Grand Bahama, Nassau, Eleuthera, Harbor Island, Abaco and Spanish Wells between September 2008 and January 2010. Women were eligible if they had been diagnosed with invasive primary breast cancer at any age, in any year and if at least one parent was born in the Bahamas [1]. Accrual centers were set up at the Princess Margaret Hospital Oncology Centre, at the Cancer Society in Nassau, at the Cancer Association in Freeport, at Cancer Societies in Eleuthera and Abaco, at the Government Clinic of St George's Cay and at a private home in Harbor Island. In addition, breast cancer patients who were born in the Bahamas were eligible for the study in Miami, at the University of Miami, Jackson Memorial Hospital and the Mount Sinai Cancer Center. All women provided written consent. The study protocol was approved by the ethics review board of the University of Miami, Jackson Memorial Hospital, Mount Sinai Cancer Center, Princess Margaret Hospital, Doctors Hospital and the Bahamian Ministry of Health. In total, 214 women from 204 different families agreed to participate. A saliva sample was taken from each woman and a medical and family history was taken. The family history asked about cancer diagnoses in first- and second-degree relatives. In the event that additional cases in more distant relatives were reported these were recorded on the pedigree. Of 165 women who previously tested negative for mutations in our first study, 156 underwent full testing.

Mutation analysis

Saliva was collected using the Oragene® DNA sample collection kit, (OG-250 format, DNA Genotek, Kanata, ON, Canada) and extracted following the manufacturer's instructions. DNA was then quantified using the NanoDrop ND-1000 Spectrophotometer (ThermoScientific Inc., Wilmington, DE).

Sequencing

All 22 coding exons (exons 2–24) of BRCA1 and 26 coding exons (exons 2–27) of BRCA2 were amplified in 34 and 47 amplicons respectively. The primers have been designed to cover all coding exons and their adjacent consensus splicing sites. The amplified DNA fragments were sequenced using the BigDye Terminator Cycle Sequencing kit on an ABI 3500xl DNA Analyzer (Applied Biosystems Co., Foster City, CA). Generated sequencing chromatograms by the sequencer instrument were analyzed for variant detection using Mutation Surveyor software (SoftGenetics LLC, State College, PA). All sequences were compared to the BRCA1 (U14680.1) and BRCA2 (U43746.1) reference sequences for variant detection.

MLPA

We screened BRCA1 and BRCA2 genes for large insertions/deletions using MLPA method. The BRCA1 (Cat#: P002) and BRCA2 (Cat#: P090) MLPA kits (MRC-Holland Inc., Amsterdam, the Netherlands) contain 35- and 43 pairs of probes respectively that bind to different exons of the genes. Probes were hybridized to the genomic DNA and then adjacent probe pairs were ligated and amplified. The fragment analysis of the amplified probes was implemented on an ABI 3500XL DNA analyzer. Generated fragment analysis data were analyzed for detecting large insertions/deletions using GeneMarker software (SoftGenetics LLC). The identified insertions/deletions were confirmed by a second MLPA test using different probe sets (P087 for BRCA1 and P077 for BRCA2) from the initial probe sets used for screening.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We completed full genetic sequencing for 156 women with breast cancer for whom a founder mutation in BRCA1 had not been found previously. Among these 156 women, a mutation was identified in 9. Four women carried the same nucleotide deletion in BRCA2 (8128delA) and there were four unique mutations in BRCA1 and BRCA2 (1127insGT, 3477delGT, 1538elAAGA and 2190delA). In addition, we found a large deletion encompassing exons 8 and 9 of BRCA1 in one patient through MLPA analysis.

There were 214 patients studied from 204 different families. The patients represent prevalent cases of cancer and were not selected on the basis of family history, and five families were represented by more than one case. In total, including the 49 mutations previously identified, we have now extended the number of mutations in the panel of 214 women to 58, which represents 27% of all the cases (Table 1). There was one family with five cases included in the study, one family with four cases and three families with two cases each. The other 199 families included one case each. If we restrict the testing to the 204 unrelated women (one per family) there were 49 mutations detected among 204 patients (24.0%).

Table 1. Distribution of 58 mutations found in 214 breast cancer patients
GeneExonMutationNumber of patientsPercentage of patients with mutationNumber of families with mutationPreviously reported
BRCA1 IVS13+1G>A305224Yes
BRCA1154730insG6103Yes
BRCA121T5443G595Yes
BRCA1 IVS16+6T>C353Yes
BRCA12185delAG232Yes
BRCA111943ins10353Yes
BRCA1113477delGT121Yes
BRCA1112190delA121Yes
BRCA18 and 9deletion121Yes
BRCA2178128delA474No
BRCA2101127insGT121No
BRCA2101538delAAGA121Yes

The mutation prevalence was 52% in 63 women diagnosed at age 39 and under, was 26% in 74 women diagnosed from age 40 to 49 and was 10% in 77 women diagnosed at age 50 and above (Table 2). The mean age of diagnosis of the women with a mutation was 38.2 years (range 22–70 years) and the mean age of diagnosis of the women without a mutation was 47.6 years (range 25–80 years). A mutation was present in 33% of 142 patients with a family history of breast and ovarian cancer and in 15% of 72 patients without a family history (Table 3).

Table 2. Prevalence of mutations by age of diagnosis
Age groupNumber of subjectsNumber of mutationsMutation prevalence (%)
AllBRCA1BRCA2
20–291398169.2
30–39502423148.0
40–49741916325.7
50–59595418.4
60+181105.6
All ages2145852627
Table 3. Mutation prevalence, by family history of cancer
Family historyNumber of probandsNumber of mutationsPercentage with mutation
Breast cancer only1083734
Ovarian cancer only500
Breast and ovarian cancer20945
Neither cancer721115
Unknown9111
Total2145827

Of the 58 mutations, 53 were founder mutations (91%). A single mutation (BRCA1 IVS13+1G>A) represented 52% of all the mutations. Five other mutations were seen in three or more women (BRCA1: IVS13+1G>A, 943ins10, IVS16+6T>C, 5443T>G, 4730insG and BRCA2: 8128delA). There were five patients with a unique (non-founder) mutation (BRCA1: 3477delGT, 2190delA, del exons 8–9 and BRCA2: 1538elAAGA, 1127insGT). Of these, three had a strong family history of breast cancer.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the Bahamas, we estimate that from 24% to 27% of all breast cancer patients carry a mutation in BRCA1 or BRCA2. We have previously described six recurrent BRCA1 mutations in breast cancer patients from the Bahamas (IVS13+1G>A, 943ins10, IVS16+6T>C, 5443T>G, 4730insG, 185delAG) [1]. The majority of these (IVS13+1G>A, 943ins10, IVS16+6T>C and 5443T>G) are African founder mutations. [2] We used DNA sequencing and MLPA to show the full spectrum of BRCA mutations in the Bahamas. Six additional mutations were identified (8128delA, 1127insGT, 1538delAAGA, 3477delGT, 2190delA and a large deletion of exons 8 and 9) and are reported here. Three mutations were found in the BRCA2 gene (8128delA, 1127insGT, 1538delAAGA). A novel BRCA2 mutation (8128delA) was seen in four patients and thus is the seventh recurrent mutation increasing the prevalence of founder mutations in the Bahamian population to 25% of unselected breast cancer patients. The two additional BRCA2 mutations were seen in one study participant each. BRCA2 mutation1127insGT is a novel mutation, whereas 1538delAAGA has been reported in three patients in Nigeria [3] and one patient in Barbados and both Latin American and Western European patients [BIC (Breast Cancer Information Core) database]. Three additional mutations were found in BRCA1 (3477delGT, 2190delA and a large deletion of exons 8 and 9). The large deletion of exons 8 and 9 has been reported previously in one African-American patient [4]. The 3477delGT BRCA1 mutation has been reported twice in families from Central and Western Europe and the 2190delA BRCA1 mutation was seen in several families from Western Europe. There were 87 women with a positive family history of breast or ovarian cancer who did not carry one of the seven founder mutations. Of these, a unique mutation was found in three women (3.4%). In the event of a positive family history and a negative founder result, additional testing may be warranted.

The prevalence of mutations among breast cancer cases in the Bahamas is higher than that observed in any other country. We have previously estimated the mutation frequency among unselected breast cancer patients to be 12% in Ashkenazi Jews, 6% in French-Canadians and 6% in Poles [5-8]. Recently, a study in Nigeria yielded 11% mutation rate in 434 unselected breast cancer patients, although this included a diverse group of mutations [3]. In Canada and the United States, approximately 3% of all breast cancer cases are due to a mutation in one of two cancer susceptibility genes, BRCA1 and BRCA2 [9]. The unique heritage of Bahamian women makes this small country an ideal location to conduct studies of genetic testing and personalized medicine.

On the basis of studies of other populations, we estimate that between 2% and 4% of all Bahamian women will carry a founder mutation in BRCA1 or BRCA2 (for example, mutations are present in 12% of unselected Ashkenazi Jewish breast cancer cases and in 1.5% of the unselected Jewish women) [6, 10]. It is important that the mutation prevalence be measured prior to offering genetic testing outside of a personal or family history of breast cancer. Two population studies of unselected individuals from high founder rate ethnic groups have shown success with respect to interest in testing and uptake of cancer risk reduction strategies [10-12].

All women with a history of breast cancer were eligible for the study, but the patients who enrolled may have done so in response to a family history of breast cancer and may not be representative of all cases in the country. Of note, 128 of the 214 patients had a family history of breast cancer (60%). The 27% estimate is based on 58 mutations identified in 214 different patients, but five families contributed more than one case. If we consider only unrelated individuals (one case per family) the mutation frequency was 24%

The genetic composition of the Bahamas may be distinct from that of neighboring populations because of historical patterns of migration and relative reproductive isolation. These factors may be particularly relevant for other island nations with a small number of founders. Our group is currently investigating whether other islands in the Caribbean have these founder mutations in common or those unique to their island.

In conclusion, we identified one of seven founder mutations in BRCA1 and BRCA2 in 27% of breast cancer patients in the Bahamas. It is possible that mutations in other genes are present in one or more of the 156 women in our study without a mutation in BRCA1 or BRCA2. If so, then the hereditary proportion of Bahamian breast cancer patients might be even higher.

On the basis of our results, a seven mutation testing panel is predicted to be 91% sensitive for BRCA mutations and should be offered to all breast and ovarian cancer patients in the Bahamas. Patients who have a strong family history of breast and ovarian cancer, who test negative for the founder mutations in this panel should be offered more extensive testing.

We have now initiated a program in the Bahamas which offers genetic testing to all women with breast cancer. Those who test positive will be offered options of increased surveillance and preventive surgery. It is also reasonable to offer predictive testing to unaffected patients with a family history of breast or ovarian cancer if there is no affected family member available for testing. Finally, given the public health consequences of the high founder mutation rate reported here, consideration should be given to population-wide genetic screening and development of a national breast cancer prevention program in the Bahamas. It is important to estimate the prevalence of these mutations among Bahamian women at large and to estimate the penetrance of these mutations in the population.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by Komen for the Cure, the Ride for Hope and the Bahamas Breast Cancer Initiative.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References