Affiliations of authors: N. L. S. Tang, C.-P. Pang, K.-W. Choy, L. K. Law, M. Hjelm (Department of Chemical Pathology), W. Yeo, P. Johnson (Department of Clinical Oncology), P. K. Lam, W. W. K. King (Department of Surgery), M. Suen (Department of Anatomical and Cellular Pathology), Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China.
Correspondence to:Nelson L. S. Tang, F.R.C.P.A., Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Special Administrative Region, China (e-mail: nelsontang{at}cuhk.edu.hk).
Inherited mutations in the tumor susceptibility genes BRCA1 and BRCA2 account for about 70%-90% of familial breast cancer in most Caucasian populations while BRCA1 mutations alone contribute to about half of the cases (1-3). Epidemiologic data from Caucasians show that BRCA1 mutations occur in the general population at a frequency between 0.05% and 0.2%. BRCA1 mutations account for about 5% of all breast cancers and up to 12% of early-onset breast cancers (3,4). The relative contribution of BRCA2 may be slightly lower (2,3).
The BRCA1 tumor suppressor gene is composed of 22 coding and two noncoding exons (5,6). The BRCA1 protein may play a role in cell cycle regulation (7) and control of DNA repair (8,9). More than 300 heritable mutations in the BRCA1 gene have been found spread throughout the whole coding sequence (10). All known BRCA1 mutations are germline and more than 85% are nonsense or frameshift mutations leading to premature termination of protein translation (2). Ancient and founder BRCA1 mutations have been identified, such as 185delAG mutation in exon 2, which is found in almost 1% of the Ashkenazi Jewish population (11,12).
Studies of the BRCA1 gene in Oriental populations are few. There is no information on the frequency of BRCA1 mutation in the Chinese population. However, a lower prevalence of BRCA1 mutations has been suggested in the Japanese population (13-15). Although the incidence of breast cancer among Chinese women is about half of that seen in Caucasians, the proportion of familial cases and incidence of early-onset cancer are similar (16,17). Here, we report our investigation of mutations in BRCA1 among unselected consecutive samples of archived tumor tissues to determine an unbiased estimation of the prevalence of BRCA1 mutations among Chinese patients with breast cancer.
Frozen tumor tissues were obtained for BRCA1 mutation analysis from 130 female Chinese patients, who underwent mastectomy for carcinoma of breast at the Prince of Wales Hospital in Hong Kong during the period of 1992 through 1993. As informed consent was not available for these stored samples, mutation analysis was performed in an anonymous manner. The patient identifier codes of these specimens had been removed before mutation analysis was performed. This procedure conforms to the consensus statement on use of stored samples in genetic research (18). Estrogen receptor and progesterone receptor statuses were determined by immunohistochemistry employing commercially available antibodies (DAKO, Glostrup, Denmark).
In addition, truncating and missense mutations identified from this series of 130 tissue samples were also screened for in another group of 56 patients with early-onset breast cancers who were diagnosed with cancer before 45 years of age. This prospective group of patients had consented to participate in an ongoing BRCA1/BRCA2 genetics study. The study protocol was approved by a local Institutional Research Ethics Committee.
The entire BRCA1 coding region was covered by 35 amplicons amplified by polymerase chain reaction (PCR) from genomic DNA (19). Mutations were screened by single-strand conformational polymorphism (SSCP) analysis under three different electrophoresis conditions. PCR products showing mobility shift on SSCP were sequenced by cycle sequencing in both sense and antisense directions (Life Technologies, Inc., Gaithersburg, MD). The chi-squared test was used to compare frequency of alleles. Fisher's exact test was used to compare clinical parameters between patients with and without BRCA1 mutation. Exact 95% confidence intervals (CIs) of prevalence data were determined by StatXact software (CYTEL, Cambridge, MA).
Three nonsense (589delCT, Q356X, and Q1458X) and one presumptive disease-related
(P1150S) BRCA1 mutations were found in five of 130 patients, which represented a prevalence
of 3.8% (95% confidence interval [CI] =
1.3%-8.8%). Three nonsense mutations were identified in four patients (Table 1). Mutation 589delCT was found in two patients aged 24 years and 34
years, respectively, with infiltrating ductal carcinoma (IDC). The other two mutations were
novel.
The Q1458X mutation was found in a 44-year-old patient having a medullary carcinoma. Q356X
was found in an 80-year-old patient with IDC. A missense mutation P1150S was detected in a
31-year-old woman with IDC. Fifty of 130 patients had breast cancer before the age of 45 years,
and the prevalence of BRCA1 mutation among this subgroup of early-onset patients was
8.0% (95% CI = 2.2%-19.2%). Additional screening
specifically for these four mutations among another group of 56 patients with early-onset breast
cancer further identified one more patient who was a heterozygote for 589delCT. The hometowns
of these three unrelated patients sharing 589delCT mutation were different cities within the
Guangdong Province of Southern China, but, as far as we know, the patients were not related.
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Six novel and 10 previously reported sequence variants were found (Table 1). Twelve variants show no change to the coding sequence (233G
A, 667
- 3delT, 1511C
T, 2201C
T, 2430T
C, 2816C
G, 4427T
C,
5106 - 68G
A, 5272 + 66G
A, 5429G
A, 5526 + 32A
T,
and 5711 + 31G
A) and are considered
as nondeleterious variants. Three missense mutations (2731C
T, 3232A
G, and
4956A
G) are polymorphisms reported previously. Eight heterozygote patients (6.2%
of 130 patients) had a novel 2685T
C allele, which predicts a tyrosine to histidine
substitution in codon 856 (Table 1
). Further investigation using Bpu1102I restriction
analysis as the variant created a new Bpu1102I site and found a similar prevalence
(4.5%) among 110 cancer-free women aged greater than 65 years. No difference in the
allele frequency was found between the patient with breast cancer and the control subject.
Four BRCA1 mutations are now known to recur in Oriental populations. Mutation 589delCT
was found in three unrelated patients in this study. This may indicate that it is a prevalent
mutation in Southern Chinese women but further study with a large sample size is required to
confirm this observation. Analysis of intragenic polymorphism of these cases showed that they
shared the rare alleles of a few common polymorphisms, including 2430TC,
2731C
T, 4427T
C,
4956A
G, and 5106 - 68G
A. These features may suggest a shared haplotype
for 589delCT; however, it is not definitive because we could not analyze the haplotype of other
family members. Other prevalent mutations included the following: P1150S (15), 2509delAA (14) at codon 797, and L63X in exon 5 (20). None of the known BRCA1 mutations that occur with a high
frequency in other populations were found in the Chinese patients. Our data do not establish that
any particular mutation in BRCA1 occurs at sufficiently high frequency in Chinese women to
qualify as a founder mutation, although 589delCT is a potential candidate. All BRCA1 mutations
identified previously existed in the germline, and no somatic BRCA1 mutation among patients
with breast cancer has been described to date (10). Although we only
studied DNA extracted from breast tissue and did not analyze germline DNA in the retrospective
series of 130 samples, mutations reported here were presumably germline. However, we cannot
rule out the possibility of somatic mutations in at least some of the cases.
The most prominent clinical feature among the five patients with BRCA1 mutations was that
four of the five had disease onset before 45 years of age (Table 2). The
majority of BRCA1 mutation-negative cancers were found in patients above 45 years of age.
Two BRCA1 mutation-positive patients had a family history of breast cancer, in which two
first-degree relatives of one patient were affected. There was only one affected family member in
other cases. The most common tumor was IDC among these patients. Medullary carcinoma is a
rare tumor but
recent studies also showed that it occurred in a higher proportion among BRCA1 mutation
carriers than in other patients with breast cancer (21,22). Only one case of
medullary cancer in this series was observed, which was in a heterozygote for Q1458X. It
appeared that fewer tumors with BRCA1 mutations expressed estrogen and progesterone
receptors, which was consistent with findings in larger series published earlier (23,24). However, the small number of cases with BRCA1 mutations precluded us from
making any statistical inference.
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Manuscript received August 25, 1998; revised January 19, 1999; accepted March 17, 1999.
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