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1393
Frequency of BRCA1/BRCA2 Mutations in a
Population-Based Sample of Young Breast Carcinoma
Cases
Kathleen E. Malone, Ph.D.1,2
Janet R. Daling, Ph.D.1,2
Cassandra Neal, B.S.3
Nicola M. Suter, Ph.D.3
Cecilia O’Brien, B.A.1
Kara Cushing-Haugen, M.S.1
Thora J. Jonasdottir, D.V.M.3
Jennifer D. Thompson, B.A.3
Elaine A. Ostrander, Ph.D.3
1
Public Health Sciences Division, Fred Hutchinson
Cancer Research Center, Seattle, Washington.
2
School of Public, Health and Community Medicine, Department of Epidemiology, University of
Washington, Seattle, Washington.
3
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.
Supported in part by grants and contracts R01CA-63705, R01-CA-41416, N01-CP-95671, R01CA-59736, and R21-CA-66186 from the National
Cancer Institute.
The authors thank the study participants and area
physicians for their generous contributions to this
study, Kay Byron for programming assistance,
Julie Bittner for project coordination efforts, and
Neil Wiegand for technical assistance.
This article repesents a multidisciplinary effort. Dr.
Ostrander led the molecular analysis and Dr. Malone led the epidemiologic and statistical component. Both authors contributed equally to this
work.
Address for reprints: Elaine A. Ostrander, Ph.D.,
Clinical Research Division, Fred Hutchinson Cancer
Research Center, 1100 Fairview Ave. N., MP-381,
P.O. Box 19024, Seattle, WA 98109-1024
Received June 28, 1999; revision received December 13, 1999; accepted December 13, 1999.
© 2000 American Cancer Society
BACKGROUND. There is a clear and growing need for data regarding BRCA1 and
BRCA2 mutation frequencies among breast carcinoma cases not specifically ascertained on the basis of extreme family history profiles. Toward this end, the authors
previously reported results with regard to BRCA1 in breast carcinoma patients
drawn from a population-based study. In the current study the authors present
new findings concerning BRCA2 mutation frequency in this same population, as
well as summary data regarding the combined contribution of these two genes.
METHODS. Subjects were drawn from two population-based, case-control studies
of breast carcinoma in young women conducted in western Washington State and
focused on 1) women diagnosed with breast carcinoma before age 35 years (n ⫽
203); and 2) women with a first-degree family history of breast carcinoma who were
diagnosed before age 45 years (n ⫽ 225). Similarities and differences between
BRCA2 carriers and BRCA1 carriers were analyzed in terms of age at diagnosis,
family history status, and disease features.
RESULTS. Of cases diagnosed before age 35 years, all of whom were unselected for
family history, 9.4% carried germline mutations (3.4% for BRCA2 and 5.9% for
BRCA1). Of cases diagnosed before age 45 years who had a first-degree family
history of breast carcinoma, 12.0% carried germline mutations (4.9% for BRCA2
and 7.1% for BRCA1). Increased frequencies of mutations were observed in cases
with a personal or family history of early age at diagnosis and in those with four or
more family members affected with breast carcinoma. BRCA2 mutations were less
common than BRCA1 mutations in families with any history of ovarian carcinoma.
CONCLUSIONS. Overall, given current constraints on health care resources, these
data suggest that screening for germline mutations in these breast carcinoma
susceptibility genes may have the greatest impact on overall health care if it is
prioritized toward high and moderate risk populations. Cancer 2000;88:
1393–1402. © 2000 American Cancer Society.
KEYWORDS: breast carcinoma, BRCA1, BRCA2, population.
R
apid advances in the development and availability of BRCA1 and
BRCA2 genetic testing have generated the need to better understand the contributions of these genes to the overall incidence of
breast carcinoma in the general population. Most insights to date
regarding the frequency of mutations in the breast carcinoma susceptibility genes have been drawn from unusual cancer-rich families
that are not representative of the family history profiles of women in
the general population who develop breast carcinoma.
Chromosomal mapping of the BRCA1 and BRCA2 genes relied
exclusively on the analysis of rare, deliberately selected, “high-risk”
families, each of which had multiple generations with multiple mem-
1394
CANCER March 15, 2000 / Volume 88 / Number 6
bers affected with breast and/or ovarian carcinoma.1-3
Mutation carrier frequency subsequently has been assessed in several distinct collections of families for
both BRCA1 and BRCA2.4-10 These studies provided
important insights regarding the type and frequency
of mutations found in very high risk families and the
likely gene penetrance in the context of a pronounced
family history. However, it is unclear the extent to
which findings in high risk families extrapolate to
breast carcinoma patients who lack such pronounced
high risk profiles but who nevertheless may be at risk
as BRCA1 and/or BRCA2 mutation carriers. Previously
we have shown that of women drawn from a population-based cohort, 6.2% of breast carcinoma cases
diagnosed before age 35 years and 7.2% of breast
carcinoma cases diagnosed before age 45 years who
had a first-degree family history of breast carcinoma
were BRCA1 mutation carriers.11 Similar results have
been reported in a study of African-American and
white women.12 Recently, several studies have assessed the frequency of single founder mutations in
BRCA2 within populations believed to carry only one
or two mutations, namely Ashkenazi Jewish13,14 and
Icelandic breast carcinoma patients.15 However, to
our knowledge, to date no studies have assessed comprehensively the role of BRCA2 mutations in the entire
BRCA2 coding region in breast carcinoma cases from
the general population, nor have studies profiled the
differences between BRCA1 and BRCA2 carriers drawn
from the general population in terms of family history
and features of disease, or documented the combined
contributions of BRCA1 and BRCA2.
The current study addresses these needs by 1)
assessing the type and frequency of BRCA2 germline
mutations in two potentially “at-risk” subgroups of
women with breast carcinoma who were drawn from
the general population; and 2) comparing these findings with our previously reported results of BRCA1
mutation analysis in the same group of women. This
approach allows us to describe how features of proband or family history characterize the comparatively
lower or higher proportions of BRCA1 and BRCA2 carriers among the subsets of women studied. Due to the
population-based study design, the results derived offer a more generalizable approximation of the actual
BRCA1 and/or BRCA2 carrier frequency in these two
potentially “at risk for carrying a mutation” groups, an
assessment that to date has been possible only
through statistical extrapolation.
MATERIALS AND METHODS
The subjects for this study were drawn from two previously completed, population-based, case– control
studies of breast carcinoma conducted in western
Washington State.16,17 Cases (women with breast carcinoma) were identified through the Cancer Surveillance System, a population-based cancer registry, and
controls (similarly aged women without breast carcinoma) were identified through random digit dialing.
The first study ascertained all incident cases of breast
carcinoma diagnosed between January 1, 1983 and
April 30, 1990 in white women born after 1944 who
were residents of King, Pierce, and Snohomish counties at the time of diagnosis. The second study targeted
all incident cases diagnosed before age 45 years between May 1, 1990 and December 31, l992 among
women of all races residing in the 3 county areas.
Interviews were completed on 845 cases (83.6% of
those eligible) and 961 controls (75.5% of those eligible) in the first study and 648 cases (87% of those
eligible) and 610 controls (78.7% of those eligible) in
the second study. Supplemental funding to the first
study allowed for subsequent collection of blood specimens from 592 cases and a subset of control women
(n ⫽ 165). In the second study, blood was collected
from 545 of the cases and 473 of the controls.
Information regarding potential risk factors for
breast carcinoma, including family history, was obtained through a structured interview. A subsequent
follow-up study allowed us to update the pedigree
information that was provided originally. This study
was approved by the Institutional Review Board at the
Fred Hutchinson Cancer Research Center and informed consent was obtained from all subjects prior
to participation.
The molecular analyses described herein focused
on 3 subgroups of women drawn from the 2 case–
control studies: 1) cases with a first-degree family history of breast carcinoma (i.e., an affected mother
and/or sister; 2) cases diagnosed before age 35 years;
and 3) controls with a first-degree family history of
breast carcinoma. These particular groups were targeted because the presence of family history and/or a
young age at diagnosis are believed to be indicative of
an increased probability of carrying a BRCA1/BRCA2
mutation.
Molecular Analyses
Genomic DNA was purified from either frozen buffy
coats or immortalized lymphoblastoid cell lines. Polymerase chain reaction was performed under conditions described previously11 using primers that had
been described previously by others.6,18,19 Novel primers designed to provide redundant coverage of the
gene in some regions also were constructed. All variant bands occurring in ⬍ 5% of samples were sequenced as described previously.11,20
BRCA1/BRCA2 Mutations in Young Women/Malone et al.
Statistical Analyses
The Fisher exact test was used to evaluate among
tested women whether the proportions with mutation
varied by age, disease stage, vital status, and features
of family history and to assess differences in age, disease stage, vital status, and family history between
women who were and were not tested. Binomial exact
95% confidence intervals (95% CI) were calculated for
the proportions with a mutation. P values and 95% CIs
were calculated using Stata statistical software (Stata
Corporation, College Station, TX).
RESULTS
Study Population
BRCA2 mutation status was assessed in 2 groups of
women with breast carcinoma designated as “at-risk”:
225 women diagnosed with breast carcinoma before
age 45 years who also had a family history of a firstdegree relative with breast carcinoma and 203 women
diagnosed before age 35 years. These groups overlapped; altogether, 386 cases were tested. The racial
distribution of the tested women was as follows: 96.6%
were white, 1% were African-American, 1.6% were
Asian/Pacific Islander, 0.3% were American Indian/
Aleutian, and 0.6% were classified as “other”. No other
specific information regarding ethnic background or
the country of origin of the subject’s parents and/or
grandparents was collected. The tested cases included
23 cases not available at the time of our initial BRCA1
analysis in this group.11
In both case groups studied, the proportions
tested among women who were eligible did not vary
substantively by age at diagnosis or family history as
described elsewhere in more detail.11 Tested cases
were more likely to be alive at last follow-up and were
less likely to have had advanced stage disease at diagnosis than those unavailable for testing, largely due to
the lag between the interview and blood collection in
the first case– control study. In addition, 71 controls
age ⬍ 45 years who had a family history of a firstdegree relative with breast carcinoma were assessed
for mutations in both BRCA1 and BRCA2.
Distribution and Frequency of Disease-Associated
Mutations
Among the 225 cases ages 21– 44 years with a family
history of a first-degree relative with breast carcinoma
who were tested, 11 (4.9%; 95% CI 2.5– 8.6) had germline BRCA2 mutations (Table 1) compared with 16
(7.1%; 95% CI 4.1–11.3) who had germline BRCA1 mutations. BRCA2 mutation frequency was highest
among women age ⬍ 30 years and decreased by age at
diagnosis. Similar results were observed for BRCA1.
1395
Four BRCA2 mutations (21.0%) and 2 BRCA1 mutations (10.5%) were observed in the 19 women who had
both a mother and sister with breast carcinoma. Cases
who had a first-degree relative diagnosed with breast
carcinoma before age 45 years had a higher frequency
of mutations for both BRCA2 (6.6%) and BRCA1
(11.8%) than did cases whose only first-degree affected relative(s) had breast carcinoma at or after age
45 years (4.2%, respectively, for both genes). In addition, the mutation frequency for both genes was
higher in cases from families with ⱖ 4 affected members including the proband (10.7%, respectively, for
both genes) versus those with fewer affected relatives
(BRCA2: 3.0% and BRCA1: 5.9%).
Among the 203 women diagnosed with breast carcinoma before age 35 years who were tested, 7 (3.4%;
95% CI 1.4 –7.0) had a BRCA2 mutation (Table 2) compared with 12 (5.9%; 95% CI 3.1–10.1) who had a
BRCA1 mutation. BRCA2 mutations were slightly less
frequent in women ages 30 –34 years (3.2%) versus
women age ⬍ 30 years (4.4%). This is distinct from
BRCA1, in which mutation frequency decreased noticeably with age (11.1% in women age ⬍ 30 years vs.
4.4% in women ages 30 –34 years). Among women
under 35, mutation frequency in both BRCA1 and
BRCA2 was related to family history of breast carcinoma. Of the 96 cases diagnosed with breast carcinoma before age 35 years who had no family history of
breast carcinoma, none carried a BRCA2 mutation and
only 1 carried a BRCA1 mutation. Of the 42 cases
diagnosed with breast carcinoma before age 35 years
who had a mother and/or sister with breast carcinoma, 3 (7.1%) had a BRCA2 mutation. By comparison, 5 women (11.9%) had a BRCA1 mutation. The
mutation frequencies were lower for both genes in
those cases age ⬍ 35 years who only had an aunt
and/or grandmother with breast carcinoma (2.2% for
BRCA2 and 6.7% for BRCA1). Of the 4 cases diagnosed
before age 35 years who had both an affected mother
and sister, 2 had a BRCA2 mutation and 1 carried a
BRCA1 mutation. For both genes, mutation frequency
was higher in cases with at least 1 relative with breast
carcinoma diagnosed before age 45 years versus those
cases with ⱖ 1 relatives with breast carcinoma, none
of whom were diagnosed before age 45 years.
Of the 11 BRCA2 mutations that occurred in
women with a family history of a first-degree relative
with breast carcinoma, none involved women with an
additional family history of ovarian carcinoma (data
not shown). In contrast, BRCA1 mutations were more
common in women with a family history of a firstdegree relative with breast carcinoma who also had a
relative with ovarian carcinoma (20% in those with a
family history of ovarian carcinoma in a first-degree
1396
CANCER March 15, 2000 / Volume 88 / Number 6
TABLE 1
Distribution of BRCA1 and BRCA2 Mutations According to Disease Characteristics and Family History Features among Breast Carcinoma Cases
with a First-Degree Family History of Breast Carcinoma
All
tested
cases
BRCA1 diseaseassociated
mutations
BRCA2 diseaseassociated
mutations
Characteristics
No.
No.
(%)
95% CIa
P valueb
No.
(%)
95% CIa
P valueb
All cases with first-degree family history
225
16
(7.1)
(4.1–11.3)
—c
11
(4.9)
(2.5–8.6)
—c
Age at diagnosis (yrs)
21–29
30–34
35–39
40–44
13
29
87
96
3
2
8
3
(23.1)
(6.9)
(9.2)
(3.1)
(5.0–53.8)
(0.8–22.8)
(4.0–17.3)
(0.6–8.9)
2
1
6
2
(15.4)
(3.4)
(6.9)
(2.1)
(1.9–45.4)
(0.1–17.8)
(2.6–14.4)
(0.2–7.3)
Family history of breast ca
Mother and/or sister had breast ca
Both mother and sister had breast ca
Mother had breast ca
Sister had breast ca
Aunt and/or grandmother also had breast ca
225
19
188
56
97
16
2
13
5
8
(7.1)
(10.5)
(6.9)
(8.9)
(8.2)
(4.1–11.3)
(1.3–33.1)
(3.7–11.5)
(3.0–19.6)
(3.6–15.6)
—c
—c
—c
—c
—c
11
4
11
4
6
(4.9)
(21.0)
(5.8)
(7.1)
(6.2)
(2.5–8.6)
(6.0–45.6)
(3.0–10.2)
(2.0–17.3)
(2.3–13.0)
—c
—c
—c
—c
—c
Mother/sister had breast ca ⬍ age 45 yrs
Mother/sister had breast ca ⱖ age 45 yrs
76
143
9
6
(11.8)
(4.2)
(5.6–21.3)
(1.6–8.9)
0.05
5
6
(6.6)
(4.2)
(2.2–14.7)
(1.6–8.9)
0.52
At least 1 relative had breast ca ⬍ age 45 yrs
Affected relatives all had breast ca ⱖ age 45 yrs
89
132
10
6
(11.2)
(4.5)
(5.5–19.7)
(1.7–9.6)
0.07
5
6
(5.6)
(4.5)
(1.8–12.6)
(1.7–9.6)
0.76
1–3 relatives with breast cad
⬎ 3 relatives with breast cad
169
56
10
6
(5.9)
(10.7)
(2.9–10.6)
(4.0–21.9)
0.24
5
6
(3.0)
(10.7)
(1.0–6.8)
(4.0–21.9)
0.03
0.04
0.10
95% CI: 95% confidence interval; ca: carcinoma.
a
95% confidence interval for the proportion with mutations.
b
P value for differences in mutation frequency between groups.
c
P value not presented because there is no comparison or categories are not mutually exclusive.
d
Proband’s breast carcinoma is included.
relative and 33.3% in those with only a second-degree
relative with ovarian carcinoma; P ⬍ 0.001) than in
those with no family history of ovarian carcinoma
(4.1%). In the 7 cases diagnosed before age 35 years
who had a mother and/or sister with ovarian carcinoma, we observed 1 BRCA2 mutation (14.3%) and no
BRCA1 mutations. One BRCA2 mutation (9.1%) was
detected among the cases diagnosed before age 35
years who had only a second-degree relative with
ovarian carcinoma. In this same group, 3 BRCA1 mutations (27.3%) were found.
To maximize precision, several characteristics
were examined among all 386 cases tested (Table 3).
Family history of bilateral breast carcinoma did not
appear to be an indicator of either BRCA2 or BRCA1
status, and combined BRCA2 and BRCA1 mutation
frequency was identical across family histories that
included bilateral breast carcinoma (9.4%) versus
those known not to include any bilateral breast carcinoma (9.5%). Of the 33 cases with a family history of
both breast and ovarian carcinoma, none carried a
BRCA2 mutation, but 8 (24.2%) were BRCA1 mutation
carriers (data not shown). Of the 7 cases with a family
history of ovarian but not breast carcinoma, there
were no BRCA2 mutations and only 1 BRCA1 mutation
(14.3%). Finally, of the 245 cases with a family history
of breast but not ovarian carcinoma, there were 13
BRCA2 mutation carriers (5.3%) and 12 BRCA1 mutation carriers (4.9%).
Lastly, we examined mutation frequency jointly
according to the number of family members with
breast carcinoma and the presence/absence of ovarian carcinoma in the entire series of 386 cases tested.
The majority of cases (n ⫽ 286) in this study fell into
the category of ⬍ 4 family members affected with
breast carcinoma with no family history of ovarian
carcinoma; 7 (2.4%) carried BRCA2 mutations and 11
(3.8%) had BRCA1 mutations. Of the 48 cases with ⱖ 4
family members affected with breast carcinoma but
no family members with ovarian carcinoma, there was
only 1 BRCA1 mutation (2.1%) observed compared
with 6 BRCA2 mutations (12.5%). Of 28 cases with ⬍ 4
BRCA1/BRCA2 Mutations in Young Women/Malone et al.
1397
TABLE 2
Distribution of BRCA1 and BRCA2 Mutations According to Disease Characteristics and Family History among Breast Carcinoma Cases Diagnosed
before Age 35 Years
All
tested
cases
BRCA1 diseaseassociated
mutations
Characteristics
No.
No.
(%)
95% CIa
P valueb
No.
(%)
95% CIa
P valueb
All cases diagnosed before age 35 yrs
203
12
(5.9)
(3.1–10.1)
—c
7
(3.4)
(1.4–7.0)
—c
Age at diagnosis (yrs)
21–29
30–34
45
158
5
7
(11.1)
(4.4)
(3.7–24.0)
(1.8–8.9)
0.14
2
5
(4.4)
(3.2)
(0.5–15.1)
(1.0–7.2)
0.65
96
42
1
5
(1.0)
(11.9)
(0.0–5.7)
(4.0–25.6)
0
3
—
(7.1)
(0.0–3.8)d
(1.5–19.5)
45
8
12
3
1
2
(6.7)
(12.5)
(16.7)
(1.4–18.3)
(0.3–52.6)
(2.1–48.4)
1
1
2
(2.2)
(12.5)
(16.7)
(0.1–11.8)
(0.3–52.6)
(2.1–48.4)
Both mother and sister had breast ca
Mother had breast ca
Sister had breast ca
4
33
13
1
4
2
(25.0)
(12.1)
(15.4)
(0.6–80.6)
(3.4–28.2)
(1.9–45.4)
—c
—c
—c
2
3
2
(50.0)
(9.1)
(15.4)
(6.8–93.2)
(1.9–24.3)
(1.9–45.4)
—c
—c
—c
Mother/sister had breast ca ⬍ age 45 yrs
Mother/sister had breast ca ⱖ age 45 yrs
20
20
3
2
(15.0)
(10.0)
(3.2–37.9)
(1.2–31.7)
1.00
3
0
(15.0)
—
(3.2–37.9)
(0.0–16.8)d
0.23
at least 1 relative had breast ca ⬍ age 45
yrs
Relative(s) had breast ca ⱖ age 45 yrs
32
48
5
3
(15.6)
(6.2)
(5.3–32.8)
(1.3–17.2)
0.26
3
1
(9.4)
(2.1)
(2.0–25.0)
(0.0–11.1)
0.30
1–3 relatives with breast cae
⬎ 3 relatives with breast cae
177
14
9
1
(5.1)
(7.1)
(2.4–9.4)
(0.2–33.9)
0.54
4
1
(2.3)
(7.1)
(0.6–5.7)
(0.2–33.9)
0.32
Family history of breast ca
None
Mother and/or sister had breast ca
Aunt and/or grandmother only had
breast ca
Other relative
Adopted, family history unknown
BRCA2 diseaseassociated
mutations
0.02
0.01
95% CI: 95% confidence interval; ca: carcinoma.
a
95% confidence interval for the proportion of cases with mutations.
b
P value for differences in the mutation frequency between groups.
c
P value not presented because there is no comparison or categories are not mutually exclusive.
d
One-sided 97.5% confidence interval.
e
Proband’s breast carcinoma is included.
family members with breast carcinoma but at least 1
relative with ovarian carcinoma, no BRCA2 mutations
were found, although BRCA1 mutations were observed
in 4 cases (14.3%). Of the 12 cases from families with ⱖ
4 family members with breast carcinoma plus at least
1 family member with ovarian carcinoma, 5 (41.7%)
had mutations in BRCA1 whereas none were observed
to have a BRCA2 mutation.
There were no women who carried mutations in
both genes. In addition, no disease-associated mutations were observed in the 71 controls with a firstdegree family history who were tested (data not
shown).
Frameshift Mutations
The majority of mutations observed for both genes in
our studies are small insertions or deletions resulting
in premature stop codons (Fig. 1). Nearly all BRCA1
mutations observed in these women, except for the
stop codons at 1728 and 1854, have been noted previously in various studies of high risk families and are
listed in the Breast Cancer Information Core (BIC).
However, in the case of BRCA2, we noted that 4 of 14
frameshifts had not been reported previously in the
BIC. These included small deletions of 1–7 base pairs
that resulted in premature stop codons at 3019, 1739,
and 2845, respectively. We also noted a deletion of a
single amino acid resulting from a 3-base pair deletion
at nucleotide 4081, resulting in the loss of a lysine at
codon 1285. Although we believe too little is known
regarding the functional domains of the protein to
classify this change with absolute certainty, we provisionally have classified it as a disease-associated mutation.
1398
CANCER March 15, 2000 / Volume 88 / Number 6
TABLE 3
Distribution of BRCA1 and BRCA2 Mutations among all 386 Breast Carcinoma Cases Tested
All
tested
cases
BRCA1
diseaseassociated
mutations
BRCA2
diseaseassociated
mutations
BRCA1 and
BRCA2
mutations
combined
Characteristics
No.
No.
(%)
95% CIa
P valueb
No.
(%)
95% CIa
P valueb
No.
(%)
95% CIa
P valueb
All cases tested
386
23
(6.0)
(3.8–8.8)
—c
15
(3.9)
(2.2–6.3)
—c
38
(9.8)
(7.1–13.3)
—c
Family history of breast ca
At least one relative had breast ca ⬍ age
45 yrs
Affected relatives all had breast ca ⱖ age
45 yrs
99
12
(12.1)
(6.4–20.2)
0.03
5
(5.0)
(1.7–11.4)
0.77
17
(17.2)
(10.3–26.1)
0.05
162
7
(4.3)
(1.8–8.7)
7
(4.3)
(1.8–8.7)
14
(8.6)
(4.8–14.1)
314
60
15
6
(4.8)
(10.0)
(2.7–7.8)
(3.8–20.5)
7
6
(2.2)
(10.0)
(0.9–4.5)
(3.8–20.5)
22
12
(7.0)
(20.0)
(4.4–10.4)
(10.8–32.3)
137
64
69
9
3
7
(6.6)
(4.7)
(10.1)
(3.0–12.1)
(1.0–13.1)
(4.2–19.8)
4
3
5
(2.9)
(4.7)
(7.2)
(0.8–7.3)
(1.0–13.1)
(2.4–16.1)
13
6
12
(9.5)
(9.4)
(17.4)
(5.1–15.7)
(3.5–19.3)
(9.3–28.4)
334
14
12
2
(3.6)
(14.3)
(1.9–6.2)
(1.8–42.8)
13
1
(3.9)
(7.1)
(2.1–6.6)
(0.2–33.9)
25
3
(7.5)
(21.4)
(4.9–10.8)
(4.7–50.8
25
3
7
0
(28.0)
—
(12.1–49.4)
(0.0–70.8)e
1
0
(4.0)
—
(0.1–20.4)
(0.0–70.8)e
8
0
(32.0)
—
(14.9–53.5)
(0.0–70.8)e
286
28
48
12
11
4
1
5
(3.8)
(14.3)
(2.1)
(41.7)
(1.9–6.8)
(4.0–32.7)
(0.0–11.1)
(15.2–72.3)
7
0
6
0
(2.4)
—
(12.5)
—
(1.0–5.0)
(0.0–12.3)e 0.02
(4.7–25.2)
(0.0–26.5)e
18
4
7
5
(6.3)
(14.3)
(14.6)
(41.7)
(3.8–9.8)
(4.0–32.7)
(6.1–27.8)
(15.2–72.3)
No. of family members with breast ca
1–3
4⫹
d
Family history of bilateral breast ca
Unilateral only
Bilateral
Bilateral status unknown
Family history of ovarian ca
None
Mother and/or sister had ovarian ca
Aunt and/or grandmother only had
ovarian ca
Other relative only had ovarian ca
Number with breast ca & presence/absence
of ovarian ca in relativesd
⬍ 4 breast ca, no ovarian ca
⬍ 4 breast ca, yes ovarian ca
4⫹ breast ca, no ovarian ca
4⫹ breast ca, yes ovarian ca
0.12
0.47
⬍ 0.001
⬍ 0.001
0.009
0.32
0.65
0.005
0.24
0.001
0.001
95% CI: 95% confidence interval; ca: carcinoma.
a
95% confidence interval for the proportion of cases with mutations.
b
P value for differences in the mutation frequency between groups.
c
P value not presented because there is no comparison or categories are not mutually exclusive.
d
Proband’s breast carcinoma is included.
e
One-sided 97.5% confidence interval.
Missense Changes
Missense changes for both genes are noted in Figure 1.
Although several of these changes were found within
domains predicted to have functional significance
(i.e., the isoleucine to methionine at amino acid 379 in
the p53-binding domain21), only four also are observed in conserved positions in the protein when the
sequence of human, mouse, and either dog or rat is
considered; for BRCA1 these are a methionine to threonine change at amino acid 18, which abuts the transcriptional RING finger, and a glycine to valine change
at amino acid 1788 within the BRCT domain. For
BRCA2 these include an asparagine to glycine change
at amino acid 2811 and the glutamic acid to alanine
change at amino acid 2856, both with the BRCT domains. No unique missense changes were noted in the
control (women without breast carcinoma) samples
for BRCA2.
Polymorphisms
Single stranded confirmation polymorphism (SSCP)
analysis variants that occurred in ⬍ 5% of individuals
were sequenced. For both genes a number of changes
that are believed to be common polymorphisms not
associated with disease predisposition were noted.
These included changes in DNA sequence that did not
change the resulting amino acid or that were within
intron sequences and are unlikely to affect splicing of
BRCA1/BRCA2 Mutations in Young Women/Malone et al.
1399
FIGURE 1. The diagram includes designation of areas of potential functional significance together with all missense, nonsense, and frameshift changes noted
in the cases and controls tested. Findings in controls are illustrated by circles and cases are shown as squares. Deletions of a single repeat within a reiterated sequence
are counted as a deletion of the first repeat unit. (a) BRCA1 (1863AA) functional domains and mutations. (b) BRCA2 (3418) functional domains and mutations.
1400
CANCER March 15, 2000 / Volume 88 / Number 6
exons. In addition, we noted a nonsense change in two
cases and three controls, at base 10204, which generated a stop signal in BRCA2 at codon 3326. This previously has been described22 as being a polymorphic
stop that is not associated with disease.
DISCUSSION
It is clear that a comprehensive understanding of
BRCA1 and BRCA2 carrier frequency will require a
variety of study designs. Estimates of gene carrier frequency from studies of women with pronounced family histories involving multiple generations of affected
relatives vary noticeably, from 20 –50%.5-10,23-27 Such
studies provide critical data relevant to women from
these types of families, but these studies may vary by
family size, degree of relatedness among relatives, and
the overall prevalence of breast carcinoma in the families and thus are not easily manipulated to provide
risk assessment information regarding women with
more modest family histories. Similarly, studies derived from high risk breast carcinoma screening clinics also have been extremely useful, but estimates
derived from such studies often reflect gene frequencies of select groups of women seeking specialized
services rather than frequencies likely to be encountered in the population at large.28,29
In this report, we have expanded our earlier study
of BRCA1 mutations in two subgroups of cases drawn
from a population-based case– control study of early
onset breast carcinoma in western Washington to now
include an analysis of BRCA2 mutation frequency.11 In
addition, we provide a comparative analysis of BRCA2
versus BRCA1 carriers with regard to features of disease and family history.
The results of the current study show that overall,
BRCA2 mutations were less frequent than were BRCA1
mutations. Of women diagnosed with breast carcinoma before age 35 years who were tested, 3.4% were
BRCA2 carriers and 5.9% were BRCA1 carriers. Of cases
diagnosed before age 45 who were selected because of
their family history of one or more affected first-degree relatives, 4.9% were BRCA2 carriers and 7.1%
carried a BRCA1 mutation. For both groups of cases
there were variations in the BRCA1/BRCA2 mutation
frequency that appeared to increase with decreasing
age at diagnosis, the presence of more than three
relatives with breast carcinoma, and, in the case of
BRCA1 mutation frequency, a family history of ovarian
carcinoma.
Among all 386 cases studied overall, 9.8% carried
a mutation in either gene. The mutation frequencies
observed in this investigation are compatible with
those derived from statistical analyses of interview
data collected from a number of population-based
studies.30-32 Such studies suggested that mutations in
autosomal dominant, high penetrance genes could
account for up to 36% of breast carcinomas in women
age ⬍ 30 years30 and approximately 5–11% in women
age ⬍ 40 years.31,32 Mutation frequency is expected to
decrease dramatically in older women, with the proportion of breast carcinoma cases in the general population due to autosomal dominant breast carcinoma
susceptibility genes being 2.2% between ages 40 – 49
years and 1.1% between ages 50 –70 years.33
Some findings in the current study were particular
to one or the other gene. For instance, although the
likelihood of being a BRCA1 versus BRCA2 carrier was
similar for women with no family history of ovarian
carcinoma who had ⬍ 4 family members affected with
breast carcinoma (3.8% and 2.4%, respectively), it was
much higher for BRCA2 in women with ⱖ 4 family
members with breast carcinoma (2.1% vs. 12.5% for
BRCA1 and BRCA2, respectively). In addition, in the
current data set, a family history of ovarian carcinoma
was more predictive of BRCA1 than BRCA2 mutations.
BRCA1 mutations were significantly more common in
women with a family history of ovarian carcinoma
(21.4% or 9 of 42 women with a family history of
ovarian carcinoma) than in women without such a
history (3.6%) (P ⬍ 0.001) (data not shown). BRCA2
mutations were found in only 2 of 42 women with a
family history of ovarian carcinoma (4.8%) versus 13 of
334 women without a family history of ovarian carcinoma (3.8%) (P ⫽ 0.67) (data not shown), suggesting
that, in this data set, ovarian carcinoma is not a strong
predictor of BRCA2 mutations.
In addition, prior investigations have suggested
that male breast carcinoma may be another hallmark
for the presence of BRCA2 mutations.18,23 Five of the
386 cases tested reported a family history of male
breast carcinoma, none of whom had a BRCA1 mutation and 1 of whom (20%) had a BRCA2 mutation.
Although the rarity of male breast carcinoma makes it
a relatively infrequently occurring indicator for consideration of genetic testing, our finding of a BRCA2
mutation within the five cases with a male relative
with breast carcinoma is consistent with prior studies.
Finally, one additional factor that appeared important when considering the likelihood of being a
BRCA1 versus BRCA2 carrier was the age of onset in
relatives. In cases with at least 1 affected family member diagnosed before age 45 years, we noted that
12.1% and 5% of cases were BRCA1 and BRCA2 mutation carriers, respectively. But in cases whose affected
relatives were all diagnosed after age 45 years, BRCA1
and BRCA2 mutations occurred with similar frequency
(4.3% each). It is interesting to note that in the single
largest investigation of high risk breast carcinoma (or
BRCA1/BRCA2 Mutations in Young Women/Malone et al.
breast-ovarian carcinoma) families published to date,
a study of 237 breast carcinoma families originally
identified for mapping purposes, Ford et al.33 showed
that the overall lifetime risk of breast carcinoma in
BRCA1 and BRCA2 carriers was similar. However,
there was some suggestion of lower risk in BRCA2
carriers age ⬍ 50 years.33 Similar findings were reported by Oddoux et al.13 and Schubert et al.26 Therefore, the results presented in the current study simply
may represent the delayed penetrance for BRCA2 carriers over BRCA1 in younger women. However, because we have not specifically studied at women age ⬎
45 years, we cannot address this question directly.
Ford et al. also have shown that in the same cohort breast carcinoma was linked to BRCA1 in an
estimated 52% of families, to BRCA2 in 32% of families, and to neither gene in 16% of families.33 In the
data presented in the current study we observed that
the ratio of BRCA1 to BRCA2 carriers among the
women tested was 1.5 to 1, suggesting that in the
general population, as in high risk families, BRCA2
mutations occur approximately half as frequently as
BRCA1 mutations.
The above conclusions must be considered in
light of potential limitations in our study design and
the sensitivity of our mutational screening strategy. A
study such as the current one, of women drawn from
the general population, offers the advantage of a
broad generalizability of results for the many women
who have modest to minimal family histories of cancer. However, the generalizability of these findings
also may be influenced by the extent to which women
whose samples were available for testing may not be
representative of all eligible women in the study.11 If
there was an association between mutation status and
survival, with mutations in BRCA1 or BRCA2 leading to
either increased or reduced survival, our results could
overestimate or underestimate the true frequency of
mutations. No mutations were found among the controls with a family history of a first-degree relative with
breast carcinoma. This is not unexpected, given the
estimates of mutation frequency in the general population, the minimal family history that many controls
had, and the relatively small number of controls with
a family history of a first-degree relative with breast
carcinoma that were available for analysis.
In addition there are technical considerations.
When performed correctly, SSCP and techniques like
it can detect ⬎ 80% of mutations.34,35 Indeed, in an
analysis of 237 families, each with at least 4 cases of
breast carcinoma, Ford et al.33 assessed and compared
the sensitivity of completely sequencing the coding
region of the BRCA1 gene with the sensitivity of other
gel-based genomic screening techniques and found
1401
them to be the same. Nevertheless, some sequence
changes will be missed by gel-based techniques such
as SSCP, the majority of which are single base pair
changes that lead to missense and nonsense changes.
Considerable care was taken in the current study to
optimize primers and gel conditions so that even subtle variants were detected routinely, but in the absence of complete gene sequencing for every woman it
must be assumed that some mutations are missed. An
additional factor that limits the generalizability of
these findings is the fact that mutations affecting the
expression, splicing, or stability of the transcript will
not be detected by the majority of screening methods
currently in use. Finally, in the absence of a definitive
functional assay, the significance of most missense
changes remains unclear. Although we estimate that
as many as four BRCA1 or BRCA2 changes observed in
the current study might have been disease-associated
based solely on their position in conserved domains of
potential functional significance, this cannot be stated
with certainty in the absence of a clear functional
assay, and these changes remain as unclassified variants. Therefore, little information can be provided to
patients regarding the significance of single amino
acid changes in disease predisposition.
Overall the current study data suggest that, even
in two groups of women selected for their perceived
increased risk of carrying germline mutations, the majority do not carry such mutations. It appears likely,
given current constraints on health care resources,
that BRCA1 and BRCA2 screening will have its strongest impact when targeted to specific, at-risk populations.
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