GERMLINE MUTATIONS OF BRCA1 AND BRCA2 GENES
– FOUNDER EFFECTS AND CONTRIBUTION TO
OVARIAN CARCINOMA IN FINLAND
Laura Sarantaus
Department of Obstetrics and Gynaecology
Helsinki University Central Hospital
University of Helsinki
Helsinki, Finland
Academic Dissertation
To be publicly discussed, with the permission of the Faculty of Medicine of
the University of Helsinki, in Auditorium 2 of Biomedicum Helsinki,
Haartmaninkatu 8, Helsinki, on December 14, 2002, at 12 noon.
Helsinki 2002
SUPERVISED BY
Docent Heli Nevanlinna, PhD
Department of Obstetrics and Gynaecology
Helsinki University Central Hospital
University of Helsinki
REVIEWED BY
Professor Päivi Peltomäki, MD, PhD
Department of Medical Genetics
University of Helsinki
Docent Ulla Puistola, MD, PhD
Department of Obstetrics and Gynaecology
Oulu University Hospital
University of Oulu
OFFICIAL OPPONENT
Docent Johanna Schleutker, PhD
Institute of Medical Technology
Tampere University Hospital
University of Tampere
ISBN 952-91-5312-0 (Print)
ISBN 952-10-0785-0 (PDF)
http://ethesis.helsinki.fi
Helsinki 2002
Multiprint Oy
3
TABLE OF CONTENTS
LIST OF ORIGINAL PUBLICATIONS..................................................................................6
ABBREVIATIONS ..................................................................................................................7
ABSTRACT .............................................................................................................................9
INTRODUCTION ..................................................................................................................11
REVIEW OF THE LITERATURE ........................................................................................13
1 General features of ovarian carcinoma ................................................................................13
2 General features of breast carcinoma...................................................................................14
3 Genes involved in carcinogenesis........................................................................................14
4 Inherited predisposition to cancer........................................................................................15
5 Inherited predisposition to breast and ovarian carcinoma ...................................................16
6 BRCA1 and BRCA2 genes ...................................................................................................18
6.1 Structure and expression .........................................................................................18
6.2 BRCA1 and BRCA2 gene-encoded protein products and their proposed
functions.................................................................................................................18
6.3 Inherited germline mutations ..................................................................................20
6.3.1 Spectrum .......................................................................................................20
6.3.2 Ethnic differences in mutation spectra ..........................................................22
6.3.3 Prevalence .....................................................................................................23
6.4 Risk of cancer in BRCA1 and BRCA2 mutation carriers.........................................29
6.5 Prediction of presence of a BRCA1/BRCA2 germline mutation in a family ...........31
6.6 Characteristics of BRCA1 and BRCA2 mutation-associated breast and ovarian
carcinomas .............................................................................................................32
6.6.1 Breast carcinomas .........................................................................................32
6.6.2 Ovarian carcinomas.......................................................................................33
6.7 Prognosis of patients with BRCA1 or BRCA2 mutation-associated breast or
ovarian carcinoma ..................................................................................................34
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7 Population history of Finland and its influence on the Finnish gene pool ..........................34
7.1 Settlement in Finland ..............................................................................................34
7.2 Finnish disease heritage ..........................................................................................35
7.3 Inherited diseases other than those included in the Finnish disease heritage..........36
7.4 Linkage disequilibrium and haplotypes ..................................................................36
AIMS OF THE STUDY.........................................................................................................39
MATERIALS AND METHODS ...........................................................................................40
1 Ethical issues .......................................................................................................................40
2 Patients and families (I-IV) .................................................................................................40
3 Previously identified BRCA1 and BRCA2 germline mutations studied here (I-IV) ............42
4 Collection of cancer and genealogical data (I-IV)...............................................................42
5 Extraction of DNA (I-IV) ....................................................................................................44
6 Genotyping (I, II) ................................................................................................................44
7 Haplotype construction (I, II) ..............................................................................................46
8 Detection of BRCA1 and BRCA2 germline mutations (III, IV) ...........................................47
8.1 Screening for previously identified mutations (III, IV)...........................................47
8.1.1 Allele-specific oligonucleotide (ASO) hybridization (III, IV)......................47
8.1.2 Restriction fragment length polymorphism (RFLP) analysis (III, IV) ..........48
8.1.3 Agarose gel electrophoresis (III)...................................................................48
8.2 Scanning for novel mutations (III)..........................................................................48
8.2.1 Protein truncation test (PTT) (III).................................................................48
8.2.2 Southern blot hybridization (III) ...................................................................49
8.3 Direct sequencing (III, IV) ......................................................................................50
9 Statistical methods (I-IV) ....................................................................................................50
9.1 General (I, III, IV) ...................................................................................................50
9.2 Luria-Delbrück equation (I, II)................................................................................50
9.3 Logistic regression (III)...........................................................................................51
RESULTS...............................................................................................................................52
1 Studies on recurrent BRCA1 and BRCA2 mutations (I, II) ..................................................52
1.1 Mutation-associated haplotypes ..............................................................................52
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1.2 Estimated number of generations from a common ancestor for families sharing
a conserved core haplotype ....................................................................................57
1.3 Geographical origins of the families .......................................................................58
2 Breast and ovarian carcinoma phenotypes of BRCA1 and BRCA2 mutation carriers (I) ....60
3 BRCA1 and BRCA2 germline mutations in unselected Finnish ovarian carcinoma
patients (III)........................................................................................................................62
3.1 Mutations detected ..................................................................................................62
3.2 Personal and family history of breast and ovarian carcinoma of the mutation
carriers....................................................................................................................63
3.3 Relationship between mutation carrier status and personal and family history
of breast and ovarian carcinoma ............................................................................64
4 BRCA1 and BRCA2 germline mutations in Finnish ovarian carcinoma families (IV) ........65
4.1 Mutations detected ..................................................................................................65
4.2 Characteristics of mutation-positive and -negative ovarian carcinoma families ....66
DISCUSSION.........................................................................................................................67
1 Studies on recurrent BRCA1 and BRCA2 mutations (I, II) ..................................................67
2 Contribution of BRCA1 and BRCA2 germline mutations to ovarian carcinoma in
Finland (III, IV), and breast and ovarian carcinoma phenotypes of Finnish BRCA1
and BRCA2 mutation carriers (I, III, IV)............................................................................73
SUMMARY AND CONCLUSIONS.....................................................................................79
ACKNOWLEDGEMENTS....................................................................................................81
REFERENCES .......................................................................................................................83
ORIGINAL PUBLICATIONS..............................................................................................107
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LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications, which are referred to in the text by
their Roman numerals.
I Sarantaus L*, Huusko P*, Eerola H, Launonen V, Vehmanen P, Rapakko K, Gillanders
E, Syrjäkoski K, Kainu T, Vahteristo P, Krahe R, Pääkkönen K, Hartikainen J,
Blomqvist C, Löppönen T, Holli K, Ryynänen M, Bützow R, Borg Å, Wasteson Arver
B, Holmberg E, Mannermaa A, Kere J, Kallioniemi O-P, Winqvist R*, and Nevanlinna
H*: Multiple founder effects and geographical clustering of BRCA1 and BRCA2
families in Finland. European Journal of Human Genetics 8: 757-763, 2000.
II Barkardottir RB, Sarantaus L, Arason A, Vehmanen P, Bendahl P-O, Kainu T,
Syrjäkoski K, Krahe R, Huusko P, Pyrhönen S, Holli K, Kallioniemi O-P, Egilsson V,
Kere J, and Nevanlinna H: Haplotype analysis in Icelandic and Finnish BRCA2 999del5
breast cancer families. European Journal of Human Genetics 9: 773-779, 2001.
III Sarantaus L, Vahteristo P, Bloom E, Tamminen A, Unkila-Kallio L, Butzow R, and
Nevanlinna H: BRCA1 and BRCA2 mutations among 233 unselected Finnish ovarian
carcinoma patients. European Journal of Human Genetics 9: 424-430, 2001.
IV Sarantaus L, Auranen A, and Nevanlinna H: BRCA1 and BRCA2 mutations among
Finnish ovarian carcinoma families. International Journal of Oncology 18: 831-835,
2001.
*Equal contribution
Publication I is also included in the thesis of Pia Huusko (Predisposing genes in hereditary
breast and ovarian cancer, Acta Universitatis Ouluensis D Medica 541, University of Oulu,
Oulu, 1999).
The original publications have been reproduced with the permission of the copyright holders.
7
ABBREVIATIONS
A adenine
ABCSG Anglian Breast Cancer Study Group
AKT2 murine thymoma viral (v-akt) oncogene homologue 2 gene
ASO allele-specific oligonucleotide
BCLC Breast Cancer Linkage Consortium
BIC Breast Cancer Information Core
BRCA1 breast cancer 1 gene
BRCA2 breast cancer 2 gene
C cytosine
cDNA complementary deoxyribonucleic acid
CGH comparative genomic hybridization
cM centiMorgan
dCTP deoxycytidine triphosphate
del deletion
DNA deoxyribonucleic acid
ERBB2 avian erythroblastic leukaemia viral (v-erb-b2) oncogene homologue 2 gene
ESR oestrogen receptor
FCR Finnish Cancer Registry
FIGO Fédération Internationale de Gynécologie et d’Obstetrique
G guanine
g number of generations
GDB Genome Database
HBOC hereditary breast-ovarian cancer
HNPCC hereditary non-polyposis colorectal cancer
ins insertion
kb kilobase
kDa kiloDalton
KRAS Kirsten rat sarcoma 2 viral (v-Ki-ras2) oncogene homologue gene
LD linkage disequilibrium
Mb megabase
MLH1 mutL (E. coli) homologue 1 gene
mRNA messenger ribonucleic acid
MYC avian myelocytomatosis viral (v-myc) oncogene homologue gene
8
NCBI National Center for Biotechnology Information
nt nucleotide
OCCR ovarian cancer cluster region
PCR polymerase chain reaction
PGR progesterone receptor
PTEN phosphatase and tensin homologue gene
PTT protein truncation test
q long arm of the chromosome
RFLP restriction fragment length polymorphism
RNA ribonucleic acid
SD standard deviation
STK11 serine/threonine kinase 11 gene
T thymine
TP53 tumour protein p53 gene
TSG tumour suppressor gene
999del5 999delTCAAA
5145del11 5145delTTAACTAATCT
9
ABSTRACT
Two major genes, BRCA1 and BRCA2, germline mutations of which predispose to both
breast and ovarian carcinoma, have been identified. At the time this study began, 11 distinct
germline mutations in BRCA1 and seven in BRCA2 had been described in Finnish breast and
breast-ovarian carcinoma families. Eleven of these 18 mutations had been detected in more
than one Finnish family, and they had been found to account for the vast majority of all
BRCA1/BRCA2 mutation-positive families identified in the screening of the entire coding
regions of the genes. The aims of the present study were to examine ancestral origins and
geographical distribution of families with recurrent Finnish BRCA1 and BRCA2 mutations, to
study breast and ovarian carcinoma phenotypes of Finnish BRCA1 and BRCA2 mutation
carriers, and to evaluate the prevalence of BRCA1 and BRCA2 founder mutations in Finnish
ovarian carcinoma patients and ovarian carcinoma families.
Haplotype analysis was used to study the origins of families with recurrent mutations,
and time from a common ancestor for the families was estimated by modifications of the
Luria-Delbrück equation. All BRCA1/BRCA2 mutation-positive families identified in Finland
were included in the phenotype analysis examining the distribution of ages at breast and
ovarian cancer diagnosis, and the proportion of ovarian carcinoma. The contribution of
BRCA1 and BRCA2 founder mutations to ovarian carcinoma was evaluated by studying the
prevalence of previously identified Finnish BRCA1 and BRCA2 germline mutations in
unselected ovarian carcinoma patients and in a population-based series of families with at
least two cases of ovarian carcinoma in first-degree relatives. In addition, a subset of ovarian
carcinoma patients was screened for novel BRCA1/BRCA2 germline mutations. The
relationship between mutation carrier status and personal and family history of breast and
ovarian carcinoma was studied by logistic regression analysis.
Haplotype analyses revealed that all carriers of the same recurrent BRCA1/BRCA2
mutation, except for those with the BRCA2 999del5 mutation, shared a common core
haplotype. In the 999del5 mutation-positive families, two distinct core haplotypes were seen.
The mutation-associated haplotypes shared by carriers of the same mutation indicate that
mutation alleles are identical by descent, i.e., founder mutations. The two 999del5 mutation-
associated haplotypes may be due to gene conversion, which is supported by the geographical
clustering of the families as well as by the population history of Finland. Finnish families
with one of the 999del5 mutation-associated haplotypes shared a four-marker (0.5 cM)
haplotype with Icelandic families with the same mutation, which may indicate a common
ancient origin for the Finnish and Icelandic 999del5 mutation-positive families. Nevertheless,
10
distinct mutational events cannot be ruled out. Estimations of time from a common ancestor
for the Finnish families varied widely, ranging from 6 to 32 generations. For some mutations,
birthplaces of the parents and grandparents were clustered in a very restricted area, while for
other mutations, a wider distribution was seen. The high coverage of founder mutations of all
BRCA1/BRCA2 mutations in Finland and the narrow mutation spectra observed in certain
geographical areas have a significant impact on BRCA1/BRCA2 mutation testing in Finnish
breast and ovarian carcinoma families.
Analysis of breast and ovarian carcinoma phenotypes revealed that the proportion of
ovarian carcinoma was significantly higher in BRCA1 mutation-associated families than in
those with BRCA2 mutations. Moreover, in the BRCA1 mutation-positive families, the
proportion of ovarian carcinoma was significantly higher in families carrying mutations in
exon 11 as compared with those carrying mutations 3´ of this exon. For breast carcinoma, the
distribution of ages at diagnosis was similar in BRCA1 and BRCA2 mutation-positive
families, while for ovarian carcinoma, the mean age at diagnosis was significantly younger in
families with BRCA1 mutations.
In unselected ovarian carcinoma patients, the frequency of BRCA1 and BRCA2
mutations was 4.7% and 0.9%, respectively. No novel mutations were identified, and seven
founder mutations accounted for 12 of the 13 mutations detected. The most significant
predictor of a BRCA1 or BRCA2 mutation was presence of both breast and ovarian carcinoma
in the same patient. Moreover, family history of breast carcinoma was strongly related to
mutation carrier status. In Finnish ovarian carcinoma families, the BRCA1/BRCA2 mutation
frequency was 26%. All families with strong family history of ovarian carcinoma (i.e., three
affected cases) or early-onset (<50 years) breast carcinoma were mutation-positive, while all
families with later-onset breast carcinoma and most (9/11) families with two cases of ovarian
carcinoma only were mutation-negative. A combination of chance clustering of sporadic
cases, non-genetic familial factors and incomplete sensitivity of mutation detection may
account for BRCA1/BRCA2 mutation-negative ovarian carcinoma families. However,
unidentified ovarian cancer-susceptibility genes, possibly with low penetrance, may segregate
in some families.
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INTRODUCTION
Breast cancer is the most common and ovarian cancer the fourth most common cancer among
women in Finland, with 3578 and 522 new cases diagnosed in 1999, respectively [the Finnish
Cancer Registry (FCR), 2002]. Approximately 10% of all ovarian carcinomas and 7% of all
breast carcinomas are estimated to be associated with dominantly inherited germline
mutations in cancer-susceptibility genes (Claus et al., 1996). To date, two major genes,
BRCA1 and BRCA2, germline mutations of which predispose to both breast and ovarian
carcinoma, have been identified (Miki et al., 1994; Wooster et al., 1994). More than 1000
distinct germline alterations have been identified in each gene, most of them appearing
uniquely in a single family [the Breast Cancer Information Core (BIC) database]. However, in
several ethnic groups and populations, recurrent mutations have been described (Szabo and
King, 1997; Neuhausen, 1999). The proportion of unique versus recurrent BRCA1/BRCA2
mutations varies among populations, reflecting historical influences of migration, population
structure, and geographical and cultural isolation (Szabo and King, 1997). Germline
mutations of BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, although
risk estimates obtained from different studies are variable [Ford et al., 1994, 1998; Easton et
al., 1995; Thorlacius et al., 1998; the Breast Cancer Linkage Consortium (BCLC), 1999;
Anglian Breast Cancer Study Group (ABCSG), 2000; Antoniou et al., 2000, 2002; Satagopan
et al., 2001]. There is also evidence for a modifying effect of other genes as well as non-
genetic factors on the risks of breast and ovarian cancer in BRCA1 and BRCA2 mutation
carriers (Hopper et al., 1999; Antoniou et al., 2000, 2002; Nathanson and Weber, 2001).
Furthermore, the location of the mutation in BRCA1 and BRCA2 may influence breast and
ovarian carcinoma risks (Gayther et al., 1995, 1997b; Risch et al., 2001; Thompson and
Easton, 2001, 2002).
In the Finnish population, 11 recurrent mutations have been found to account for the
vast majority (84%) of BRCA1/BRCA2 mutations identified in the screening of the entire
coding regions of the genes (Vehmanen et al., 1997a, 1997b; Huusko et al., 1998). Therefore,
a reasonable estimate of the BRCA1/BRCA2 mutation burden in various Finnish study
populations can be achieved rapidly and cost-efficiently by screening samples for the known
Finnish BRCA1/BRCA2 mutations. In different populations, inherited mutations of BRCA1
and BRCA2 account for a varying fraction of hereditary breast and ovarian carcinoma (Szabo
and King, 1997). Only a small proportion of familial aggregation of breast carcinoma appears
to be explained by BRCA1 and BRCA2 germline mutations in most populations (Szabo and
King, 1997), and there is evidence that other still undiscovered breast cancer-susceptibility
12
genes exist (Serova et al., 1997; Ford et al., 1998; Kainu et al., 2000; Antoniou et al., 2001;
Cui et al., 2001). BRCA1 and BRCA2 germline mutations may, however, be sufficient to
explain the majority of hereditary ovarian carcinoma (Gayther et al., 1999; Antoniou et al.,
2000). The aims of this thesis were thus to examine the ancestral origins and geographical
distribution of families with recurrent Finnish BRCA1 and BRCA2 mutations, to study breast
and ovarian carcinoma phenotypes of Finnish BRCA1 and BRCA2 mutation carriers, and to
evaluate the prevalence of BRCA1 and BRCA2 founder mutations in Finnish ovarian
carcinoma patients and ovarian carcinoma families.
13
REVIEW OF THE LITERATURE
1 General features of ovarian carcinoma
Ovarian cancer is the sixth most common cancer among women world-wide (Parkin et al.,
1999). In Finland, 522 new cases of ovarian cancer were diagnosed in 1999, making it the
fourth most common cancer among women (the FCR, 2002). The cumulative incidence of
ovarian cancer by the age of 75 years is 1.4% (Auranen et al., 1996b).
Of all malignant ovarian tumours, carcinomas, i.e., tumours originating from the
surface epithelium of the ovary, account for approximately 90% (Russell, 1994; Holschneider
and Berek, 2000). Ovarian carcinoma is predominantly a disease of peri- and postmenopausal
women (Russell, 1994; Holschneider and Berek, 2000), and the mean age at diagnosis is 62
years (Auranen et al., 1996a). The most common histological subtypes of ovarian carcinoma
are serous, endometrioid, and mucinous carcinoma, representing 40–50%, 15–25%, and 5–
15% of all cases, respectively (Russell, 1994; Heintz et al., 2001). Less common histological
subtypes include clear cell carcinoma, undifferentiated carcinoma, transitional cell
carcinoma, malignant Brenner tumour, and malignant mixed epithelial tumour (Russell,
1994).
The overall prognosis of patients with ovarian carcinoma is poor, which is related to the
high proportion of women being diagnosed with advanced stage disease (stages III and IV)
(Heintz et al., 2001). Stage of disease at diagnosis according to the Fédération Internationale
de Gynécologie et d’Obstetrique (FIGO) staging system is one of the most significant
prognostic factors of the disease (Friedlander, 1998). The five-year survival rate for patients
with FIGO stage I, II, III, and IV tumours is 85–89%, 57–67%, 24–42%, and 12–17%,
respectively (Nguyen et al., 1993; Heintz et al., 2001). Other prognostic indicators include
histological type and grade, residual tumour size, performance status, and patient’s age
(Friedlander, 1998).
One of the strongest risk factors for the disease is a family history of ovarian and/or
breast cancer, and the risk depends on the number of affected first- and second-degree
relatives and their age at diagnosis (Schildkraut et al., 1989; Parazzini et al., 1992; Stratton et
al., 1998; Holschneider and Berek, 2000). Other factors associated with an increased risk
include infertility and nulliparity (Edmondson and Monaghan, 2001; Ness et al., 2002), while
factors associated with a decreased risk include multiparity, lactation, oral contraceptive use,
tubal ligation, and hysterectomy (Whittemore et al., 1992; Edmondson and Monaghan, 2001).
14
2 General features of breast carcinoma
Breast cancer is the most frequently occurring cancer among women world-wide, comprising
21% of all female cancers (Parkin et al., 1999). In Finland, 3578 new breast cancer cases
were diagnosed in women and 14 in men in 1999 (the FCR, 2002). Incidence rates rise
rapidly with increasing age, but the rate of increase declines around menopause (Pike et al.,
1983; the FCR, 2002). Average age at diagnosis is 61 years (Dickman et al., 1999), and about
one in ten Finnish women will develop breast cancer during her lifetime (the FCR, 2002).
The majority of malignant breast tumours are carcinomas. Infiltrating ductal carcinoma
is by far the most common histological type of invasive breast carcinoma, accounting for
about 70% of all cases (Berg and Hutter, 1995). The five-year relative survival rate for
Finnish breast cancer patients is 80% (Dickman et al., 1999). However, the prognosis varies
widely between patients, and for those with localized disease, regional metastases, and distant
metastases, the five-year relative survival rates are 93%, 69%, and 22%, respectively
(Dickman et al., 1999).
The aetiology of breast cancer is closely linked to oestrogen, with a prolonged or
increased exposure being suggested to increase breast cancer risk (Pike et al., 1983;
Henderson and Feigelson, 1998). Many known risk factors for breast cancer are related to the
reproductive life of women: early age at menarche, late onset of menopause, late age at first
full-term pregnancy, and nulliparity (Henderson and Feigelson, 1998; McPherson et al., 2000;
Hulka and Moorman, 2001). A family history of breast cancer and/or ovarian cancer is one of
the strongest risk factors for the disease (Sattin et al., 1985; Schildkraut et al., 1989; Madigan
et al., 1995; Pharoah et al., 1997); the risk increases as the number of affected first- and
second-degree relatives increases and their age at diagnosis decreases, and if there are cases
of bilateral breast cancer among relatives (Sattin et al., 1985; Slattery and Kerber, 1993;
Pharoah et al., 1997). Other risk factors for breast cancer include exposure to ionizing
radiation, postmenopausal obesity, and history of atypical epithelial hyperplasia (McPherson
et al., 2000; Hulka and Moorman, 2001). Factors that confer protection consist of multiparity,
early age at first full-term pregnancy, lactation, and physical activity (Henderson and
Feigelson, 1998; Hulka and Moorman, 2001).
3 Genes involved in carcinogenesis
Carcinogenesis is a multistep process during which genetic and epigenetic alterations
accumulate in a cell, resulting in the progressive transformation of normal cells through steps
15
of initiation, promotion, and progression into cancer cells. Genes involved in cancer affect the
normal functions of such cellular processes as cell proliferation and differentiation, apoptosis,
deoxyribonucleic acid (DNA) repair, genomic stability, senescense, cell-cell communication,
cell-matrix interactions, angiogenesis, tumour invasion, motility, and metastasis (Nowell,
1976; Compagni and Christofori, 2000; Hanahan and Weinberg, 2000; Evan and Vousden,
2001; Ponder, 2001). Three major groups of genes are known to be involved in cancer: proto-
oncogenes, classical tumour suppressor genes (also known as gatekeeper tumour suppressor
genes), and caretaker tumour suppressor genes (Weinberg, 1989; Kinzler and Vogelstein,
1997; Ponder, 2001). At present, around 30 tumour suppressor genes (TSGs) and over 100
proto-oncogenes have been identified (Futreal et al., 2001).
Proto-oncogenes are involved in the control of normal cell proliferation, apoptosis, and
differentiation, and their inappropriate activation may turn them into oncogenes. At the
cellular level, these genes are dominant, i.e., activation of one allele (gain-of-function) is
sufficient to give a growth advantage to the cell (Ponder, 2001). Conversely, gatekeeper and
caretaker TSGs act recessively at the cellular level, i.e., inactivation of both alleles (loss-of-
function) is required for an altered cell phenotype (Weinberg, 1989; Ponder, 2001). However,
recent evidence for haplo-insufficiency at some tumour suppressor gene loci, e.g., BRCA1,
BRCA2, PTEN, and STK11, exists (Fero et al., 1998; Kwabi-Addo et al., 2001; Buchholz et
al., 2002; Miyoshi et al., 2002). Gatekeeper TSGs act directly to prevent tumour growth by
suppressing proliferation, inducing apoptosis, or promoting differentiation, and their loss of
function is rate-limiting for a particular step in tumourigenesis, whereas caretaker TSGs act
indirectly to suppress neoplasia, and their inactivation leads to genetic instability which
results in a greatly increased mutation rate of all genes, including gatekeeper TSGs and proto-
oncogenes (Kinzler and Vogelstein, 1997). This sub-classification of TSGs has, however,
become arbitrary as some genes, including BRCA1, BRCA2, and TP53, have been shown to
have both gatekeeper and caretaker tumour suppressor functions (Macleod, 2000; Zheng et
al., 2000). Furthermore, a new class of TSGs has been proposed: landscapers that are
predicted to act by modulating the local stromal microenvironment such that the neoplastic
conversion of epithelia is promoted (Kinzler and Vogelstein, 1998; Liotta and Kohn, 2001).
4 Inherited predisposition to cancer
Most genetic alterations that lead to cancer are somatic and are found only in indivual’s
cancer cells. However, 1–2% of all cancers are associated with inherited cancer-
predisposition syndromes, arising in individuals who carry an inherited germline mutation of
16
a cancer-susceptibility gene in every cell of their body (Fearon, 1997; Ponder, 2001). The
lifetime risk of cancer for these individuals is high (up to 50–80%), but the likelihood of
developing cancer depends on the particular mutant allele, on other genetic and non-genetic
factors (risk modifiers), and on the complex interplay of all these factors, which remains
poorly understood (Fearon, 1997; Ponder, 2001). In most inherited cancer-predisposition
syndromes, inheritance follows an autosomal dominant mode and cancer susceptibility is due
to inactivating loss-of-function mutations in gatekeeper and caretaker TSGs, rather than
activating gain-of-function alterations in proto-oncogenes (Fearon, 1997; Kinzler and
Vogelstein, 1997). Inherited cancer-predisposition syndromes are characterized by multiple
affected family members, early age at cancer onset, and multiple primary cancers. Some of
the syndromes also feature other rare conditions, particularly congenital abnormalities
(Fearon, 1997). However, in some families segregating a mutant allele of a major inherited
cancer-susceptibility gene, no striking features of inherited cancer-predisposition syndromes
are seen, possibly due to small family size, uncertain family history, or incomplete
penetrance. In addition to hereditary cancers that occur in association with rare inherited
cancer-predisposition syndromes, an unknown fraction of cancers are due to cosegregation of
mutant alleles of minor cancer-susceptibility genes, conferring low to moderate cancer risk;
these mutant alleles are estimated to be relatively common in the general population, and
thus, may confer a higher population-attributable risk for cancer (Ponder, 2001).
5 Inherited predisposition to breast and ovarian carcinoma
Familial association of breast and ovarian carcinoma was first suggested in the 1970s, when
large families with an excess of both breast and ovarian carcinoma, transmitted through
several generations, were identified (Lynch et al., 1972, 1978). Large families with an excess
of only breast or ovarian cancer were also described, and they were called site-specific breast
or site-specific ovarian cancer families (Lynch et al., 1972, 1981). A significant genetic
correlation detected between breast and ovarian carcinoma provided further support for the
existence of hereditary breast-ovarian cancer (HBOC) syndrome, and predisposition to these
two cancers was suggested to be due partly to mutations in the same gene and partly to
mutations in different genes (Schildkraut et al., 1989). In segregation analyses, breast cancer
was found to follow an autosomal dominant mode of inheritance in some families (Newman
et al., 1988; Claus et al., 1991), and in 1990, the first breast cancer-susceptibility gene was
mapped by genetic linkage to chromosome 17q21 in families with multiple cases of early-
onset breast cancer (Hall et al., 1990). Soon thereafter, linkage to the same chromosomal
17
region was reported in breast-ovarian cancer families (Narod et al., 1991, 1995; Easton et al.,
1993) and in families with site-specific ovarian cancer (Steichen-Gersdorf et al., 1994). The
first breast-ovarian cancer-susceptibility gene BRCA1 (breast cancer 1) was identified by
positional cloning methods in 1994 (Miki et al., 1994). During the same year the second
breast cancer-susceptibility locus, BRCA2 (breast cancer 2), was localized to chromosome
13q12–q13 by linkage studies of families with multiple cases of early-onset breast cancer that
were not linked to BRCA1 (Wooster et al., 1994). Male breast cancer was found to be present
in many BRCA2-linked families (Thorlacius et al., 1995, 1996; Gudmundsson et al., 1996;
Tavtigian et al., 1996). The BRCA2 gene was identified in 1995 by positional cloning
methods (Wooster et al., 1995), and its complete coding sequence and exonic structure were
described in 1996 (Tavtigian et al., 1996).
Approximately 10% of all ovarian carcinomas and 7% of all breast carcinomas are
estimated to be associated with dominantly inherited germline mutations in breast/ovarian
cancer-susceptibility genes (Claus et al., 1996). Moreover, a large twin study has shown that
heritable factors are of importance in about 30% of all breast cancers (Lichtenstein et al.,
2000). Germline mutations in the BRCA1 and BRCA2 genes seem to account for the majority
of families with multiple cases of both breast and ovarian cancer and of those with site-
specific ovarian cancer, but only for a small proportion of site-specific breast cancer families
(Steichen-Gersdorf et al., 1994; Ford et al., 1995, 1998; Rebbeck et al., 1996; Håkansson et
al., 1997; Schubert et al., 1997; Serova et al., 1997; Vehmanen et al., 1997a; Zelada-Hedman
et al., 1997; Boyd, 1998; Kainu et al., 2000; Eerola et al., 2001a). In addition, a number of
other rare hereditary cancer-predisposition syndromes include breast and/or ovarian
carcinoma in their clinical presentation; breast cancer has been identified as a component of
Li-Fraumeni syndrome, Cowden disease, Peutz-Jeghers syndrome, ataxia-telangiectasia, and
cutaneous malignant melanoma (Kamb et al., 1994; Arver et al., 2000; Borg et al., 2000),
while ovarian carcinoma manifests in hereditary non-polyposis colorectal cancer (HNPCC)
syndrome and in Peutz-Jeghers syndrome (Arver et al., 2000). However, these syndromes
explain only a small proportion of all hereditary breast and ovarian cancers (Arver et al.,
2000). The residual inherited susceptibility to breast cancer may be partly due to rare
mutations in one or a few additional major breast cancer-susceptibility genes conferring a
high risk of disease (high-penetrance alleles) (Serova et al., 1997; Kainu et al., 2000; Cui et
al., 2001), and evidence for both dominantly and recessively inherited risk has been presented
(Antoniou et al., 2001, 2002; Cui et al., 2001). Nevertheless, several common, low-
penetrance alleles with multiplicative effects on breast cancer risk have been proposed to be
responsible for a large fraction of hereditary breast cancers (Antoniou et al., 2001, 2002). The
possibility that additional ovarian cancer-susceptibility genes exist has been suggested as well
18
(Sekine et al., 2001a). In the Finnish population, a recessive mode of inheritance of ovarian
carcinoma has been proposed (Auranen and Iselius, 1998).
6 BRCA1 and BRCA2 genes
6.1 Structure and expression
The BRCA1 gene covers 81 kilobases (kb) of genomic DNA on chromosome 17q21 and has
24 exons, 22 of which are encoding (Miki et al., 1994; Smith et al., 1996). The BRCA2 gene
is distributed over roughly 70 kb of genomic DNA on chromosome 13q12, and of its 27
exons, 26 are encoding (Wooster et al., 1995; Tavtigian et al., 1996). Both genes have a large
exon 11 (comprising 61% and 48% of the whole coding sequences of BRCA1 and BRCA2,
respectively) and have translational start sites in exon 2 (Miki et al., 1994; Tavtigian et al.,
1996). In BRCA1, exon 4 is not translated (Miki et al., 1994; Smith et al., 1996). The
genomic regions of BRCA1 and BRCA2 have unusually high (47%) densities of repetitive
DNA elements (Smith et al., 1996; Welcsh and King, 2001).
The human BRCA1 and BRCA2 genes are expressed in a wide variety of tissues, with
the highest levels of messenger ribonucleic acid (mRNA) expression seen in the testis,
thymus, and breast (Miki et al., 1994; Tavtigian et al., 1996). Studies on mice have shown
that Brca1 and Brca2 mRNA levels are highest in rapidly proliferating cell types, particularly
those undergoing differentiation (Marquis et al., 1995; Rajan et al., 1997), and their
expression levels vary during the cell cycle, peaking at the G1/S boundary (Rajan et al.,
1996). In the mouse mammary gland, expression of Brca1 and Brca2 mRNA is induced
during puberty and pregnancy, when oestrogen levels are dramatically increased, and
following treatment of ovariectomized animals with 17β-oestradiol and progesterone
(Marquis et al., 1995; Rajan et al., 1997). In human breast cancer cell lines, BRCA1 and
BRCA2 mRNA levels are also co-ordinately elevated in response to oestrogen (Spillman and
Bowcock, 1996; Marks et al., 1997).
6.2 BRCA1 and BRCA2 gene-encoded protein products and their proposed functions
The 7.8 kb BRCA1 mRNA encodes a protein with 1863 amino acids and a predicted
molecular weight of 208 kiloDaltons (kDa) (Miki et al., 1994). The BRCA2 transcript is 12
kb long and encodes a protein with 3418 amino acids and a predicted molecular weight of
19
384 kDa (Tavtigian et al., 1996). Shorter, alternatively spliced isoforms have been identified
as well (Miki et al., 1994; Lu et al., 1996; Wilson et al., 1997; Zou et al., 1999).
The BRCA1 and BRCA2 proteins bear little resemblance to one another or to other
known proteins (Venkitaraman, 2002). Nevertheless, there are striking similarities in their
expression patterns, and they both appear to be involved in the process of proliferation and
differentiation in multiple tissues, notably in the mammary gland in response to ovarian
hormones (Marquis et al., 1995; Rajan et al., 1996, 1997; Spillman and Bowcock, 1996;
Marks et al., 1997). Several functional domains and structural motifs have been identified in
BRCA1 and BRCA2, and they have been found to interact with each other and with various
other proteins, including transcription factors and proteins involved in DNA double-strand
break repair (Zheng et al., 2000; Welcsh and King, 2001; Venkitaraman, 2002). Their
localization varies according to the phase of the cell cycle; during S phase, they are localized
to discrete, subnuclear foci, and after DNA damage, they rapidly relocalize to sites of DNA
synthesis (Scully et al., 1997; Chen et al., 1998; Yarden et al., 2002). BRCA1 appears to be
activated during late G1 and S phases and following DNA damage, when it has been shown to
undergo hyperphosporylation (Thomas et al., 1997).
Cells deficient in BRCA1/Brca1 or BRCA2/Brca2 accumulate chromosomal
abnormalities (Tirkkonen et al., 1997; Abbott et al., 1998; Lee et al., 1999; Xu et al., 1999;
Moynahan et al., 2001) and are hypersensitive to genotoxic agents (Sharan et al., 1997;
Gowen et al., 1998; Scully et al., 1999; Moynahan et al., 2001). This suggests that BRCA1
and BRCA2 may function as caretakers whose loss leads to genetic instability and increases
the probability that inactivation of gatekeeper TSGs and activation of proto-oncogenes will
occur, eventually leading to tumour formation (Kinzler and Vogelstein, 1997). Inactivation of
the TP53 tumour suppressor gene or other genes critical in cell-cycle checkpoint control have
been found to be frequent in tumours of Brca1/Brca2-deficient mice (Lee et al., 1999; Xu et
al., 1999). In human BRCA1 mutation-associated breast and ovarian carcinomas, inactivation
of TP53 has been suggested to be more common than in corresponding sporadic tumours
(Crook et al., 1998; Ramus et al., 1999; Buller et al., 2001; Greenblatt et al., 2001).
Furthermore, BRCA1 and BRCA2 have been shown to suppress proliferation of breast and
ovarian cancer cell lines, suggesting that they act directly to suppress tumour growth, hence
possessing gatekeeper tumour suppressor functions as well (Thompson et al., 1995; Holt et
al., 1996; Somasundaram et al., 1997; Randrianarison et al., 2001; Wang et al., 2002).
Although the precise functions of BRCA1 and BRCA2 remain unclear, there is strong
evidence that they are involved in the DNA damage response pathway, and they have been
proposed to play roles in transcriptional regulation, cell-cycle checkpoint control, DNA
20
damage repair, and recombination (Zheng et al., 2000; Welcsh and King, 2001;
Venkitaraman, 2002).
The tissue-specificity of BRCA1/BRCA2 mutation-associated carcinogenesis has been
proposed to be related to their oestrogen responsiveness (Hilakivi-Clarke, 2000; Welcsh and
King, 2001). Oestrogens induce cell proliferation and stimulate development of tissues
involved in reproduction. However, they may also predispose cells to DNA damage during
periods of rapid cellular proliferation. Furthermore, oestrogens have been reported to be able
to induce direct and indirect free radical-mediated DNA damage (Cavalieri et al., 2000;
Liehr, 2000). BRCA1 and BRCA2 have been suggested to function in protecting breasts and
ovaries from genetic instability during oestrogen-induced periods of rapid cellular
proliferation (Fan et al., 1999; Hilakivi-Clarke, 2000).
6.3 Inherited germline mutations
6.3.1 Spectrum
Since the identification of the BRCA1 and BRCA2 genes (Miki et al., 1994; Wooster et al.,
1995; Tavtigian et al., 1996), more than 1000 distinct sequence variants have been described
in each gene (the BIC database). Alterations are distributed throughout the coding regions,
and disease-associated mutations are mainly frameshift, nonsense, or splice site mutations
leading to formation of truncated protein products (Ellisen and Haber, 1998; the BIC
database). Missense variants have been identified as well, but their effect on carcinogenesis is
not as easy to determine as in the case of protein-truncating mutations, which are considered
to be functionally deleterious (Shattuck-Eidens et al., 1997; Spain et al., 1999). However, in
BRCA2 exon 27, four sequence variants that result in a stop codon have been proposed to be
non-disease-associated (Mazoyer et al., 1996; Wagner et al., 1999; the BIC database).
Significance of some common missense variants has been studied by comparing the
frequencies of the variants in large series of breast/ovarian cancer cases and matched
controls, and most of them do not appear to confer an increased risk of breast or ovarian
cancer (Durocher et al., 1996; Dunning et al., 1997; Healey et al., 2000; Deffenbaugh et al.,
2002). In families with rare missense alterations, cosegregation of the variants with breast and
ovarian cancer has been used to evaluate their significance on carcinogenesis, but many
families do not have appropriate pedigree structure or sufficient samples for such an analysis
(Shattuck-Eidens et al., 1997; Vallon-Christersson et al., 2001; Fackenthal et al., 2002).
Some missense variants have been proposed to have an effect on protein function based on
21
their location within functional domains or evolutionally conserved regions of the proteins
(Castilla et al., 1994; Wu et al., 1996; Stoppa-Lyonnet et al., 1997; Roth et al., 1998; Janezic
et al., 1999; Wagner et al., 1999; Brzovic et al., 2001; Smith et al., 2001). Recently,
functional studies of BRCA1 have given further support for the hypothesis that missense
alterations located within functional domains may play a role in disease predisposition
(Scully et al., 1999; Vallon-Christersson et al., 2001). Only a small number of missense
variants in either gene have been described as deleterious mutations (Górski et al., 2000;
Sekine et al., 2001a; Vallon-Christersson et al., 2001; de La Hoya et al., 2002; Meindl and
German Consortium for Hereditary Breast and Ovarian Cancer, 2002; the BIC database), and
the clinical significance of a number of amino acid substitutions in BRCA1 and BRCA2
remains still to be resolved (the BIC database).
The observed mutation spectrum is surely influenced by techniques used in mutation
screening. Most studies searching for germline mutations of BRCA1 and BRCA2 have
analysed the coding regions and splice sites of the genes, and used techniques based on
polymerase chain reaction (PCR), e.g., direct sequencing, single-strand conformation
polymorphism (SSCP) analysis, conformation-sensitive gel electrophoresis (CSGE),
denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HA), and protein
truncation test (PTT) (the BIC database). However, by standard screening methods, only 63%
of breast cancer families showing linkage to BRCA1 in a large BCLC study could be
identified as mutation-positive (Ford et al., 1998). Large genomic rearrangements and
regulatory mutations are not detected by standard approaches and may thus account for a
proportion of cases without identified mutations. Recently, several studies have examined the
presence of large genomic rearrangements of BRCA1 and BRCA2 in breast/ovarian cancer
families; within BRCA1 and its regulatory regions, several germline rearrangements (ranging
from 0.5 to 23.8 kb) have been described (Petrij-Bosch et al., 1997; Puget et al., 1997, 1999;
Swensen et al., 1997; Rohlfs et al., 2000; Unger et al., 2000), while in BRCA2, only two such
alterations have been identified (Miki et al., 1996; Nordling et al., 1998). Many of these
rearrangements are likely to be due to Alu-mediated homologous recombination, and they
have been presumed to be less frequent in BRCA2 than in BRCA1 because of the lower
density of Alu sequences in the BRCA2 gene (20% versus 42%, respectively) (Smith et al.,
1996; Welcsh and King, 2001). Moreover, it has recently been proposed that some missense
and silent BRCA1/BRCA2 variants may lead to exon skipping and deleterious protein-
truncating mutations through disruption of critical exonic splicing enhancer sequences (Liu et
al., 2001; Fackenthal et al., 2002).
22
6.3.2 Ethnic differences in mutation spectra
Although the majority of germline alterations identified in BRCA1 and BRCA2 (57% and
63%, respectively) are unique (the BIC database), several recurrent mutations have been
described in a number of ethnic groups and populations (the BIC database), e.g., in
Ashkenazi Jews (Roa et al., 1996; Struewing et al., 1997; Fodor et al., 1998), French
Canadians (Simard et al., 1994; Tonin et al., 1998), Icelanders (Johannesdottir et al., 1996;
Thorlacius et al., 1997), Finns (Vehmanen et al., 1997a; Huusko et al., 1998), Swedes
(Håkansson et al., 1997; Bergman et al., 2001), the Dutch (Peelen et al., 1997; Petrij-Bosch et
al., 1997; Verhoog et al., 2001), Belgians (Peelen et al., 1997; Goelen et al., 1999), Russians
(Gayther et al., 1997a), Polish (Górski et al., 2000), and Hungarians (Ramus et al., 1997; van
der Looij et al., 2000). Some of the recurrent BRCA1/BRCA2 mutations are population-
specific, while others are found in a number of different populations and ethnic groups
(Neuhausen et al., 1996, 1998, 1999; Szabo and King, 1997; the BIC database). The
proportion of recurrent mutations to unique mutations varies in different populations and
subpopulations, reflecting historical influences of migration, population structure, and
geographical and cultural isolation (Szabo and King, 1997). Studies on mutation-associated
haplotype sharing between families carrying the same BRCA1/BRCA2 mutation have been
carried out to determine whether recurrent mutations are identical by descent, i.e., founder
mutations, or whether they represent distinct mutational events (Simard et al., 1994;
Friedman et al., 1995; Berman et al., 1996; Neuhausen et al., 1996, 1998; Petrij-Bosch et al.,
1997; Rohlfs et al., 2000; Bergman et al., 2001; Ikeda et al., 2001; Meindl and German
Consortium for Hereditary Breast and Ovarian Cancer, 2002).
Among Icelanders, only one mutation has been identified in each of the BRCA1 and
BRCA2 genes; the mutation located in BRCA2 accounts for the majority (76%) of Icelandic
breast cancer families with multiple affected persons (Thorlacius et al., 1997), while the
mutation located in BRCA1 has been observed in only two families (Bergthorsson et al.,
1998). In the Polish population, six distinct BRCA1 mutations and one BRCA2 mutation have
been described, and three recurrent BRCA1 mutations account for most (83%)
BRCA1/BRCA2 mutation-positive families (Górski et al., 2000). In the western part of
Sweden, one BRCA1 mutation have been reported to account for as much as 77% of
identified BRCA1/BRCA2 mutations (Bergman et al., 2001). In Finland, around 30 different
BRCA1/BRCA2 mutations have been described (Vehmanen et al., 1997a, 1997b; Huusko et
al., 1998; Syrjäkoski et al., 2000; Vahteristo et al., 2001; unpublished data), and six BRCA1
mutations and five BRCA2 mutations account for most (84%) BRCA1/BRCA2 mutations
23
identified in the screening of the entire coding sequences of the genes (Vehmanen et al.,
1997a, 1997b; Huusko et al., 1998).
6.3.3 Prevalence
In outbred populations, the prevalence of BRCA1 and BRCA2 mutation carriers has been
estimated to be 0.048–0.29% (1/2083–1/345) and 0.082%–0.34% (1/1220–1/291),
respectively (Ford et al., 1995; Whittemore et al., 1997; Peto et al., 1999; Antoniou et al.,
2000, 2001, 2002). In populations with strong BRCA1/BRCA2 founder effects, mutant alleles
have been detected with higher carrier frequencies; among Ashkenazi Jews, approximately
2.5% (1/40) of individuals carry one of the three common BRCA1/BRCA2 founder mutations
(185delAG or 5382insC in BRCA1, or 6174delT in BRCA2) (Roa et al., 1996; Struewing et
al., 1997; Fodor et al., 1998), and in the Icelandic population, 0.4–0.6% (1/250–1/167) of
individuals carry one BRCA2 founder mutation [999delTCAAA (999del5)] (Johannesdottir et
al., 1996; Tavtigian et al., 1996; Thorlacius et al., 1997).
Based on early linkage analyses, BRCA1 and BRCA2 germline mutations were
estimated together to account for the large majority of families with multiple cases of breast
and/or ovarian cancer; of breast-ovarian cancer families, 80–100% were proposed to be
linked to BRCA1 (Easton et al., 1993; Narod et al., 1995), while of site-specific breast cancer
families about 45% were estimated to be linked to BRCA1 and about 35% to BRCA2 (Easton
et al., 1993; Wooster et al., 1994). Most families with ovarian cancer only were suggested to
be linked to BRCA1 (Steichen-Gersdorf et al., 1994). Later, the contribution of both BRCA1
and BRCA2 to hereditary breast cancer was evaluated in the large collaborative BCLC study
(Ford et al., 1998). The families included in this study contained at least four cases of either
female breast cancer diagnosed before the age of 60 years or male breast cancer diagnosed at
any age (ovarian cancer was not used as a selection criterion), and breast cancer was
estimated to be associated with BRCA1 and BRCA2 mutant alleles in 52% and 32% of the
families, respectively (Ford et al., 1998). Nevertheless, when the families were divided into
subgroups according to additional criteria, such as the presence of both breast and ovarian
cancer in a family, the proportions of families linked to BRCA1 or BRCA2 differed strikingly
in various subgroups. Most (81%) breast-ovarian cancer families were due to BRCA1, while
the majority (76%) of families with both male and female breast cancer were estimated to be
due to BRCA2. In families with four or five cases of female breast cancer and no cases of
ovarian cancer, only a minority was linked to BRCA1 or BRCA2 (28% and 5%, respectively)
(Ford et al., 1998).
24
The prevalence of BRCA1 and BRCA2 germline mutations in breast and/or ovarian
cancer families has been examined in a number of different populations and ethnic groups
(Table 1). These studies are, however, hard to compare as definitions of family history as well
as mutation screening methods used have been highly variable. Nevertheless, germline
mutations of BRCA1/BRCA2 have been detected in most populations in 20–50% of breast
and/or ovarian cancer families (Table 1), implying that the contribution of BRCA1/BRCA2
germline mutations to hereditary breast and/or ovarian cancer predisposition is not as high as
originally estimated based on linkage analyses of extended high-risk families.
25
Table 1. Prevalence of BRCA1 and BRCA2 germline mutations in series of breast and/or ovariancancer families.
Mutation frequency (%)Reference Population Selection criteria No. of
patients
Type of mutation
screening BRCA1 BRCA2 BRCA1/2Vahteristoet al., 2001
Finnish >3 bc or oc (in 1st/2nd-degreerelatives)
148 W, BRCA1 andBRCA2 (n=95);F, 11 in BRCA1 and8 in BRCA2 (n=53)
10.8 8.8 19.6
Ligtenberget al., 1999
Dutch >3 bc or oc (in 1st/2nd/3rd-degree relatives)
104 W, BRCA1 andBRCA2
25.0 4.8 29.8
Thorlaciuset al., 1996
Icelandic >3 bc (in 1st-degree relativesor in >3 generations), or >1male bc
21 F, 1 in BRCA2 76.2
Håkanssonet al., 1997
Swedish/Danish
>3 bc (in 1st-degreerelatives), with >1 diagnosedat age <50 y, or 2 bc (in 1st-degree relatives), with >1diagnosed at age <40 y, or>1 bc diagnosed at age <30 y
106 W, BRCA1and BRCA2
22.6 11.3 34.0
de La Hoyaet al., 2002
Spanish >3 bc or oc (in 1st/2nd-degreerelatives), with >1 diagnosedat age <50 y
102 W, BRCA1and BRCA2
18.6 12.7 31.4
Tonin et al.,1998
FrenchCanadian
>3 female/male bc (femalesdiagnosed at age <65 y) oroc, with >2 cases in1st/2nd/3rd-degree relatives ofthe index case
97 F, 4 in BRCA1and 4 in BRCA2
24.7 17.5 42.3
Frank et al.,1998
American >2 bc or oc (in 1st/2nd-degreerelatives), with >1 bcdiagnosed at age <50 y
238 W, BRCA1and BRCA2
26.5 13.0 39.5
Ikeda et al.,2001
Japanese >2 bc (in 1st-degreerelatives), no ovarian cancer
101 W, BRCA1and BRCA2
7.9 20.8 28.7
Meindl andGermanConsortiumfor HBOC,2002
German >2 bc, with >2 diagnosed atage <50 y, no ovarian cancer
328 W, BRCA1and BRCA2
24.4 12.5 36.9
Meindl andGermanConsortiumfor HBOC,2002
German >1 bc and >1 oc 250 W, BRCA1and BRCA2
42.4 9.6 52.0
Martin etal., 2001
American >1 bc and >1 oc 100 W, BRCA1and BRCA2
45.0 11.0 56.0
Gayther etal., 1997a
Russian >2 oc (in 1st-degree relatives) 19 W, BRCA1 73.7
Gayther etal., 1999
British >2 oc (in 1st/2nd-degreerelatives)
112 W, BRCA1and BRCA2
35.7 7.1 42.9
Gayther etal., 1999
British 2 oc (in 1st/2nd-degreerelatives), no breast cancer
50 W, BRCA1and BRCA2
16.0 4.0 20.0
Sekine etal., 2001b
Japanese >2 oc (in 1st/2nd-degreerelatives), no breast cancer
55 W, BRCA1and BRCA2
40.0 3.6 43.6
Moslehi etal., 2000
AshkenaziJewish(Israel andN. America)
>2 oc (in 1st/2nd-degreerelatives), no breast cancer
30 P, BRCA1 (ex 2, 11,20) and BRCA2 (ex10, 11)
30.0 30.0 60.0
bc, breast carcinoma; ex, exon; F, founder mutation(s); HBOC, Hereditary Breast and Ovarian Cancer; N., north; oc, ovariancarcinoma; P, partial coding sequence; W, whole coding sequence; y, years
26
In Finland, BRCA1 and BRCA2 germline mutations have been detected in 11% and 9%
of families with three or more cases of breast or ovarian cancer in first- or second-degree
relatives (Vehmanen et al., 1997a, 1997b; Vahteristo et al., 2001). The highest (80%)
frequency of mutations has been observed in families with both ovarian cancer and early-
onset (<40 years) breast cancer, while in families with later onset (>40 years) breast cancer
only, the mutation frequency has been lowest (1.5%) (Table 2) (Vahteristo et al., 2001).
Table 2. Frequency of BRCA1/BRCA2 germline mutations in Finnish breast andbreast-ovarian cancer families according to family history of breast and/or ovariancancer (modified from Vahteristo et al., 2001).
Criteriaa No. of families Mutation frequency (%)
3 affected 74 10.8Only breast cancer, none under 40 y 47 2.1Only breast cancer, some under 40 y 15 6.7Breast and ovarian cancer, none under 40 y 9 33.3Breast and ovarian cancer, some under 40 y 3 100
4 affected 35 22.9Only breast cancer, none under 40 y 15 0Only breast cancer, some under 40 y 7 14.3Breast and ovarian cancer, none under 40 y 11 36.4Breast and ovarian cancer, some under 40 y 3 100
> 5 affected 39 33.3Only breast cancer, none under 40 y 6 0Only breast cancer, some under 40 y 10 20.0Breast and ovarian cancer, none under 40 y 9 11.1Breast and ovarian cancer, some under 40 y 14 71.4
Total 148 19.6Only breast cancer, none under 40 y 68 1.5Only breast cancer, some under 40 y 32 12.5Breast and ovarian cancer, none under 40 y 28 28.6Breast and ovarian cancer, some under 40 y 20 80.0
y, years; aIn first- or second-degree relatives
Studies on population- and hospital-based series of female breast cancer patients
unselected for family history of cancer have reported BRCA1 and BRCA2 germline mutations
in 0.4–16.5% and 0.2–24.0% of patients, respectively (Johannesdottir et al., 1996; Krainer et
al., 1997; Thorlacius et al., 1997; Fodor et al., 1998; Malone et al., 1998; Hopper et al., 1999;
Peto et al., 1999; Tang et al., 1999; Warner et al., 1999; ABCSG, 2000; Anton-Culver et al.,
2000; Syrjäkoski et al., 2000; van der Looij et al., 2000; Loman et al., 2001; Tonin et al.,
2001; Liede et al., 2002). The highest BRCA1 mutation frequencies have been reported
among Ashkenazi Jewish (16.5%) and Icelandic (15.8%) breast cancer patients diagnosed
before the age of 50 years (Warner et al., 1999), while the highest BRCA2 mutation frequency
(24.0%) has been described in Icelandic breast cancer patients diagnosed before the age of 40
years (Thorlacius et al., 1997). In general, BRCA1/BRCA2 mutation frequencies are higher
27
among cases diagnosed at an earlier age. Among Ahkenazi Jews and Icelanders,
BRCA1/BRCA2 mutations have been detected in 7–12% (Fodor et al., 1998; Warner et al.,
1999) and 8–9% (Johannesdottir et al., 1996; Thorlacius et al., 1997), respectively, of breast
carcinomas unselected for age at diagnosis and family history of cancer. In 1035 unselected
Finnish breast cancer patients, the combined frequency of BRCA1 and BRCA2 mutations was
14.3%, 9.8%, 5.0%, 3.1%, 2.3%, and 2.0% among patients diagnosed at the age of <35 years,
<40 years, <45 years, <50 years, <55 years, and <75 years, respectively (Syrjäkoski et al.,
2000; some of the data is unpublished). Overall, the frequency of BRCA2 mutations (1.4%)
was notably higher than that of BRCA1 mutations (0.4%) in unselected Finnish breast cancer
patients (Syrjäkoski et al., 2000).
In unselected ovarian carcinoma patients, the frequency of BRCA1 and BRCA2
mutations has been reported to be 0–27.4% and 0–13.9%, respectively (Table 3). The highest
proportion of unselected ovarian cancer patients carrying BRCA1/BRCA2 mutations has been
reported among Ashkenazi Jews (25–41%) (Moslehi et al., 2000; Tobias et al., 2000). In
admixed American, Canadian, and British populations, BRCA1 and BRCA2 mutations have
been reported in unselected ovarian carcinoma patients with frequencies varying from 2% to
9% (Stratton et al., 1997; Rubin et al., 1998; Janezic et al., 1999; Anton-Culver et al., 2000;
Risch et al., 2001; Smith et al., 2001) and 1% to 4% (Takahashi et al., 1996; Rubin et al.,
1998; Risch et al., 2001), respectively. Early-onset ovarian carcinoma does not seem to be
related to BRCA1/BRCA2 mutations, as no mutations were detected in a study of patients
diagnosed below the age of 30 years (Stratton et al., 1999).
28
Table 3. Prevalence of BRCA1 and BRCA2 germline mutations in population- and hospital-basedseries of ovarian carcinoma patients unselected for family history of cancer.
Reference Population Type of sample Age, y Mutation frequency (%)No. of
patients
Type of mutation
screening BRCA1 BRCA2 BRCA1/2Stratton etal., 1999
British Population-based <30 101 W, BRCA1;P, BRCA2 (OCCR)
0 0 0
Stratton etal., 1997
British Hospital-based <70 374 W, BRCA1 3.5
Smith et al.,2001
American(throughout)
Hospital-based 20–74 258 W, BRCA1 4.7
Takahashi etal., 1996
American(PA andtissue bank)
Hospital-based Allages
130 W, BRCA2 3.1
Anton-Culveret al., 2000
American(CA)
Population-based Allages
120 W, BRCA1(n=107);F, 7 in BRCA1(n=13)
3.3
Janezic et al.,1999
American(CA)
Population-based Allages
107 W, BRCA1 1.9
Rubin et al.,1998
American(PA)
Hospital-based Allages
116 W, BRCA1and BRCA2
8.6 0.9 9.5
Berchuck etal., 1998
American(NC)
Hospital-based Allages
103 W, BRCA1 3.9
Risch et al.,2001
Canadian(Ontario)
Population-based Allages
515 P, BRCA1 (ex11)and BRCA2 (ex 10,11);F, 7 in BRCA1and 4 in BRCA2
7.6 4.1 11.7
Tonin et al.,1999
FrenchCanadian
Hospital-based Allages
99 F, 3 in BRCA1 and4 in BRCA2
5.1 3.0 8.1
Johannesdottiret al., 1996
Icelandic Hospital-based Allages
38 F, 1 in BRCA2 7.9
van der Looijet al., 2000
Hungarian Hospital-based Allages
90 F, 3 in BRCA1and 2 in BRCA2
11.1 0
Khoo et al.,2000
Chinese Hospital-based Allages
53 forBRCA1and 48
forBRCA2
W, BRCA1;P, BRCA2 (ex 11)
11.3 2.1
Liede et al.,2002
Pakistani Hospital-based Allages
120 P, BRCA1 (ex 2,11, 12, 15, 20) andBRCA2 (ex 10, 11,22)
13.3 2.5 15.8
Tobias etal., 2000
AshkenaziJewish(USA)
Hospital-based Allages
92 F, 2 in BRCA1and 1in BRCA2
17.4 7.6 25.0
Moslehi etal., 2000
AshkenaziJewish(Israel and N.America)
Hospital-based Allages
208 P, BRCA1 (ex 2,11, 20) and BRCA2(ex 10, 11)
27.4 13.9 41.3
CA, California; ex, exon; F, founder mutation(s); N., north; NC, North Carolina; OCCR, ovarian cancer cluster region; P, partialcoding sequence; PA, Pennsylvania; W, whole coding sequence
29
6.4 Risk of cancer in BRCA1 and BRCA2 mutation carriers
The initial risk estimates, based on the large, original breast and breast-ovarian cancer
families collected for BRCA1/BRCA2 linkage analyses, were generally very high. For BRCA1
mutation carriers, the cumulative risk of female breast and ovarian cancer by the age of 70
years was estimated to be 71–87% and 42–63%, respectively (Ford et al., 1994; Easton et al.,
1995; Narod et al., 1995). For BRCA2 mutation carriers, the cumulative risk of female breast
cancer by the age of 70 years was estimated to be similar (67–84%) to that of BRCA1
mutation carriers, but the risk of ovarian cancer was estimated to be substantially lower (16–
27% by the age of 70 years) (Schubert et al., 1997; Ford et al., 1998; the BCLC, 1999). The
risk of subsequent breast and ovarian cancer has also been reported to be very high in breast
cancer patients with BRCA1 or BRCA2 mutations; 52–64% of BRCA1/BRCA2 mutation
carriers were affected with contralateral breast cancer by the age of 70 years, and 29–44% of
BRCA1 mutation carriers and 8–16% of BRCA2 mutation carriers developed subsequent
ovarian cancer by the same age (Ford et al., 1994; the BCLC, 1999; Eerola et al., 2001a). The
cumulative risk of male breast cancer by the age of 70 years has been estimated to be 3–6%
for BRCA2 mutation carriers (Easton et al., 1997; Thompson and Easton, 2001).
Studies based on population- and hospital-based series of breast/ovarian cancer patients
and unaffected individuals have reported considerably lower breast and ovarian cancer risk
estimates for BRCA1 and BRCA2 mutation carriers than studies consisting of families with
multiple cases of breast and/or ovarian cancer. In the Icelandic population, the cumulative
risk of female breast cancer for BRCA2 999del5 mutation carriers has been estimated to be
37% by the age of 70 years (Thorlacius et al., 1998). Among Ashkenazi Jews, the risk of
breast cancer by the same age has been reported to be 46–60% for BRCA1 mutation carriers
(185delAG or 5382insC) and 26–28% for carriers of the BRCA2 6174delT founder mutation
(Warner et al., 1999; Satagopan et al., 2001); the risk of ovarian cancer has been estimated to
be 16% for those who carry any of the three Ashkenazi Jewish founder mutations (Struewing
et al., 1997). Also in admixed British and Australian populations, where BRCA1/BRCA2
mutation spectra are wider, lower risk estimates have been observed (Hopper et al., 1999;
Peto et al., 1999; ABCSG, 2000; Antoniou et al., 2000, 2002); the cumulative risk of breast
cancer by the age of 70 years has been estimated to be 35–47% for BRCA1 mutation carriers
and 50–56% for BRCA2 mutation carriers. Estimates of ovarian carcinoma risk by the age of
70 years have varied between 26% and 66% for BRCA1 mutation carriers, while for BRCA2
mutation carriers, substantially lower ovarian carcinoma risk estimates (9–10% by the age of
70 years) have been reported (ABCSG, 2000; Antoniou et al., 2000, 2002).
30
In addition, several studies have suggested that the risk of prostate cancer is increased
in both BRCA1 (Arason et al., 1993; Ford et al., 1994; Struewing et al., 1997; Warner et al.,
1999) and BRCA2 mutation-associated families (Struewing et al., 1997; Thorlacius et al.,
1997; the BCLC, 1999; Johannsson et al., 1999; Warner et al., 1999; Eerola et al., 2001a).
Nevertheless, BRCA1/BRCA2 germline mutations appear to have a minor role in familial
prostate cancer (Langston et al., 1996; Sinclair et al., 2000).
The risk of cancer may vary according to the location of the mutation in BRCA1 and
BRCA2 (Gayther et al., 1995, 1997b; Risch et al., 2001; Thompson and Easton, 2001, 2002).
In BRCA1, mutations located in the first two-thirds of the coding sequence have been
suggested to be associated with a higher ovarian cancer risk relative to breast cancer risk than
mutations in the last third of the gene [optimal breakpoint between nucleotides (nt) 4422 and
4448 (Gayther et al., 1995), or at nt 4191 (Thompson and Easton, 2002)]. However, others
have observed no difference in ovarian cancer risk according to the location of the mutation,
while the risk of breast cancer has been found to increase towards the 3´ end of the gene
(Risch et al., 2001). Moreover, the breast cancer risk associated with mutations in the central
region of the BRCA1 gene (nt 2401–4190) has been reported to be lower than in mutations in
the outer two regions (Thompson and Easton, 2002). In BRCA2, mutations located in the
central portion of the gene, in an area termed the ovarian cancer cluster region (OCCR)
[optimal location either between nt 3035 and 6629 (Gayther et al., 1997b), or between nt
3059 and 4075, and 6503 and 6629 (Thompson and Easton, 2001)], have been reported to be
associated with a higher ratio of ovarian to breast cancer cases than mutations located outside
the region (Gayther et al., 1997b; Thompson and Easton, 2001). The distinctive phenotype
associated with mutations located in the OCCR relative to other BRCA2 mutations has been
suggested to be predominantly due to a reduced risk of breast cancer rather than an increased
risk of ovarian cancer (Thompson and Easton, 2001). Furthermore, the risk of prostate cancer
has been suggested to be higher for non-OCCR mutations than for mutations located within
the OCCR (34% and 19%, respectively, by the age of 80 years) (Thompson and Easton,
2001).
Evidence exists for a modifying effect of other genes as well as non-genetic factors on
the risks of breast and ovarian cancer in BRCA1 and BRCA2 mutation carriers (Hopper et al.,
1999; Antoniou et al., 2000, 2002; Nathanson and Weber, 2001), which may explain some of
the differences in risk estimates obtained from studies based on population- and hospital-
based series of breast and ovarian cancer patients and those based on families selected on the
basis of multiple occurrence of breast/ovarian carcinoma. Several association studies have
reported that allelic variation in genes involved in steroid hormone signalling pathways (e.g.,
androgen receptor and progesterone receptor genes) may affect cancer penetrance in BRCA1
31
and BRCA2 mutation carriers (Rebbeck et al., 1999a, 2001; Runnebaum et al., 2001). Non-
genetic factors that may influence breast/ovarian cancer risk of BRCA1 and/or BRCA2
mutation carriers include pregnancy (Johannsson et al., 1998; Jernström et al., 1999; Modan
et al., 2001), smoking (Brunet et al., 1998), bilateral prophylactic oophorectomy (Rebbeck et
al., 1999b, 2002) and mastectomy (Hartmann et al., 1999), and use of tamoxifen (an anti-
oestrogenic drug) (Narod et al., 2000; King et al., 2001; Duffy and Nixon, 2002).
6.5 Prediction of presence of a BRCA1/BRCA2 germline mutation in a family
As screening of BRCA1 and BRCA2 mutations is laborious and expensive, and genetic
mutation testing is emotionally stressful for families, it is important to find clinical risk
factors that could best predict the presence of a BRCA1/BRCA2 mutation in a family so that
mutation screening could be directed to potential mutation carrier families. According to the
guidelines of the American Society of Clinical Oncology (1996) and the French National Ad
Hoc Committee (Eisinger et al., 1998), the chance of detecting a mutation should be above
10% and 25%, respectively. Many studies have been performed to develop models to
estimate the likelihood that a woman carries a BRCA1/BRCA2 germline mutation based on
her personal and family history of breast and ovarian cancer (Berry et al., 1997; Couch et al.,
1997; Shattuck-Eidens et al., 1997; Frank et al., 1998; Malone et al., 1998; Parmigiani et al.,
1998; Hodgson et al., 1999; Ligtenberg et al., 1999; Ikeda et al., 2001; Loman et al., 2001;
Vahteristo et al., 2001; de La Hoya et al., 2002). In general, the stronger the family history of
breast and/or ovarian cancer and the younger the age at breast cancer diagnosis, the more
likely the family is to carry a BRCA1 or BRCA2 mutation. However, most women with a
family history of breast and/or ovarian cancer are not members of large families with multiple
cases of breast and ovarian cancer but instead have only a few affected family members
(Couch et al., 1997; Goelen et al., 1999; Ligtenberg et al., 1999; Ikeda et al., 2001).
Nevertheless, also in these cases, the presence of ovarian cancer in a family, bilateral breast
cancer, the presence of both breast and ovarian cancer in the same woman, and the age at
diagnosis of the youngest breast cancer patient are useful in predicting the presence of a
BRCA1/BRCA2 mutation (Berry et al., 1997; Couch et al., 1997; Frank et al., 1998; Malone et
al., 1998; Hodgson et al., 1999; Ligtenberg et al., 1999; Loman et al., 2001). Furthermore, the
histopathological phenotype of breast cancer has been suggested to be useful in predicting
BRCA1 mutation carrier status (Eisinger et al., 1999; Cortesi et al., 2000; Lidereau et al.,
2000).
32
6.6 Characteristics of BRCA1 and BRCA2 mutation-associated breast and ovarian
carcinomas
6.6.1 Breast carcinomas
Evidence has been found for phenotypic differences between breast carcinomas occurring in
women carrying germline mutations in BRCA1 and BRCA2 and those occurring in non-
carriers (Phillips, 2000). Both BRCA1 and BRCA2 mutation-associated breast carcinomas are
diagnosed on average at a younger age than sporadic ones (Marcus et al., 1996; Noguchi et
al., 1999), and bilateral breast carcinoma is more common among BRCA1/BRCA2 mutation
carriers than among patients with sporadic breast carcinoma (Ford et al., 1994; Easton et al.,
1995; Verhoog et al., 1998; the BCLC, 1999; Noguchi et al., 1999). BRCA1 mutation-
associated breast carcinomas are more often highly proliferating, aneuploid, and of high
histological grade than sporadic control tumours (Marcus et al., 1996; the BCLC, 1997;
Jóhannsson et al., 1997; Lakhani et al., 1998; Armes et al., 1999; Noguchi et al., 1999).
Furthermore, BRCA1 mutation-associated breast tumours have been found to be more
frequently oestrogen receptor- (ESR) and progesterone receptor- (PGR) negative, as well as
negative for the ERBB2 oncoprotein (Jóhannsson et al., 1997; Loman et al., 1998; Verhoog et
al., 1998; Armes et al., 1999; Noguchi et al., 1999). Finally, they have been observed to have
a higher frequency of sporadic TP53 mutations and/or overexpression of the TP53 protein as
compared with sporadic control tumours (Crook et al., 1998; Armes et al., 1999; Noguchi et
al., 1999; Greenblatt et al., 2001).
For BRCA2 mutation-associated breast carcinomas, no distinct phenotype has been
documented (Armes et al., 1999; Noguchi et al., 1999; Phillips, 2000). Nevertheless,
compared with BRCA1 mutation-associated tumours, they tend to be more often ESR- and
PGR-positive (Loman et al., 1998; Armes et al., 1999; Verhoog et al., 1999, Duffy, 2002
#1650), and show no clear increase in the frequency of TP53 mutations and/or
overexpression of the TP53 protein (Armes et al., 1999; Noguchi et al., 1999; Phillips, 2000).
A comparative genomic hybridization (CGH) study has shown that the total number of
somatic genetic alterations is almost two times higher in breast tumours from both BRCA1
and BRCA2 mutation carriers than in control tumours (Tirkkonen et al., 1997). Furthermore,
BRCA1 and BRCA2 tumours have been found to be characterized by distinct CGH changes,
suggesting that specific genetic pathways may operate in the progression of these tumours
(Tirkkonen et al., 1997). Recently, using complementary DNA (cDNA) microarray
technology, Hedenfalk et al. (2001) determined global gene expression profiles in BRCA1
and BRCA2 mutation-associated breast carcinomas as well as in sporadic tumours and found
33
that these three groups of tumours expressed significantly different groups of genes and could
thus be distinguished from each other.
6.6.2 Ovarian carcinomas
Both BRCA1 and BRCA2 mutation-associated ovarian carcinomas are typically of serous
histology, moderate to high grade, and advanced stage (Rubin et al., 1996; Aida et al., 1998;
Boyd et al., 2000; Moslehi et al., 2000; Werness et al., 2000; Risch et al., 2001). Compared
with sporadic carcinomas, mucinous subtype is underrepresented in BRCA1 and BRCA2
mutation carriers (Boyd et al., 2000; Moslehi et al., 2000; Werness et al., 2000; Risch et al.,
2001). Moreover, epithelial ovarian tumours of low malignant potential, i.e., borderline
ovarian tumours, do not seem to be associated with BRCA1 or BRCA2 mutations (Gotlieb et
al., 1998; Werness et al., 2000; Risch et al., 2001). In BRCA1 mutation carriers, ovarian
carcinomas are diagnosed on average at a younger age than sporadic ones, while for BRCA2
mutation-associated ovarian carcinomas, the mean age at diagnosis does not differ from that
of sporadic tumours (Boyd et al., 2000; Moslehi et al., 2000; Risch et al., 2001).
Somatic alterations of the TP53 gene and/or overexpression of the TP53 protein have
been suggested to be more common in BRCA1/BRCA2 mutation-associated ovarian
carcinomas than in sporadic tumours (Ramus et al., 1999; Buller et al., 2001). Nevertheless,
some have found no difference in TP53 expression between BRCA1/BRCA2 mutation-
positive and -negative ovarian tumours (Zweemer et al., 1999; Ravid et al., 2000). Mutations
of KRAS and amplification of ERBB2, MYC, and AKT2, which are commonly seen in
sporadic ovarian carcinoma, have not been observed in BRCA1/BRCA2 mutation-associated
ovarian carcinomas (Rhei et al., 1998; Tanner et al., 2000).
A study using CGH has reported genetic similarity between BRCA1/BRCA2 mutation-
associated and sporadic ovarian carcinomas of serous histology both in total number of
somatic genetic alterations and their location, suggesting a common main pathway in tumour
progression (Tapper et al., 1998). Recently, a cDNA microarray study of BRCA1/BRCA2
mutation-positive and -negative ovarian tumours of similar stage, grade, and histology
observed a great contrast in gene expression profiles betweeen tumours of BRCA1 mutation
carriers and those of BRCA2 mutation carriers (Jazaeri et al., 2002). Among sporadic
tumours, ”BRCA1-like” and ”BRCA2-like” subgroups were seen, suggesting that mutations in
BRCA1 and BRCA2 may lead to carcinogenesis through distinct molecular pathways that also
appear to be involved in sporadic cancers (Jazaeri et al., 2002).
34
6.7 Prognosis of patients with BRCA1 or BRCA2 mutation-associated breast or ovarian
carcinoma
Most studies on survival of breast cancer patients with BRCA1 mutations (Gaffney et al.,
1998; Jóhannsson et al., 1998; Verhoog et al., 1998; Eerola et al., 2001b) or BRCA2
mutations (Gaffney et al., 1998; Verhoog et al., 1999, 2000; Eerola et al., 2001b) have
suggested that their prognosis does not differ significantly from that of sporadic cases.
For ovarian carcinoma, conflicting results have been presented, with BRCA1 or BRCA2
mutation-associated patients proposed to have better (Rubin et al., 1996; Aida et al., 1998;
Boyd et al., 2000; Ben David et al., 2002), equal, or worse prognosis (Jóhannsson et al.,
1998; Pharoah et al., 1999) than sporadic patients.
7 Population history of Finland and its influence on the Finnish gene pool
7.1 Settlement in Finland
According to the dual-origins hypothesis, Finland was inhabited by two main migratory
waves: an earlier wave of eastern Uralic speakers from about 4000 years ago was followed by
a wave about 2000 years ago from the south via the Gulf of Finland (Kittles et al., 1998;
Peltonen et al., 1999). Nevertheless, it seems more likely that Finland has been inhabited
continuously since the last glacial period around 10 000 years ago by small immigrant groups
mainly from the south and east but also from the west (Jutikkala and Pirinen, 1996; Peltonen
et al., 1999; Norio, 2000). Both Y-chromosomal haplotypes and mitochondrial sequences
show a decrease in genetic diversity of the Finns as compared with other European
populations, indicating a bottleneck in the founding of the Finnish population (Sajantila et al.,
1996).
Agriculture arrived in Finland approximately 4000 to 5000 years ago (Jutikkala and
Pirinen, 1996), and it first spread slowly into the southern and western coastal areas. The
early settlement region (Figure 1) has had permanent inhabitation for at least 2000 years,
while the settling of the central and northern parts of the country (late settlement region)
began only in the 16th century (Nevanlinna, 1972; Norio et al., 1973). The spread of the
population towards the east and north occurred partly due to a royal decree that ordered
people from the region of South Savo to settle in uninhabited areas. This resulted in repeated
sampling of small numbers of settlers from the main population (multiple founder effects)
and formation of small, rural subisolates which remained relatively stable until the Second
35
World War and industrialization (Nevanlinna, 1972; Norio et al., 1973; Peltonen et al., 1999).
Since then, migration to towns has increased, but gene flow between rural communities has
remained limited (Norio et al., 1973). Wars, great famine (during 1696–1698), and epidemics
prevented continuous population growth until the 18th century, after which the population
expanded rapidly from 250 000 to its present size of about 5.1 million (Norio et al., 1973;
Peltonen et al., 1999). Due to linguistic, religious, cultural, and geographical barriers, the
main expansion of the Finnish population occurred in remarkable isolation (Nevanlinna,
1972; Norio et al., 1973). This rapid expansion of the Finnish population in a subisolate
structure allowed random genetic drift to play with allele frequencies, resulting in allele
enrichments and losses in rural subisolates (Nevanlinna, 1972; Norio et al., 1973, 2000).
Figure 1. Map of Finland indicating the early and late settlement areas. The early settlement regionhas had permanent inhabitation for at least 2000 years, while the late settlement region was inhabitedonly after the 16th century by internal migration movement mainly from the region of South Savo.Adapted from Norio et al. (1973).
7.2 Finnish disease heritage
The population history of Finland is reflected in the unique disease pattern known as ”the
Finnish disease heritage”. This concept was introduced in 1973 by Drs. Norio, Nevanlinna,
and Perheentupa (Norio et al., 1973), and it refers to diseases that are more prevalent in the
Finnish population than in other populations. On the other hand, some diseases that are
common elsewhere are extremely rare in Finland. At present, more than 30 monogenic
Earlysettlement
Latesettlement
Savo
36
disorders are included in the Finnish disease heritage. Most disorders are autosomal
recessive, but two autosomal dominant, and two X-chromosomal recessive disorders have
been described (de la Chapelle and Wright, 1998; Norio, 2000; Kere, 2001). To date, all but
two of the disease loci have been mapped, and the gene and its common mutations are known
for 22 diseases (Kere, 2001). Diseases of the Finnish disease heritage are typically caused by
one major mutation originating from a common ancestor, i.e., a founder mutation, but for
some diseases, multiple founder mutations have been described (Peltonen et al., 1999; Kere,
2001). Distinct geographical clustering of affected individuals is indicative of founder effects
and is seen in many diseases of the Finnish disease heritage even today. Nevertheless, the
most common recessive disorders are fairly equally distributed throughout the country, or
their distribution closely resembles the present-day population density of Finland (Peltonen et
al., 1995, 1999; Norio, 2000). It is noteworthy that there appears to be some clustering of
these diseases, reflecting regional subisolates (Norio, 2000).
7.3 Inherited diseases other than those included in the Finnish disease heritage
Also in inherited diseases that are not overrepresented in Finland as compared with other
populations, distinct founder effects have been described (Kere, 2001). For example, in
familial hypercholesterolemia, four Finnish founder mutations in the low-density lipoprotein
receptor gene are responsible for three-quarters of the cases in Finland, and the distribution of
these mutations varies in different parts of the country (Koivisto et al., 1992, 1995). In
HNPCC, two founder mutations in the MLH1 gene account for approximately half of all
Finnish HNPCC families (Nyström-Lahti et al., 1995, 1996), and distinct geographical
clustering of the ancestral origins of families with these mutations has been observed (Moisio
et al., 1996).
7.4 Linkage disequilibrium and haplotypes
Founder mutations are characterized by linkage disequilibrium (LD), i.e., non-random
association of alleles at closely linked loci. Due to recombination, LD gradually decays as a
function of the number of generations, and the time required for disappearance of LD
between alleles depends crucially on the genetic distance between the loci (Jorde, 1995).
Around disease alleles of the Finnish disease heritage, significant LD has been detected over
large genetic distances, varying between 2 and 13 centiMorgans (cM) (Peltonen et al., 1999).
37
Moreover, in HNPCC, marked LD over a genetic distance of 18 cM around a MLH1 mutant
allele has been reported in Finnish families (Moisio et al., 1996). Although LD can be
observed over wide genetic intervals, identical haplotypes (combinations of alleles) around
disease alleles in affected individuals from different families are systematically found only
across very restricted areas (Peltonen et al., 1999; Kere, 2001). For example, carriers of the
Finnish MLH1 founder mutation shared an identical haplotype of 2 cM (Moisio et al., 1996).
LD mapping and monitoring of shared haplotypes have been successfully used in fine-
mapping of disease loci in the Finnish population (Hästbacka et al., 1992, 1994; Lehesjoki et
al., 1993; Nyström-Lahti et al., 1994; Höglund et al., 1995; Schleutker et al., 1995; Peltonen
et al., 2000a). Power to detect LD tends to be greatest when a single mutant allele that
accounts for a large proportion of affected individuals has arisen recently on a relatively
uncommon haplotype background. Locus and allelic heterogeneity, which is common in
complex diseases, decreases the power to detect LD (Jorde, 2000). LD is also influenced by
numerous forces, e.g., mutation, natural selection, gene flow, demographic history,
chromosomal location, and gene conversion, which complicate the relationship between LD
and physical distance (Laan and Pääbo, 1997; Zavattari et al., 2000; Daly et al., 2001;
Pritchard and Przeworski, 2001). Finnish subpopulations have been used in the genetic
studies of multifactorial diseases, such as multiple sclerosis (Kuokkanen et al., 1997),
schizophrenia (Hovatta et al., 1999; Ekelund et al., 2000; Paunio et al., 2001), and asthma
and atopy (Laitinen et al., 2001). In most cases, these linkage-based approaches have resulted
in the initial positioning of several susceptibility loci, but no individual genes have yet been
identified based on these studies.
The extent of LD around mutation alleles can give some clues of the time elapsed since
a mutation was introduced into a population (Peltonen et al., 1995; de la Chapelle and
Wright, 1998). To estimate the age of a mutation, several statistical approaches have been
developed (Slatkin and Rannala, 2000; Rannala and Bertorelle, 2001). The equation of Luria
and Delbrück (1943), originally introduced to estimate mutation rates in rapidly growing
bacterial cultures, has been adapted to estimate genetic distances between disease loci and
linked markers in the Finnish population (Hästbacka et al., 1992, 1994; Lehesjoki et al.,
1993; Höglund et al., 1995; Schleutker et al., 1995). The same equation can be applied to
estimate the age of a mutation since its appearance in the Finnish population (Höglund et al.,
1996), the expansion of which is considered to have occurred rapidly and in remarkable
isolation (Nevanlinna, 1972; Norio et al., 1973). Distribution of birthplaces of mutation
carriers’ ancestors is also of importance in estimating the time elapsed since a mutation was
introduced into a population (Peltonen et al., 1995; Norio, 2000). The most common
disorders of the Finnish disease heritage that are more or less equally distributed throughout
38
the country (e.g., aspartyl-glucosaminuria and congenital nephrosis) show significant LD
spanning only 2–3 cM, and these mutations may have been introduced into the Finnish
population thousands of years ago (Peltonen et al., 1995, 1999; Norio, 2000). In contrast,
diseases with more restricted geographical distribution show LD extending over larger
genetic distances (10–13 cM) (Peltonen et al., 1995, 1999; Norio, 2000). For instance,
mutations that cause congenital chloride diarrhoea and variant late infantile neuronal ceroid
lipofuscinosis have been estimated to have started to spread in Finnish subpopulations 13–30
generations (i.e., ca. 400–500 years) ago (Höglund et al., 1996; Varilo et al., 1996). In
HNPCC, where LD extended 18 cM around a MLH1 mutant allele and affected members of
different families shared a common haplotype of 2 cM, the spread of the mutation was
estimated to have started 16–43 generations ago (Moisio et al., 1996).
39
AIMS OF THE STUDY
At the time this work began, 11 distinct germline mutations in BRCA1 and seven in BRCA2
had been identified in Finnish breast and breast-ovarian cancer families (Vehmanen et al.,
1997a, 1997b; Huusko et al., 1998). Of these 18 different BRCA1/BRCA2 mutations, 11 had
been described in more than one Finnish family. These recurrent mutations accounted for the
vast majority (84%) of all BRCA1 and BRCA2 mutations found in the screening of the entire
coding regions of the genes (Vehmanen et al., 1997a, 1997b; Huusko et al., 1998).
The aims of this thesis were to study:
1) ancestral origins and geographical distribution of families with recurrent Finnish
BRCA1/BRCA2 germline mutations (I, II)
2) breast and ovarian cancer phenotypes of Finnish BRCA1 and BRCA2 germline mutation
carriers (I)
3) the prevalence of BRCA1 and BRCA2 founder mutations in Finnish ovarian carcinoma
patients (III) and ovarian carcinoma families (IV)
40
MATERIALS AND METHODS
1 Ethical issues
Appropriate research permissions were obtained from the Ministry of Social Affairs and
Health in Finland, and from the Ethics Committees of the Department of Obstetrics and
Gynaecology, and the Department of Oncology, Helsinki University Central Hospital,
Finland. All index patients and family members donating blood samples signed a written
informed consent.
2 Patients and families (I-IV)
BRCA1 and BRCA2 mutation-positive Finnish families included in Studies I and II had been
identified at the University Hospitals of Helsinki, Kuopio, Oulu, and Tampere (Vehmanen et
al., 1997a, 1997b; Huusko et al., 1998; Syrjäkoski et al., 2000; Vahteristo et al., 2001). In
Study I, 34 BRCA1 and 37 BRCA2 mutation-positive families were examined for breast and
ovarian cancer phenotypes (Table 4). Families with recurrent BRCA1/BRCA2 mutations
(n=55) were studied for mutation-associated haplotype conservation (I, II). The number of
families with each recurrent mutation is indicated in Table 4. In addition to the Finnish
families, 18 Icelandic families with the BRCA2 999del5 mutation were included in Study II.
The Icelandic families had been identified by the research group of Dr. R. B. Barkardóttir at
the University Hospital of Iceland. For comparison of the mutation-associated haplotypes
between Finnish and Swedish BRCA1 3744delT families, and between Finnish and Austrian
BRCA1 5370C→T families, samples from four Swedish families identified by Zelada-
Hedman et al. (1997) and from one Austrian family identified by Wagner et al. (1998) were
included in Study I.
41
Table 4. Number of Finnish BRCA1/BRCA2 mutation-positive families studied for breast and ovariancancer phenotypes (I), and mutation-associated haplotype conservation (I, II), as well as mutationsscreened and methods used in mutation screening of unselected ovarian carcinoma patients (III) andovarian carcinoma families (IV).
Gene and mutation Mutationtype
No. of familiesincluded in the
phenotypeanalysis (I)
No. of familiesincluded in the
haplotypeanalysis(I or II)d
Screened in 233unselected
ovariancarcinoma
patients (III)
Screened in23 ovariancarcinoma
families (IV)
BRCA1Ex 11, 1806C→T Nonsense - - No Yese
Ex 11, 1924delA Frameshift 1 - Yesf Yese
Ex 11, 2803delAAa Frameshift 2 - Yese Yese
Ex 11, 3264delT Frameshift - - Yese Yese
Ex 11, 3604delA Frameshift 5 5 (I) Yesf Yese
Ex 11, 3744delTb Frameshift 7 7 (I) Yesf Yese
Ex 11, 3904C→A Nonsense 1 - Yesf Yese
Ex 11, 4153delAc Frameshift 1 - Yesf Yese
Int 11, 4216nt-2A→G Splice site 9 8 (I) Yesf Yese
Ex 13, 4446C→T Nonsense 3 3 (I) Yese Yese
Ex 17, 5145del11 Frameshift 1 - Yesg Yese
Ex 20, 5370C→T Nonsense 3 2 (I) Yese Yese
Ex 20, 5382insC Frameshift 1 - Yese Yese
Total 34 Total 25BRCA2Ex 9, 999del5 Frameshift 13 10 (II) Yesg Yese
Ex 11, 4081insA Frameshift 1 - Yese Yese
Ex 11, 5797G→T Nonsense - - Yesf Yese
Ex 11, 6495delGCA→C Frameshift 1 - Yese Yese
Ex 11, 6503delTT Frameshift 3 2 (I) Yese Yese
Ex 15, 7708C→T Nonsense 8 7 (I) Yese Yese
Ex 18, 8555T→G Nonsense 4 4 (I) Yesf Yese
Int 23, 9346nt-2A→G Splice site 7 7 (I) Yesf Yese
Total 37 Total 20 (I),and 10 (II)
ex, exon; int, intron; aAlso known as 2804delAA (the BIC database); bAlso known as 3745delT (the BICdatabase); cAlso known as 4154delA (the BIC database);dThe recurrent BRCA1 2803delAA mutation was notincluded in the haplotype analysis as no samples were available; Mutation detection was performed by eallele-specific oligonucleotide (ASO) hybridization, frestriction fragment length polymorphism (RFLP) analysis, orgagarose gel electrophoresis
In Study III, the prevalence of BRCA1 and BRCA2 mutations was studied in 233
unselected Finnish ovarian carcinoma patients who belonged to a cohort of 573 epithelial
ovarian carcinoma patients treated at the Department of Obstetrics and Gynaecology,
Helsinki University Central Hospital, Finland, during 1989–1998. Blood samples were
collected from the patients in 1997 and 1998 in conjunction with routine check-up visits that
occur at least once a year for a time period of 10 years after the initial treatment. At the end of
the year 1998, 220 of the 573 patients were alive, and the study cohort covered 91% of the
patients who were alive during the study period.
In Study IV, BRCA1 and BRCA2 mutations were screened in 23 Finnish ovarian
42
carcinoma families with at least two cases of epithelial ovarian carcinoma in first-degree
relatives. The families had been identified in a population-based study on cancer incidence in
first-degree relatives of Finnish ovarian carcinoma patients as described by Auranen et al.
(1996b). Briefly, all women with epithelial ovarian carcinoma diagnosed from 1980 to 1982
who were under the age of 76 years were selected from the FCR (n=863). The first-degree
relatives of these patients were identified through local parish registers and from the
Population Register Centre, and information on their cancer diagnoses was obtained from the
FCR. Data on all first-degree relatives was obtained for 559 women, 27 of which had at least
one first-degree relative with epithelial ovarian carcinoma. Formalin-fixed, paraffin-
embedded tumour and normal tissue blocks from cancer patients belonging to these 27
families had been collected from different hospitals and pathology laboratories. For
BRCA1/BRCA2 mutation analyses, samples were available from 51 individuals belonging to
23 of these 27 families. Of these 51 individuals, 41 had ovarian carcinoma, five had breast
cancer, and five had some other cancer (borderline ovarian cancer, melanoma, or cancer of
the bladder or pancreas). Additionally, blood samples were available from three sisters with
epithelial ovarian carcinoma and from their healthy brother. Fourteen of the families were
site-specific ovarian carcinoma families: 11 with two affected cases, and three with three
affected cases; breast cancer was present in nine families.
3 Previously identified BRCA1 and BRCA2 germline mutations studied here (I-IV)
The previously identified BRCA1 and BRCA2 mutations analysed in Studies I-IV are
indicated in Table 4 (Vehmanen et al., 1997a, 1997b; Huusko et al., 1998; Syrjäkoski et al.,
2000; Vahteristo et al., 2001).
4 Collection of cancer and genealogical data (I-IV)
For Study I, information on breast and ovarian cancer diagnoses and ages at onset in the 34
BRCA1 and 37 BRCA2 mutation-positive families was obtained by family questionnaires and
from hospital records and death certificates. All female breast and ovarian cancer cases in
first- and second-degree relatives were included in the phenotype analysis, except for those
who were known not to be BRCA1 or BRCA2 mutation carriers.
For the 55 Finnish families that were studied for mutation-associated haplotype
conservation (I, II), genealogical data (extending preferentially at least to grandparents or
43
great-grandparents of the index patients) were obtained by interviewing the index patients
and through church parish registers and the Population Register Centre. For the 18 Icelandic
families studied for 999del5 mutation-associated haplotype conservation (II), pedigree data
were obtained from the Icelandic Genetic Council at the University Hospital of Iceland, and
cancer diagnoses were verified through the Department of Pathology, University Hospital of
Iceland, and from the Icelandic Cancer Registry.
For the 233 unselected ovarian carcinoma patients (III), information on age at ovarian
carcinoma diagnosis, FIGO stage, tumour histology, and grade were collected from hospital
records. In case of inconsistency in the information, tumour specimens obtained from
different hospitals and pathology laboratories were reviewed for tumour histology and grade.
Distribution of clinical and tumour characteristics in our study cohort and in the general
Finnish ovarian carcinoma patient population are shown in Table 5. Information on other
cancers in index patients and on cancer cases in the first- and second-degree relatives of the
index patients were collected by questionnaire interviews.
For the 23 Finnish ovarian carcinoma families (IV), information on cancer diagnoses of
the index patients and their first-degree relatives had been obtained from the FCR (Auranen
et al., 1996b). The FIGO stage, tumour histology, grade, and ploidy were extracted from a
study by Auranen et al. (1997).
Table 5. Clinical and tumour characteristics of the 233 unselected ovarian carcinomapatients (III) and of the general ovarian carcinoma patient population.
Variable Our study cohort(n=233)
General ovariancarcinoma patient
populationa
Stage available 133 (57%)I 60 (45%) 27%II 8 (6%) 13%III 57 (43%) 47%IV 8 (6%) 13%
Grade available 155 (67%)1 69 (44%) 27%2 38 (25%) 20%3 48 (31%) 53%
Histology available 233 (100%)Serous 118 (51%) 45%Mucinous 49 (21%) 13%Endometrioid 33 (14%) 10%Mesonephroid 19 (8%) 9%Poorly differentiated 14 (6%) 23%
Mean+SD age at ovariancarcinoma diagnosis, range
55+13 years,23–96 years
58+13 years
SD, standard deviation; aAccording to the study by Venesmaa (1994)
44
5 Extraction of DNA (I-IV)
Genomic DNA was extracted from blood leucocytes using standard phenol-chloroform
methods (I-IV) and from formalin-fixed, paraffin-embedded tumour and normal tissue blocks
(IV) according to the protocol described by Isola et al. (1994).
6 Genotyping (I, II)
For genotyping of the 25 Finnish families carrying recurrent BRCA1 mutations, 11
polymorphic microsatellite markers spanning a genetic interval of 26 cM around BRCA1
were used (I). Markers and their genetic map distances (cM) are shown in Figure 2a [Weber
et al., 1990; Futreal et al., 1992; Feunteun et al., 1993; Albertsen et al., 1994; Dib et al., 1996;
Murrell et al., 1997; the Genome Database (GDB); National Center for Biotechnology
Information (NCBI)]. Sequences of the PCR primers were obtained from the GDB. The PCR
products were labelled with [α-32P]deoxycytidine triphosphate (dCTP) (DuPont/NEN),
electrophoresed on denaturing 7% polyacrylamide gels, and visualized by exposing the gels
to autoradiography. Allele sizes were determined by the M13mp18 marker (Sequenase kit,
United States Biochemical) on each gel.
45
Figure 2. Marker order and map distances in centiMorgans (cM).a) Markers used in genotyping of the Finnish BRCA1 mutation-positive families (I).b) Markers used in genotyping of the Finnish BRCA2 mutation-positive families; all 24 markers wereused in genotyping of the families with the 999del5 mutation (II), while markers indicated by anasterisk (n=17) were used in genotyping of the families with other recurrent BRCA2 mutations (I).c) Markers used in genotyping of the Icelandic BRCA2 999del5 mutation-positive families (II); all 28markers were used in genotyping of the five large, high-risk breast cancer families, while five markersindicated by an asterisk were used in genotyping of the 13 pairs of sisters with breast cancer.cen, centromere; ex, exon; tel, telomere; THRA1, avian erythroblastic leukemia viral (v-erb-a)oncogene homolog
For genotyping of the 30 Finnish BRCA2 families (I, II), polymorphic markers spanning
a genetic interval of 36 cM around BRCA2 were used; 24 markers were used for the 10
families with the 999del5 mutation (II), and 17 markers were used for the 20 families with
other recurrent BRCA2 mutations (I) (Figure 2b) (Couch et al., 1996; Dib et al., 1996;
Généthon; the GDB; NCBI). For marker loci D13S260 through D13S267, marker order and
physical distances were determined using the genomic sequence of this region (the Sanger
Centre), and PCR primer sequences were positioned with Sequencher v3.0 (Gene Codes
Corporation). The physical distances were converted to genetic distances assuming 1 cM =
Marker Marker Markercen cen cenD17S1872 D13S283* D13S283D17S946 D13S1294* D13S217D17S250 D13S217* D13S1246*THRA1 D13S1246* D13S290D17S800 D13S260* D13S1226D17S846 SLS312 SLS320D17S855 BRCA1 D13S1699* SLS385D17S902 D13S1698* SLS165D17S579 SLS163 D13S260*D17S588 D13S1697* SLS312D17S787 SLS329 D13S1699tel EX11 D13S1698
D13S1701 SLS163SLS321 D13S1697D13S171* SLS329SLS886 EX 11D13S1695* D13S1701D13S1694* SLS321D13S1696* D13S171*D13S267* SLS886D13S263* D13S1695D13S1227* D13S1694D13S1272* SLS234D13S153* D13S267*tel D13S1293
D13S219D13S220*D13S263tel
Finnish families Icelandic familiesa) b) c)
0.41.16.08.0
1.20.60.72.03.03.0
cM
BRCA20.024
0.080.130.240.230.090.32.93.74.43.1
0.530.170.080.140.480.570.040.111.51.32.03.8
cM
BRCA20.024
cM
0.53
0.080.17
0.14
0.570.48
0.080.130.240.230.090.3
2.9
3.77.5
0.14
1.9
9.6
46
0.5 megabases (Mb), which was the observed average recombination ratio at this region. In a
haplotype analysis of recurrent BRCA2 mutations, Neuhausen et al. (1998) used an
assumption of 1 cM = 0.67 Mb between marker loci D13S290 and D13S267, which is similar
to the ratio used here. For the other markers, map distances were obtained from Généthon
(NCBI). Primer sequences for all other D13S markers except D13S1696, which was obtained
from Couch et al. (1996), were obtained from the GDB. For the SLS markers, primer
sequences were kindly provided by Dr. M Stratton. Genotyping was carried out with a
fluorescent technique using an ABI377 instrument according to instructions provided by
Applied Biosystems. Allele sizes were matched based on the Centre d’Etude du
Polymorphisme Humain (CEPH) reference individual 1347-02 (Applied Biosystems), which
was used as a control on each gel. Genotype data were analysed by GeneScan v3.1 and
Genotyper v2.0 software (Applied Biosystems).
For genotyping of the 18 Icelandic families with the BRCA2 999del5 mutation (II),
polymorphic markers covering a 29 cM interval around BRCA2 were used (Figure 2c) (Couch
et al., 1996; Dib et al., 1996; Généthon; the GDB; NCBI). Of the 18 Icelandic families, five
were large, high-risk breast cancer families and 13 were pairs of sisters diagnosed with breast
cancer by the age of 60 years. For genotyping of the five large families, 28 markers were
used, while for genotyping of the families with an affected sister pair, five of these markers
were used (Figure 2c). Primer sequences, as well as marker order and map distances, were
obtained as described above. Genotyping was carried out using the protocol described by
Barkardottir et al. (1995). For direct comparison of allele sizes between the Finnish and
Icelandic 999del5 mutation-positive families, a few mutation carriers from the Icelandic
families were genotyped using the same technique used for Finnish families.
To estimate the corresponding marker allele frequencies in the general Finnish and
Icelandic populations, 42 and 96 Finnish population controls were genotyped for markers
within and flanking the BRCA1 and BRCA2 genes, respectively, and 50 Icelandic population
controls were genotyped for markers within and flanking the BRCA2 gene. To estimate the
population prevalence of the Icelandic 999del5 core haplotype, 14 Icelandic parents-child
control trios were genotyped.
7 Haplotype construction (I, II)
The haplotypes within the families were constructed with the Genehunter program (Kruglyak
et al., 1996). The haplotypes were proofread and some were reconstructed manually due to
Genehunter’s limited capability to assign haplotypes in families with complex structure. If the
47
mutation-associated haplotype could not be constructed in a family (when samples were not
available from several family members), genotypes were compared with the mutation-
associated haplotypes of the other families with the same mutation. The history of
recombinations between the families was reconstructed by assuming minimum diversity of
haplotypes; by starting from the site of the mutation and moving outwards in both directions,
historical recombinations were noted as the branching of the haplotype when two or more
different alleles were observed for a marker, thus creating a haplotype reconstruction tree.
8 Detection of BRCA1 and BRCA2 germline mutations (III, IV)
8.1 Screening for previously identified mutations (III, IV)
8.1.1 Allele-specific oligonucleotide (ASO) hybridization (III, IV)
In the ASO hybridization method, two ASO probes, one for the mutated sequence and
another for the wild-type sequence, are designed for each mutation. Mutation detection is
based on appropriate hybridization conditions in which only the probe that is a perfect match
will anneal to PCR-amplified DNA sequences (Saiki et al., 1986). The ASO hybridization
protocol described by Friedman et al. (1995) was used to screen for known BRCA1/BRCA2
mutations in unselected ovarian carcinoma patients (III) and in ovarian carcinoma families
(IV). The mutations that were screened using ASO are indicated in Table 4. Briefly, genomic
DNA was amplified by PCR, and PCR products were denatured, transferred to nylon filters
(DuPont/NEN) using a 96-well dot-blot vacuum apparatus (Bio-Dot SF, Bio-Rad), and fixed
by exposure to ultraviolet light. Two filters were prepared for detection of each mutation: one
for hybridization of the probe with the wild-type sequence and the other for the hybridization
of the probe with the mutated sequence. The 18-bp-long ASO probes were designed such that
the mutation site was located in the middle of the probe [GenBank identification numbers for
BRCA1 and BRCA2 cDNA sequences: U14680 and U43746, respectively (NCBI)]. The
probes were end-labelled with [α-32P]dCTP (DuPont/NEN) using terminal transferase
enzyme (Boehringer Mannheim) according to the manufacturer’s instructions. Hybridization
reactions were carried out at 54oC in a rotating hybrization oven (Thermo Hybaid). After
prehybridization (30–60 min), the ASO probes were added to hybridization solutions, and
after incubation (3–4 h), the filters were washed and exposed to X-ray films at -80oC for 3–18
h.
48
8.1.2 Restriction fragment length polymorphism (RFLP) analysis (III, IV)
RFLP analysis is based on the creation or destruction of a restriction enzyme cleavage site by
a mutation (Botstein et al., 1980). Here, RFLP was used to screen for nine of the known
BRCA1/BRCA2 mutations in unselected ovarian carcinoma patients (III) (Table 4). In Study
IV, RFLP was used to confirm mutations that had been detected by ASO. Briefly, genomic
DNA was amplified by PCR, PCR products were digested with appropriate restriction
enzymes (Table 6), and digestion products were analysed on 3% ethidium bromide stained
agarose gels. The RFLP analyses were designed such that incomplete digestion would lead to
a false-positive result, hence minimizing the possibility of a false-negative result.
Table 6. Restriction endonucleases and respective digestion conditions for the detectionof BRCA1 and BRCA2 mutations by RFLP.
Mutations
BRCA1 BRCA2
Enzymes Digestion conditions
1924delA, 3744delT, 3904C→A Tsp509Ia 65oC, 16 h3604delA, 4153delA MboIIa 37oC, 1 h4216nt-2A→G 8555T→G MseIa 37oC, 16 h
5797G→T NlaIIIa 37oC, 16 h9346nt-2A→G BfmIb 37oC, 16 h
RFLP, restriction fragment length polymorphism; aNew England Biolabs; bMBI Fermentas
8.1.3 Agarose gel electrophoresis (III)
Mutations that were deletions of several base pairs, i.e., BRCA1 5145del11 and BRCA2
999del5, were detected using agarose gel electorophoresis. Genomic DNA was amplified by
PCR, and PCR products were run on 3% ethidium bromide stained agarose gels.
8.2 Scanning for novel mutations (III)
8.2.1 Protein truncation test (PTT) (III)
In PTT, modified forward primers that contain a T7 RNA polymerase promoter sequence and
an eukaryotic translation initiation sequence are used in PCR, and proteins are synthesized in
a coupled in vitro transcription/translation reaction and analysed by gel electrophoresis (Roest
et al., 1993). Here, PTT was used to screen for protein-truncating mutations in BRCA1 exon
49
11 and BRCA2 exons 10 and 11 in 38 ovarian carcinoma patients (III), who reported (1) two
or more first- or second-degree relatives diagnosed with breast and/or ovarian cancer, (2) one
first-, second-, or third-degree relative with ovarian cancer, or (3) one first-degree relative
with breast cancer, or with both breast and ovarian cancer, and/or had themselves been
diagnosed with both cancers. BRCA1 exon 11 was amplified in three partly overlapping
fragments, BRCA2 exon 10 was amplified in one fragment, and BRCA2 exon 11 was
amplified in five partly overlapping fractions with previously published primers (Hogervorst
et al., 1995; Friedman et al., 1997). The PTT reactions were carried out using the TNT® T7
Coupled Reticulocyte Lysate System (Promega) according to the manufacturer’s instructions,
with [35S]methionine (Amersham Biosciences) as a radioactive label. The proteins were run
on 12.5% sodium dodecyl sulphate polyacrylamide gels and visualized by exposing the dried
gels to X-ray films at -80oC for 4–72 h.
8.2.2 Southern blot hybridization (III)
Southern blot hybridization can be used to study large genomic rearrangements,
amplifications, or deletions with sequence-specific probes (Southern, 1975). Southern
analysis using BRCA1 specific probes was performed on 11 ovarian carcinoma patient
samples and two healthy population control individuals (III) according to the protocol
published by Petrij-Bosch et al. (Petrij-Bosch et al., 1997; the BIC database) with slight
modifications (III). Briefly, genomic DNA (5 µg) was completely digested with EcoRI
restriction enzyme (New England Biolabs), separated on an 0.8% agarose gel, denatured, and
transferred onto a nylon membrane (Hybond-N+, Amersham Biosciences). The hybridization
probes were obtained by amplifying cloned, full-length BRCA1 cDNA (pcBRCA1-385, a kind
gift from Dr. L Brody) in three partly overlapping regions with primers published in the
original article (III), and by labelling purified PCR products with [α-32P]dCTP
(DuPont/NEN) using Rediprime DNA labelling system according to instructions provided by
Amersham Biosciences. Hybridizations were performed at 65oC in a rotating hybrization
oven (Thermo Hybaid); after prehybridization (40–60 min), denatured probes were added to
hybridization solutions, and after incubation (over night), the filters were washed and
exposed to autoradiography film at -80oC for 16–18 h.
50
8.3 Direct sequencing (III, IV)
Mutations detected by ASO, RFLP, or agarose gel electrophoresis were confirmed by direct
sequencing using an ABI Prism 310 Genetic Analyser and Dye Terminator Cycle Sequencing
Ready Reaction Kit according to instructions provided by Applied Biosystems. All samples
with varying bands in PTT were sequenced as well. For direct sequencing, samples were
reamplified from genomic DNA.
9 Statistical methods (I-IV)
9.1 General (I, III, IV)
Variation in the age at breast and ovarian cancer diagnosis was analysed by unpaired t-test (I,
IV). Fisher’s exact test and χ2 test were used (1) to compare the proportions of ovarian
carcinoma between BRCA1 and BRCA2 mutation-positive families (I), (2) to study the
correlation between the location of the mutation (5´ versus 3´ end of the gene) and the
proportion of ovarian carcinoma in BRCA1 mutation-positive families (I), (3) to examine the
association between BRCA1/BRCA2 mutation carrier status and young age (<50 years) at
breast cancer diagnosis (III), and (4) to determine associations between mutation carrier status
and various clinicopathological parameters of ovarian carcinomas (IV). All p-values were
two-sided. The cumulative age-specific percentages of age at diagnosis for breast and ovarian
cancer were determined using five-year intervals for the BRCA1 and BRCA2 mutation-
positive families, as well as for the general Finnish breast and ovarian cancer patient
populations (the FCR, 1997) (I).
9.2 Luria-Delbrück equation (I, II)
The number of generations (g) elapsed from a common ancestor for the BRCA1/BRCA2
families with the same haplotype was estimated by modifications of the Luria-Delbrück
equation (Luria and Delbrück, 1943; Lehesjoki et al., 1993) of pexcess = α(1 – θ)g, where α = 1
(all chromosomes carry the same mutation), and θ refers to the recombination fraction
between the gene/mutation and the marker locus. In the previous modifications of the Luria-
Delbrück equation (Lehesjoki et al., 1993; Hästbacka et al., 1994; Höglund et al., 1995),
pexcess = (paffected – pnormal)/(1 – pnormal) denoted the excess of an allele at a marker locus among
51
mutation-associated chromosomes versus normal population chromosomes. Here, mutation-
associated haplotype reconstruction trees were used to derive pexcess-values. Two
modifications were used: in the first one (modification 1), population allele frequencies were
taken into consideration and pexcess was defined as pexcess = (paffected – pnormal)/(1 – pnormal),
while in the other one (modification 2) pexcess was assumed equal to paffected. Modification 2
was used to achieve minimum and maximum estimates for time elapsed from a common
ancestor, and paffected-values were calculated at each marker either as the fraction of different
haplotype variants carrying the allele present in the most common haplotype variant
(minimum estimate) or as one of the alleles observed in different haplotype variants
(maximum estimate). Modification 1 was used to calculate minimum estimates only, and
pnormal denoted the frequency of the allele present in the most common mutation-associated
haplotype variant in normal population chromosomes. In Studies I and II, slightly different
versions of modification 1 were used; the paffected-value was calculated either as the fraction of
different haplotype variants (II) or as the fraction of families (I) carrying the allele present in
the most common haplotype variant. The average of the values obtained at different markers
was considered as the most likely time estimate in each calculation.
9.3 Logistic regression (III)
Logistic regression analysis was used to determine the odds of mutation for unselected
ovarian carcinoma patients (III); the X variable was the presence or absence of
BRCA1/BRCA2 mutation, and personal and family histories of breast and ovarian cancer were
used as explanatory variables.
52
RESULTS
1 Studies on recurrent BRCA1 and BRCA2 mutations (I, II)
1.1 Mutation-associated haplotypes
In haplotype analyses, all Finnish families carrying the same BRCA1 or BRCA2 mutation
were found to share a common core haplotype, except for those with the BRCA2 999del5
mutation (Figure 3). For the 999del5 mutation, two distinct core haplotypes were seen: a rare
one (haplotype A) was shared by three families, while a more common haplotype (haplotype
B) was present in seven families. Finnish families with haplotype A shared a four-marker
haplotype with Icelandic 999del5 mutation-positive families (Figures 3 and 4). The lengths of
the shared core haplotypes varied widely (1.6 to 26 cM) between families with different
mutations (Figures 3 and 4, and Table 7). The haplotype shared between the Icelandic and
Finnish 999del5 mutation-positive families extended approximately 0.5 cM centromeric to
the mutation site.
53
cM Markercen
10.5 D17S1872 * * 126 * * 122 126 * * * * 120 * -7.5 D17S946 132 1364.5 D17S250 164 1562.5 THRA1 1701.8 D17S800 166 1701.2 D17S846 235
D17S855 1530.4 D17S902 1441.5 D17S579 * 1107.5 D17S588 158 * 15215.5 D17S787 * * * 164 138
tel
3744delT
140142
BR
CA
1
3604delA
168 170176 174
158
6033
913
112
1000
911
132 *
152 150
158140
S41
23
5085 5 14
176
120
231 *
145148120
150 156 156168 170 174
134 136 * 136
816
S63
0
S19
53D
S30
01
14 5 188 50
85S
4123
188,
816
S
630
S30
01
S19
53D
16
62
16
62
15.5 cM
1.6 cM
cM Markercen
10.5 D17S1872 * * 112 112 120 * 1387.5 D17S946 136 136 1364.5 D17S250 154 166 1522.5 THRA1 174 168 1701.8 D17S800 174 174 1701.2 D17S846 231 239 235
D17S855 147 147 1490.4 D17S902 148 152 1481.5 D17S579 122 110 1247.5 D17S588 158 150 15815.5 D17S787 164 138 142 140 142 138
tel
5004
883
425
286
BR
CA
1
124158
*
425
5370C>T
Kb1
4216nt-2A>G 4446C>T
15
17, 1
1313
6, 2
74
Kb1
, Ko1
113
, 136
Ko1 17 274 15 883
286
5004 26
332
126
332
1
15 cM7.9 cM
26 cM
54
cM Markercen
15.5 D13S283 136 136 135 136 136 158 136 *
12.4 D13S1294 258 258 256 258 260 2748.0 D13S217 181 * 177 1794.3 D13S1246 208 214 210 2141.4 D13S260 170 1701.0 D13S1699 159 1540.8 D13S1698 164 1680.4 D13S1697 225 2290.4 D13S171 238 2381.1 D13S1695 258 2561.6 D13S1694 240 2341.7 D13S1696 221 2211.8 D13S267 261 24913.3 D13S263 167 173 161 153 159 16114.6 D13S1227 * 136 140 138 136 154 13616.6 D13S1272 * 259 255 261 257 261 261 255 25920.4 D13S153 228 224 224 232 224 228 - 226 230
tel 5
87 2
132 87 98
2
8555T>G
258
171140
9346nt-2A>G
140
47 92 3
181
BR
CA
2
47 92 314
0
983
p34
298
132
5,9
83 p
342
3.2 cM 3.2 cM
cM Markercen
15.5 D13S283 * 136 136 158 152 144 136 *
12.4 D13S1294 274 * 258 274 258 258 272 *
8.0 D13S217 173 179 175 *
4.3 D13S1246 206 212 *
1.4 D13S260 1721.0 D13S1699 1620.8 D13S1698 1640.4 D13S1697 2290.4 D13S171 2481.1 D13S1695 2561.6 D13S1694 2401.7 D13S1696 2231.8 D13S267 261 25513.3 D13S263 167 * 153 169 16914.6 D13S1227 * * * 136 129 15416.6 D13S1272 * 259 * 259 261 259 25920.4 D13S153 * 224 224 * 224 228 232 222 226
tel
378
486
257
K3 28
p185
p226 p5
1 55
p179
240221249
140
172 168162
BR
CA
2 170221236256
177 175208 214
6503delTT 7708C>T
K3
28 p51
55 486
378
p226
p17
9 p
185
9.8 cM2.7 cM
55
Figure 3. Mutation-associated haplotype reconstruction trees of the Finnish families withBRCA1/BRCA2 founder mutations. In each figure, the order of markers studied and their mapdistances in centiMorgans (cM) from the gene/mutation are shown on the left. For each mutation,haplotype reconstruction tree shows the minimum number of historical recombinations between thefamilies and the core haplotype shared by the families. Family numbers are indicated in italics. For theSwedish families with the 3744delT mutation, family numbers begin with the S letter. In 999del5mutation-associated haplotype A, the allele sizes shared between the Finnish and Icelandic familieswith the same mutation are presented in boldface; in family 158, a four-marker haplotype (164-184-225-157) at marker loci D13S1698, SLS163, D13S1697, and SLS329 was shared, while in families102 and 6003 a three-marker haplotype (184-225-157) was shared at marker loci SLS163, D13S1697,and SLS329. In haplotype A, the two different allele sizes seen at marker locus D13S1698 are likely tobe due to a mutation (Weber and Wong, 1993) or a null allele (Callen et al., 1993). cen, centromere;ex, exon; tel, telomere; THRA1, avian erythroblastic leukemia viral (v-erb-a) oncogene homolog; *,ambiguous allele; –, unknown allele
cM Markercen
15.2 D13S283 136 136 158 136 158 136 148 135 13612.1 D13S1294 258 258 258 258 2587.67 D13S217 181 173 175 181 1773.97 D13S1246 212 214 208 2141.07 D13S260 178 1700.77 SLS312 234 2420.68 D13S1699 159 1540.45 D13S1698 1680.21 SLS163 184 1840.084 D13S1697 225 2290.004 SLS329 157 1590.02 EX11 277 2770.55 D13S1701 294 3060.72 SLS321 107 1070.80 D13S171 238 2380.94 SLS886 119 1211.42 D13S1695 258 2561.99 D13S1694 234 2342.03 D13S1696 219 2192.13 D13S267 257 24913.6 D13S263 173 171 16714.9 D13S1227 138 140 140 154 13616.9 D13S1272 261 255 255 259 255 255 25520.7 D13S153 226 224 226 222 234 228 230
tel
397
158
p11 63
102
6003 11
3
130
163132
999d
el5
263
260 274
257220
136153
63 p11
113
Haplotype A Haplotype B
6003
158
102
130
397
164/168
p42 97
p42 97
3.2 cM6 cM
56
Figure 4. BRCA2 999del5 mutation-associated haplotype reconstruction tree of the Icelandic families.The order of markers studied and their map distances in centiMorgans (cM) from the mutation areshown on the left. The reconstruction tree shows the minimum number of historical recombinationsbetween and within the families and the core haplotype shared between all the families. Familynumbers are indicated in italics, and the numbers separated by colons from the family numbers referto individuals whose haplotypes have diverged from the main haplotype seen in the family. All 28markers were used in genotyping the five large, high-risk breast cancer families (2F, 4, 5A/B, 6, 7A/C),and the five markers indicated with an asterisk were used in genotyping the 13 families in which twosisters had been diagnosed with breast cancer (2, 10, 13, 15, 16, 21, 23, 24, 32, 51, 57, 59, and 67).Nine different haplotype variants, each of which is indicated with a distinct symbol, were identified inthe families based on the extent of haplotype sharing between marker loci D13S220 and D13S1246,which are marked in boldface. In families 5A/B and 7A/C, two different haplotype variants were seenin each, and for sister pairs 57 and 59, the haplotype variant remained uncertain due to missing dataon markers centromeric to the mutation. The nine different haplotype variants differed in frequency:the most common variant was present in 6 families, while 6 variants were seen in only one familyeach. A four-marker haplotype (1-1-1-1) indicated with boldface at marker loci D13S1698, SLS163,D13S1697, and SLS329 was shared between the Icelandic and Finnish families with haplotype A. cen,centromere; ex, exon; tel, telomere; –, unknown allele
cM Markercen
15.2 D13S283 - 1 2 - - 3 - - - 2 9 -7.67 D13S217 3 4 4 - - - -3.97 D13S1246* 6 1 5 4 5 - D13S290 2 - - - 1 - D13S1226 1 - - - 3 - SLS320 1 - - - 3 - SLS385 - - - 1 - SLS165 - - - 21.07 D13S260* 4 70.77 SLS312 30.68 D13S1699 30.45 D13S1698 10.21 SLS163 10.084 D13S1697 10.004 SLS329 10.02 EX 11 10.55 D13S1701 10.72 SLS321 30.80 D13S171* 60.94 SLS886 31.42 D13S1695 2 31.99 D13S1694 3 1 - SLS234 - - 3 32.13 D13S267* 1 7 2 7 - D13S1293 - - 3 - - D13S219 - - 2 4 44.0 D13S220* - - 2 4 2
13.6 D13S263 - - 1 3 8 6 8 2 1tel
6: 3
, 13
2F 67
999d
el5
7A 4 5B
243
2F7C
7C: 2
, 4
4
4: 4
4:55 5B 15
3
4
6 - -
5A
5A: 7
4
7A, 1
0 23
42
-
1
7C
-5 - 5
15 21 5A 5A: 1
, 7
6
67
2, 1
6
- -
10, 2
313
, 24
32, 5
1 2,
16
����������
��������
6, 1
3, 2
432
, 51 21
57, 5
9
?���� ����
1.7 cM
57
1.2 Estimated number of generations from a common ancestor for families sharing a
conserved core haplotype
The estimated number of generations (g) elapsed from a common ancestor for the families
sharing a conserved core haplotype was calculated with modifications of the Luria-Delbrück
equation when at least four families had the same core haplotype (Table 7). Here, families
carrying the BRCA1 3604delA mutation were an exception, as the five families shared an odd
haplotype extending 15.5 cM telomeric and 0 cM centromeric to the BRCA1 gene (Figure 3),
which did not allow us to make any estimations of time elapsed from a common ancestor.
Modifications 1 and 2 gave consistent results (Table 7); in most cases, the time estimates
obtained using modification 1 were within the minimum and maximum estimates obtained
using modification 2.
Table 7. Estimated number of generations elapsed from a common ancestor for families sharing aconserved core haplotype.
Estimated number of generations (g) and years (y)elapsed from a common ancestor for familiesa,
(No. of data points used in the calculations)
Gene and mutation,(No. of families)
Length of theshared
haplotype (cM)
Modification 1b Modification 2c
BRCA13604delA (5) 15.5 - -3744delT
Finnish families (7) 1.6 28 g; 560–700 y (8) 24–32 g; 480–800 y (8)Finnish (7) and Swedish (4)families
1.6 30 g; 600–750 y (8) 23–36 g; 460–900 y (8)
4216nt-2A→G (8) 15 < 10 g; < 250 y (2) 9 g; 180–225 y (2)4446C→T (3) 7.9 - -5370C→T (2) 26 - -BRCA2999del5
Finnish familieswith haplotype B (7) 6 6 g; 120–150 y (4) 7–12 g; 140–300 y (8)with haplotype A (3) 3.2 - -
Icelandic families (18) 1.7 21 g; 420–525 y (6) 16–40 g; 320–1000 y (9)6503delTT (2) 9.8 - -7708C→T (7) 2.7 13 g; 260–325 y (6) 10–20 g; 200–500 y (10)8555T→G (4) 3.2 7 g; 140–175 y (6) 7–9 g; 140–225 y (8)9346nt-2A→G (7) 3.2 11 g; 220–275 y (5) 7–10 g; 140–250 y (8)
s
cM, centiMorgan; g, generations; y, years; aSome additional unpublished data is presented; bThe pexcess-value ofthe Luria-Delbrück equation: pexcess = α(1 – θ)g, was defined as pexcess = (paffected – pnormal)/(1 – pnormal);
cThepexcess-value of the Luria-Delbrück equation: pexcess = α(1 – θ)g, was assumed equal to paffected; the minimum andmaximum estimates were obtained by calculating the paffected-value as the fraction of different haplotype variantscarrying the allele present in the most common haplotype variant and as one of the alleles observed in differenthaplotype variants, respectively
58
1.3 Geographical origins of the families
Plotting of the birthplaces of parents, grandparents, or great-grandparents of the index
patients on the map of Finland revealed distinct geographical clustering of the origins of
BRCA1/BRCA2 mutation-positive families (Figure 5).
59
BRCA1 3744delT BRCA1 4216nt-2A>G
BRCA1 5370C>T
BRCA2 8555T>G
BRCA1 4446C>T
BRCA2 9346nt-2A>G
BRCA2 7708C>TBRCA2 999del5 BRCA2 6503delTT
1.6 cM 7.9 cM
26 cM 6 cM for3.2 cM for
9.8 cM 2.7 cM
3.2 cM 3.2 cM
15.5 cM
BRCA1 3604delA
15 cM
Figure 5. Maps of Finland showing the origins of BRCA1 and BRCA2 mutation-positive families.Circles indicate birthplaces of grandparents or great-grandparents of the index patients. Squaresrepresent birthplaces of the parents of the index patients, and triangles represent birthplaces of theindex patients, which were marked when no information on previous generations was available. Thelengths of the shared haplotypes in centiMorgans (cM) are also indicated.
60
2 Breast and ovarian carcinoma phenotypes of BRCA1 and BRCA2 mutation carriers(I)
The number of female breast and ovarian carcinoma cases and the mean age at breast and
ovarian carcinoma diagnosis for each mutation are presented in Table 8. Altogether, 87
female breast carcinoma cases and 46 ovarian carcinoma cases were identified in the 34
BRCA1 families, which is on average 2.6 breast and 1.4 ovarian carcinoma cases per family.
In the 37 BRCA2 families, there were 123 female breast carcinoma cases, 1 male breast
carcinoma case (with the 999del5 mutation and diagnosed at age 47 years), and 21 ovarian
carcinoma cases, which is on average 3.3 female breast and 0.6 ovarian carcinoma cases per
family. The proportion of ovarian carcinoma was significantly higher, with a 2.4-fold
difference, in the BRCA1 mutation-associated families than in the BRCA2 mutation-
associated families (p<0.001, χ2 test).
Table 8. Number of breast and ovarian cancer cases and the mean age at breast and ovarian cancerdiagnosis for each mutation.
Gene and mutation No. offamilies
included inphenotypeanalysis (I)
No. offemalebreastcancercases
No. of femalebreast cancer
cases for whichage at diagnosis
was available(mean age atdiagnosis, y)
No. ofovariancancercases
No. of ovariancancer casesfor which ageat diagnosis
was available(mean age atdiagnosis, y)
BRCA1Ex 11, 1924delA 1 1 1 (44) - -Ex 11, 2803delAA 2 3 3 (56) 2 2 (62)Ex 11, 3604delA 5 7 7 (45) 10 9 (46)Ex 11, 3744delT 7 7 7 (45) 10 9 (49)Ex 11, 3904C→A 1 3 3 (49) 3 2 (59)Ex 11, 4153delA 1 1 1 (32) 1 1 (48)Int 11, 4216nt-2A→G 9 24 23 (43) 8 8 (52)Ex 13, 4446C→T 3 23 19 (46) 8 8 (53)Ex 17, 5145del11 1 4 4 (37) - -Ex 20, 5370C→T 3 12 12 (49) 3 2 (67)Ex 20, 5382insC 1 2 2 (57) 1 1 (40)
Total 34 87 82 (46) 46 42 (51)BRCA2Ex 9, 999delTCAAA 13 52 43 (47) 6 5 (60)Ex 11, 4081insA 1 2 2 (67) 2 2 (60)Ex 11, 6495delGCA→C 1 3 3 (52) - -Ex 11, 6503delTT 3 8 3 (57) 4 4 (62)Ex 15, 7708C→T 8 26 24 (45) 4 3 (56)Ex 18, 8555T→G 4 12 12 (49) 1 1 (60)Int 23, 9346nt-2A→G 7 20 19 (52) 4 4 (66)
Total 37 123 106 (48) 21 19 (61)
ex, exon; int, intron; y, years
61
A statistically significant correlation was present between the location of the mutation
and the breast and ovarian carcinoma phenotype in BRCA1 mutation-associated families; the
proportion of ovarian carcinoma was significantly higher (p<0.001, χ2 test), with a 2.3-fold
difference, in families carrying mutations in exon 11 as compared with those with mutations
towards the 3´ of this exon.
The distribution of ages at breast cancer diagnosis was similar in BRCA1 and BRCA2
mutation-associated families (mean+SD, 45.6+11.6 years vs. 48.4+13.7 years; range, 22–73
years vs. 25–95 years) (Figure 6a), while the mean+SD age at ovarian carcinoma diagnosis
was significantly younger in the BRCA1 mutation-associated families than in those with
BRCA2 mutations (51.3+9.2 years vs. 61.2+9.7 years; range, 38–77 years vs. 45–78 years;
p<0.001, unpaired t-test). In the BRCA1 families, ovarian carcinoma had been diagnosed
before the age of 50 years in almost 60% of cases; the corresponding percentages were less
than 20% for the BRCA2 mutation-associated cases and less than 30% for the general ovarian
carcinoma patient population (Figure 6b).
62
Figure 6. Cumulative age-specific percentages of age at diagnosis for (a) breast and (b) ovariancancer in the BRCA1 and BRCA2 mutation-associated families, and in the general Finnish breast andovarian cancer patient populations (i.e., all breast and ovarian cancer cases diagnosed in Finlandduring 1995) (the FCR, 1997).
3 BRCA1 and BRCA2 germline mutations in unselected Finnish ovarian carcinoma
patients (III)
3.1 Mutations detected
A germline mutation in BRCA1 or BRCA2 was found in 5.6% (13/233) of the unselected
Finnish ovarian carcinoma patients; 11 patients (4.7%) were BRCA1 mutation-positive and
two (0.9%) were BRCA2 mutation-positive. The mutations detected are presented in Table 9.
0102030405060708090
100
20-24
30-34
40-44
50-54
60-64
70-74
80-84
BRCA 1
BRCA 2
ALL 1995
a)
Cu
mul
ativ
e pe
rce
ntag
e
Age, years
0102030405060708090
100
20-24
30-34
40-44
50-54
60-64
70-74
80-84
BRCA 1BRCA 2ALL 1995
b)
Cum
ulat
ive
perc
enta
ge
Age, years
63
All 13 mutation-positive patients were carriers of the previously identified Finnish
BRCA1/BRCA2 mutations, and seven recurrent founder mutations accounted for 12 of the 13
mutations detected. The only unique mutation had been identified previously in a patient that
also belonged to the present study cohort.
Table 9. Mutations detected and personal and family history of breast and ovarian carcinoma of themutation carriers.
Patient
no.
Gene and
mutationa
Type of
cancer and
age at
diagnosis (y)
No. of 1st- and 2nd-degree
relatives with breast and/or
ovarian cancer (type of
cancer, age at diagnosis, and
degree of relatedness)
Bc at
any
agee
Bc and
oc in the
same
womanf
Bc
<50 yf
Bc and oc
in the
same
woman
and/or bc
<50 yf
BRCA1654 3604delAb Bc 41, oc 44 Yes Yes Yes Yes911 3604delAb Oc 42 1 (bc 55, 2nd) Yes No No No913 3604delAb Oc 42 1 (bc 46, 1st) Yes No Yes Yes1000 3604delAb Oc 53 2 (bc 58, oc 46, 1st; bc 50, 2nd) Yes Yes No Yes816 3744delTb Oc 43 No No No No673 4153delAb Bc 32, oc 48 Yes Yes Yes Yes723 4216nt-2A→Gc Oc 59 No No No No883 4216nt-2A→Gc Bc 52, oc 58 1 (bc n.k., 1st) Yes Yes No Yes87 4446C→Td Bc 48, oc 58 3 (bc 52, 1st; 2x bc n.k., 2nd) Yes Yes Yes Yes656 4446C→Td Bc 37, oc 40 Yes Yes Yes Yes257 5370C→Td Bc 50, oc 57 1 (bc 60, 1st) Yes Yes No Yes
BRCA2488 5797G→Td Oc 52 2 (bc d37, 1st; oc 55, 1st) Yes No Yes Yes983 9346nt-2A→Gc Oc 70 1 (bc 43, 1st) Yes No Yes Yes
11/13(85%)
7/13(54%)
7/13(54%)
10/13(77%)
bc, breast cancer; d, deceased; n.k., not known; oc, ovarian cancer; y, years; aAll mutations detected in the present study hadbeen identified previously in the Finnish population (Vehmanen et al., 1997a, 1997b; Vahteristo et al., 2001), and all but4153delA in BRCA1 are recurrent founder mutations; bFrameshift mutation; cSplice site mutation; dNonsense mutation; eIn theindex patient and/or her first- or second-degree relative(s); fIn the index patient or a first-degree relative
3.2 Personal and family history of breast and ovarian carcinoma of the mutation carriers
Personal and family history of breast and ovarian carcinoma of the 13 mutation-positive
patients is shown in Table 9; 11 patients (85%) had a personal and/or family history of breast
carcinoma, and in seven cases (54%) breast and ovarian carcinoma had been diagnosed in the
same woman. Furthermore, seven of the mutation-positive patients had a history of breast
carcinoma diagnosed before the age of 50 years. The diagnosis of both breast and ovarian
carcinoma in the same woman and/or young age (<50 years) at breast cancer diagnosis was
characteristic of most (77%) mutation carriers. In the BRCA1 mutation-associated families,
the mean+SD age at ovarian carcinoma diagnosis was 49.2+7.3 years (range 40–59 years) and
64
the mean+SD age at breast carcinoma diagnosis was 47.6+8.9 years (range 32–60 years) (in
the index patients and their first-degree relatives). Except for one mesonephroid carcinoma,
all BRCA1/BRCA2 mutation-associated ovarian carcinomas were of serous or poorly
differentiated histology.
3.3 Relationship between mutation carrier status and personal and family history of breast
and ovarian carcinoma
In the logistic regression analysis, the single most significant predictor of a BRCA1 or BRCA2
germline mutation was the presence of both breast and ovarian cancer in the index patient or
a first-degree relative (Table 10). The odds ratio also independently increased for patients
with at least two first- or second-degree relatives with breast or ovarian carcinoma, and for
patients with one first- or second-degree relative with breast carcinoma only (Table 10).
Notably, no mutations were detected in the 13 ovarian carcinoma patients who reported one
first- or second-degree relative with ovarian carcinoma only. In patients with a history of
breast carcinoma diagnosed before the age of 40 (n=4) or 50 (n=13) years, BRCA1/BRCA2
mutations were found in 75% and 54% of the patients, respectively, while mutation
frequencies were lower in patients with a history of breast carcinoma diagnosed after the age
of 50 (n=10) or 60 (n=4) years: 30% and 0%, respectively. The association between mutation
carrier status and young age (<50 years) at breast carcinoma onset was, however, not
statistically significant (p=0.40, Fisher’s exact test).
Table 10. Logistic regression analysis of the association between personal and family history ofbreast and ovarian cancer and BRCA1/BRCA2 mutation carrier status.
Variable Coefficient SE OR (95% CI)
Intercept -4.21 0.59 0.02 (0.01–0.05)Breast and ovarian cancer in the same persona 4.59 0.99 98.69 (14.11–690.15)Family history of breast and ovarian cancerb
One relative with breast cancer 1.83 0.85 6.22 (1.17–33.16)One relative with ovarian cancer -3.00 6.20 0.05 (2.65 x 10-7–9402.47)Two or more relatives with breast or ovarian cancer 2.70 1.16 14.84 (1.53–143.61)
CI, confidence interval; OR, odds ratio; SE, standard error; aIndex or a first-degree relative; bIn first- or second-degree relatives
65
4 BRCA1 and BRCA2 germline mutations in Finnish ovarian carcinoma families (IV)
4.1 Mutations detected
A germline mutation in BRCA1 or BRCA2 was detected in 5 of the 23 (22%) Finnish families
with at least two cases of ovarian carcinoma in first-degree relatives (Table 11). Two of the
families were BRCA1 mutation-positive and three were BRCA2 mutation-positive. In one
family, a novel, apparently disease-causing BRCA2 missense mutation 8702G→A in exon 21,
leading to conversion of glycine to aspartate at codon 2901, had been identified in a previous
study (Roth et al., 1998). We considered this alteration to be a disease-associated mutation, as
it has not been detected in 220 cancer-free Finnish control individuals and is located within
an evolutionally conserved region of the protein (Roth et al., 1998); thus, altogether 26%
(6/23) of the 23 Finnish ovarian carcinoma families were BRCA1 or BRCA2 mutation-
positive.
Table 11. Mutations identified in the families and clinicopathological characteristics of the ovariancarcinomas.
Ovarian carcinomaFamily and
patient no.
Gene and
mutation
Type of
cancer
Age at
diagnosis (y) Histology Stage Grade DNA ploidyBRCA1
Family 3636-1 1806C→Ta Ovarian 39 Undifferentiated III 3 A36-4 1806C→Ta Ovarian 56 Serous III 3 A36-5 1806C→Ta Ovarian 39 Undifferentiated III 3 A
Family 2929-1 3744delTb Ovarian 53 Undifferentiated I 3 A29-4 3744delTb Ovarian 49 Undifferentiated III 3 D29-5 3744delTb Ovarian 38 n.d. n.d. n.d. n.d.
BRCA2Family 10
10-1 7708C→Ta Ovarian 73 Endometrioid III 2 A10-4 7708C→Ta Ovarian 63 Transitional III 1 D
Family 3030-1 9346nt-2A→Gc Ovarian 54 Serous III 3 A30-3 n.d. Ovarian 48 n.d. n.d. n.d. n.d.30-4 9346nt-2A→Gc Breast 45
Family 3333-1 9346nt-2A→Gc Ovarian 51 Clear cell III 3 A33-4 Mutation-negative Ovarian 68 Serous III 3 A
Family 1919-1 8702G→Ad Ovarian 59 Transitional III 3 A19-4 8702G→Ad Ovarian 58 Serous III 1 A19-5 8702G→Ad Ovarian 55 Endometrioid I 2 A
A, aneuploid; D, diploid; n.d., not defined; y, years; aNonsense mutation; bFrameshift mutation; cSplice site mutation;dMissense mutation; the mutation had been previously identified in family 19 by Roth et al. (1998), and it is designated as8930G→A in the BIC database.
66
4.2 Characteristics of mutation-positive and -negative ovarian carcinoma families
Breast carcinoma was present in only one of the mutation-positive families, and that case had
been diagnosed at the age of 45 years, whereas all eight families with later-onset breast
carcinoma (range, 51–75 years; mean+SD, 61.4+8.0 years) were mutation-negative. Of the 14
site-specific ovarian carcinoma families included in the study, all three with three affected
individuals were mutation-positive, while mutations were found in only 18% (2/11) of the
families with two ovarian carcinoma cases only. In the carriers of BRCA1 mutations, the
mean age at ovarian carcinoma diagnosis was 12 years younger than in the BRCA2 mutation
carriers (45.7+8.0 years vs. 57.6+7.8 years; p=0.016, unpaired t-test) or in the BRCA1/BRCA2
mutation-negative patients (45.7+8.0 years vs. 57.5+8.3 years, p=0.002, unpaired t-test). All
the BRCA1 mutation-associated carcinomas were of undifferentiated histology, except for one
serous carcinoma, whereas carcinomas of the BRCA2 mutation carriers and non-carriers were
of various histological subtypes. Most BRCA1 and BRCA2 mutation-associated tumours
(80% and 86%, respectively) were of stage III, and all BRCA1 mutation-associated tumours
were of high grade. However, with regard to stage or grade, neither BRCA1 nor BRCA2
mutation-associated tumours differed significantly from non-BRCA1/2 mutation-associated
tumours (Fisher’s exact test; stages I and II, and III and IV were combined, and grades 1 and
2 were combined). Aneuploid tumours were more frequent in BRCA1 (80%) and BRCA2
(86%) mutation carriers than in non-carriers (31%), and the difference was statistically
significant between the BRCA2 mutation-associated cases and non-carriers (p=0.026; Fisher’s
exact test).
67
DISCUSSION
1 Studies on recurrent BRCA1 and BRCA2 mutations (I, II)
Eleven BRCA1/BRCA2 mutations have been found to account for the vast majority of all
BRCA1/BRCA2 mutations identified in the screening of the entire coding regions of the genes
(Vehmanen et al., 1997a, 1997b; Huusko et al., 1998). Here, we studied ancestral origins and
geographical distribution of families carrying these recurrent mutations. We found that the
birthplaces of the parents and grandparents of index patients were clustered in distinct
geographical regions of Finland, and all carriers of the same recurrent mutation, except for
those with the BRCA2 999del5 mutation, shared a common core haplotype, suggesting that
these mutation alleles are identical by descent, i.e., founder mutations. In the 999del5
mutation-positive families, two distinct core haplotypes were seen, which may be due to gene
conversion. This is supported by the geographical clustering of the families as well as by the
population history of Finland. Nevertheless, the possibility that the same mutation has arisen
twice cannot be ruled out. The lengths of the shared haplotypes varied widely (1.6 to 26 cM)
between families with different mutations, and the estimates of time elapsed from a common
ancestor for the families varied accordingly. For families sharing the shortest core haplotype
of 1.6 cM, the common ancestor was estimated to date back 24–32 generations (i.e., 480–800
years), while for families with a long 15 cM haplotype, the corresponding time was estimated
to be less than 10 generations (i.e., <250 years).
Birthplaces of the grandparents of index patients carrying the BRCA1 3604delA
mutation clustered in the southern, coastal region of the country, and as the mutation has also
been reported in Dutch, Belgian, and German families (Peelen et al., 1997; the BIC database),
it likely was brought into Finland from Central Europe, across the Baltic Sea. The clustering
of grandparents’ birthplaces in a very restricted area and the long haplotype of 15.5 cM
shared by the families suggest that these families had a common ancestor fairly recently. No
estimate of time elapsed from a common ancestor was obtained due to the odd haplotype
shared by the families.
The BRCA1 3744delT mutation (also known as 3745delT) has not been reported in
countries other than Finland and Sweden (Zelada-Hedman et al., 1997; the BIC database).
The Finnish and Swedish families studied here shared a short haplotype of 1.6 cM and were
estimated to have had a common ancestor 23–36 generations (i.e., 460–900 years) ago. The
common origin of the families is further supported by the low (0.57%) estimated frequency of
the haplotype in the Finnish population. In Finland, the grandparents’ birthplaces clustered in
68
Northern Ostrobothnia, although some were located in Southern and Central Finland.
According to church records, the majority of the Finnish families with this mutation have
been living in Ostrobothnia for at least 300 years, while the Swedish families clustered on the
opposite side of the Gulf of Bothnia. As the Swedish crusade to Finland began in the 12th
century, and massive colonization activity from Sweden to the southwest of Finland and to
the coast of Ostrobothnia occurred in the 12th and 13th centuries (Jutikkala and Pirinen, 1996),
the mutation might well have been introduced into the Finnish population by Swedish
colonists. Our estimate of the time elapsed from a common ancestor for the families (460–
900 years) is in accordance with these historical records.
The BRCA1 4216nt-2A→G mutation is unique to the Finns and the most frequently
observed BRCA1 mutation in Finland thus far (detected in 10 families). The birthplaces of the
grandparents were found to be enriched in Central and Northern Ostrobothnia, and this
regional concentration and the long 15 cM common haplotype suggest that the families had a
common ancestor recently. Our estimate of time elapsed since a common ancestor was less
than 10 generations (i.e., <250 years).
The BRCA1 mutations 4446C→T and 5370C→T were both clustered in Karelia. The
three Finnish families with the 4446C→T mutation shared a long haplotype of 7.9 cM, and in
the two 5370C→T mutation-positive families, the common haplotype extended over the
entire area of 26 cM studied. The numbers of families were, however, too small for time
estimations, but the long shared haplotypes and the regional clustering of parents’ and
grandparents’ birthplaces suggest that these families had a common ancestor fairly recently.
The 4446C→T mutation has been reported several (52) times in the BIC database and has
been identified in families of Dutch, Belgian, French, French Canadian, British, and North
American ancestry (Neuhausen et al., 1996; Stoppa-Lyonnet et al., 1997; Szabo and King,
1997; Tonin et al., 1998; the BIC database). The Finnish 4446C→T mutation-positive
families have been found to share a three-marker haplotype with French, French Canadian,
Dutch, and Belgian families, indicating that the mutation carriers may have a common
ancient origin (Dr. J Simard, personal communication). However, in France, some families
with the this mutation have a different common haplotype, and still another haplotype is seen
in British 4446C→T mutation-positive families, suggesting that the mutation may have arisen
de novo several times (Friedman et al., 1995; Neuhausen et al., 1996; Dr. J Simard, personal
communication). The 5370C→T mutation has been reported in Germany, Austria, and the
US (Wagner et al., 1998; Meindl and German Consortium for Hereditary Breast and Ovarian
Cancer, 2002). The Finnish families were observed to share a long haplotype of 10 cM with
the Austrian family included in the study. The mutation alleles may thus be identical by
descent in Finland and Austria, which is supported by the low estimated frequency (0.006%)
69
of the haplotype in the Finnish population.
The BRCA2 mutation 6503delT has been reported several (39) times in the BIC
database, and Swedish, Dutch, Belgian, British, American, and Canadian families have been
described to carry this mutation (Neuhausen et al., 1998; the BIC database). In Finland, it has
been identified in three families, and grandparents’ birthplaces were located in the late
settlement region, in Northern Karelia and in Northern Ostrobothnia. The Finnish families
shared a long 9.8 cM haplotype, the alleles of which were completely different from those
seen in haplotypes described elsewhere (Neuhausen et al., 1998), suggesting that the same
mutation is likely to have independent origins in different populations. The Finnish families
seem to have had a common ancestor quite recently, but as the number of families studied
was small, estimation of the time elapsed from a common ancestor was not possible.
For the BRCA2 7708C→T mutation, the birthplaces of the parents and grandparents
were scattered in the south-eastern part of the country and around Lake Ladoga. The fairly
wide distribution of the origins of the families, mainly in the area of early settlement,
excluding the coast of Bothnia, and the short (2.7 cM) identical haplotype in the mutation
alleles suggest that the common ancestor is distantly located and the mutation may have
spread from the Savo-Karelia region into the more western parts of the country. The time
from a common ancestor was estimated to date back 10–20 generations (i.e., 200–500 years).
Interestingly, the same mutation has been reported in a family of Asian ancestry but has not
been reported elsewhere (the BIC database).
The BRCA2 8555T→G mutation has not been reported in countries other than Finland,
the families with this mutation being clustered in a restricted area in Pirkanmaa and sharing a
haplotype of 3.2 cM. The number of generations from a common ancestor was estimated to
date back 7–9 generations (i.e., 140–225 years). This situation of families sharing a short
common haplotype and clustering in a restricted area in the early settlement region might
reflect the subisolate structure of the Finnish population that persisted until the Second World
War (Nevanlinna, 1972; Norio et al., 1973).
The birthplaces of the grandparents of the index patients with the BRCA2 9346nt-
2A→G mutation are located in Karelia and in Northern Finland. The mutation may have been
introduced into the area of late settlement during the internal migration movement that started
in the 16th century mainly with settlers from South Savo (Nevanlinna, 1972; Norio et al.,
1973). The families shared a 3.2 cM haplotype and were estimated to have had a common
ancestor 7–11 generations (i.e., 140–275 years) ago. This mutation is the most common
BRCA1/BRCA2 mutation identified in Finland (detected in 14 families) and has also been
reported in a Czech family but not elsewhere (the BIC database).
The surprising finding of two distinct core haplotypes (a 3.2 cM haplotype A in three
70
families, and a 6 cM haplotype B in seven families) in Finnish families with the BRCA2
999del5 mutation may be due to gene conversion. Although very little is currently known
about gene conversion in humans, it has been proposed to be quite frequent and the major
factor contributing to the decay of LD over short distances (Frisse et al., 2001; Przeworski
and Wall, 2001). The hypothesis that the two distinct Finnish 999del5 mutation-associated
haplotypes are due to gene conversion is supported by the geographical distribution of the
birthplaces of the parents and grandparents of index patients and by the population history of
Finland. Families with haplotype A, as well as those with haplotype B, clustered in the same,
restricted area in the early settlement region (in Pirkanmaa, Satakunta, and Southern
Ostrobothnia), and families with haplotype B formed another cluster in Northern Karelia, in
the late settlement region. The mutation may thus have originally arisen on haplotype A,
being then transferred to haplotype B by gene conversion. The mutation in association with
haplotype B could have been brought into Karelia when the inhabitation of the eastern and
northern parts of the country began in the 16th century by internal population movements
from the early settlement area (Nevanlinna, 1972; Norio et al., 1973). The common ancestor
of the families with the more common haplotype B was estimated to date back 6–12
generations (i.e., 120–300 years), but the number of families with haplotype A was too small
for time estimations. Previously, a similar situation has been described in Finland; a mutation
in the transglutaminase 1 gene, causing autosomal recessive congenital ichtyosis, has been
observed on two distinct haplotypes, both of which are clustered in the early settlement
region in Savo, and one of them also in Central Finland (Laiho et al., 1997).
In Iceland, the 999del5 mutation is the only BRCA2 mutations identified and has been
detected in the majority (67%) of breast cancer families with multiple affected members
(Gudmundsson et al., 1996; Thorlacius et al., 1996). The 18 Icelandic families/sister pairs
studied here shared a short conserved haplotype of 1.7 cM, and the common ancestor of the
families was estimated to date back to 16–40 generations (i.e., 320–1000 years). According to
historical records, Iceland was settled mainly by Vikings from Western Scandinavia and the
British Isles during the 9th–11th centuries (Rafnsson, 1999; Sawyer, 1999). The mutation may
thus have been brought into Iceland as early as during the settlement of the country.
Interestingly, Finnish families with haplotype A shared allele sizes with Icelandic families at
four markers spanning approximately 0.5 cM centromeric to the mutation site. This shared
haplotype appears to be rare in both populations, supporting the possibility of a common
ancient origin of the Finnish and Icelandic 999del5 mutation-positive families; the estimated
frequency of the four-marker haplotype was 1.3% and 2.2% in Finnish and Icelandic
populations, respectively, and the actual frequency of a two-marker haplotype available (164-
225 at loci D13S1698 and D13S1697) in 102 normal Finnish chromosomes was 2.0%
71
(estimated frequency 4.3%) and that of a three-marker haplotype available (1-1-1 at loci
D13S1698, SLS163, and SLS329) in 28 normal Icelandic chromosomes was 3.6% (estimated
frequency 10.0%). The ancient 999del5 mutation may have been introduced into the Finnish
and Icelandic populations during different time periods and been preserved in these
populations due to random genetic drift. For the same reason, the mutation may have been
lost in some populations where it was also introduced. If the 999del5 mutation alleles are
identical by descent in the Finnish and Icelandic families, then the common ancestor must
date back considerably further than to the time since the mutation was introduced into the
Icelandic population. Although the Finnish and Icelandic 999del5 mutation-positive families
may have a common ancient origin, and the two distinct haplotypes seen in the Finnish
families may due to gene conversion, the same mutation arising de novo more than once
cannot be ruled out. Short symmetric elements have been proposed to predispose DNA
sequences to meiotic microdeletions (Schmucker and Krawczak, 1997), and three such
elements partially overlap or closely flank the 999del5 mutation site. The 999del5 mutation
has been reported a few times elsewhere, in families of Latin American/Caribbean, Native
American/Central European/Northern European, and English origin (the BIC database).
Furthermore, three other short deletions have been identified in this region: 995delCA,
995delCAAAT, and 1002delAA (Mavraki et al., 1997; Schubert et al., 1997; the BIC
database), suggesting that the site might represent a mutational hot spot. Thus, the common
ancient origin of the 999del5 mutation in Finland and Iceland remains uncertain.
It is worth noting that haplotype analysis does not give an estimate of the time elapsed
since a mutation started to spread in a population, but it does allow estimation of the time
elapsed since there was a common ancestor for the families included in the study.
Furthermore, time estimates based on the extent of LD or shared haplotypes in a relatively
small number of mutation chromosomes are by necessity rough and subject to variation, as
the small sample size leaves the number of detected recombinations sensitive to chance.
Moreover, LD is a complex phenomenon affected not only by physical distance but also by
the distance from the centromere (Watkins et al., 1994) and the presence of recombination
hot spots (Jeffreys et al., 2001) and suppression regions (Liu and Barker, 1999; Jorde, 2000).
According to recent studies, the human genome is organized into discrete blocks of limited
haplotype diversity extending up to 100 kb, but interpopulation differences in the sizes of the
blocks have been observed (Daly et al., 2001; Patil et al., 2001; Reich et al., 2001). These
haplotype blocks have only a few (2–4) haplotypes, while greater haplotype diversity is seen
in regions spanning the blocks (Daly et al., 2001; Reich et al., 2001). The relationship
between LD and physical distance is further complicated by numerous other factors, such as
mutation, natural selection, gene flow, demographic history, stochastic events, and gene
72
conversion (Laan and Pääbo, 1997; Zavattari et al., 2000; Pritchard and Przeworski, 2001).
LD has, however, been successfully used in isolated founder populations to localize disease
loci for specific rare monogenic diseases (Ozelius et al., 1992; de la Chapelle and Wright,
1998; Peltonen et al., 1999, 2000a). The advantage of such populations for mapping
susceptibility genes underlying complex diseases has been proposed to be less striking (Eaves
et al., 2000; Peltonen et al., 2000a; Altmüller et al., 2001), and the choice of populations for
LD mapping studies of complex disease are still open issues (Kruglyak, 1999; Wright et al.,
1999). Nevertheless, isolated founder populations may be more advantageous than admixed
ones in genetic studies of complex disorders because of reduced allelic and locus
heterogeneity. Homogeneity of environmental and cultural components is also a distinct
advantage for genetic studies (Peltonen et al., 2000a, 2000b). In addition, there is recent
evidence that small, isolated subpopulations exhibit higher levels of LD around common
alleles than the larger populations from which they are derived (Zavattari et al., 2000). The
multiple local founder effects and the long shared haplotypes (1.6 to 26 cM) around mutation
alleles underlying a multifactorial disease observed here provide support for the concept that
isolated founder populations with known demographic histories may offer definite
advantages for mapping novel susceptibility genes for multifactorial disorders. In Finland,
multiple small subisolates that remained relatively stable until the Second World War and
industrialization (Nevanlinna, 1972; Norio et al., 1973; Peltonen et al., 1999) may have
allowed random genetic drift to create allelic disequilibrium.
In isolated founder populations, reduction in genetic heterogeneity is illustrated by the
significant reduction of the number of mutations found in specific disease-related genes. For
example, among Icelanders, only one mutation has been identified in each of the BRCA1 and
BRCA2 genes (Thorlacius et al., 1997; Bergthorsson et al., 1998). Since BRCA1 and BRCA2
mutations do not affect the fitness of their carriers, their fate is not determined by natural
selection, and the striking difference in the observed BRCA1/BRCA2 mutation spectra in
Finland and Iceland (the total number of distinct BRCA1/BRCA2 mutations identified being
32 and 2, respectively) may reflect differences in the settling of these countries; Finland is
likely to have been inhabited continuously since the last glacial period around 10 000 years
ago by small immigrant groups (Jutikkala and Pirinen, 1996; Peltonen et al., 1999; Norio,
2000), while Iceland is believed to have been settled mainly by Vikings quite recently, during
the 9th–11th centuries (Rafnsson, 1999; Sawyer, 1999). In Finnish families with the HNPCC
syndrome, a large number (11) of different MLH1 mutations have also been identified, but
three common founder mutations account for more than 80% of all MLH1 mutation-positive
HNPCC families (Nyström-Lahti et al., 1995, 1996; Holmberg et al., 1998; Salovaara et al.,
2000).
73
Since our studies on recurrent BRCA1 and BRCA2 mutations, a number of new families
have been found to carry the founder mutations studied here, and five more recurrent
mutations have been described: two in BRCA1 (1806C→T, and 5382insC), and three in
BRCA2 (4075delGT, 4081insA, and 5797G→T), bringing the total number of recurrent
mutations in Finland to 16 (Syrjäkoski et al., 2000; Vahteristo et al., 2001; III; IV;
unpublished data). Although a number of unique BRCA1 and BRCA2 mutations have been
identified as well, they seem to account for only a minority of all BRCA1/BRCA2 mutation-
positive families in Finland (unpublished data). In addition to 31 distinct protein-truncating
mutations and one missense mutation considered to be of clinical significance (8702G→A in
BRCA2), a number of common, benign variants have been described in each gene. The high
coverage of founder mutations of all BRCA1/BRCA2 mutations in Finland is of great
importance in clinical diagnostics of breast and/or ovarian carcinoma families, as
BRCA1/BRCA2 mutation carrier detection may be initiated with the screening of regions
where known mutations reside, and then, if no mutation is found, screening of the entire
coding regions may be required. In certain areas of Finland, BRCA1/BRCA2 mutation testing
is further facilitated by the specific and narrow mutation spectra. In contrast, in the capital
region of Helsinki, all Finnish BRCA1/BRCA2 mutations have been observed. The existence
of these founder mutations has also facilitated studies evaluating the prevalence of BRCA1
and BRCA2 germline mutations in various study populations in Finland: in unselected breast
(Syrjäkoski et al., 2000) and ovarian carcinoma (III) patients, and in ovarian carcinoma
families (IV).
2 Contribution of BRCA1 and BRCA2 germline mutations to ovarian carcinoma in
Finland (III, IV), and breast and ovarian carcinoma phenotypes of Finnish BRCA1 and
BRCA2 mutation carriers (I, III, IV)
Germline mutations of the BRCA1 and BRCA2 genes account for a varying fraction of
hereditary breast and ovarian carcinoma in different populations, and BRCA1/BRCA2
mutation spectra vary among populations as well (Szabo and King, 1997; Neuhausen, 1999).
Here, we detected a BRCA1 or BRCA2 germline mutation in 5.6% (13/233) of unselected
Finnish ovarian carcinoma patients (III), and among the Finnish ovarian carcinoma families,
the frequency of BRCA1/BRCA2 mutations was 26% (6/23) (IV). No novel mutations were
identified in Study III, and seven founder mutations accounted for 12 of the 13 mutations
detected, emphasizing the significance of BRCA1/BRCA2 founder mutations in Finland. In
Study IV, four different founder mutations accounted for the five mutation-positive families
74
observed, and in one family, a novel, apparently disease-causing BRCA2 missense mutation
had been identified in a previous study by Roth et al. (1998). Large genomic rearrangements
of BRCA1 that have recently been identified in several breast/ovarian cancer families (Petrij-
Bosch et al., 1997; Puget et al., 1997, 1999; Swensen et al., 1997; Rohlfs et al., 2000; Unger
et al., 2000) and have also been described as major BRCA1 founder mutations in the
Netherlands (Petrij-Bosch et al., 1997) do not seem to be significant in the Finnish
population, as no such mutations were detected here (III) or in another study of 80 Finnish
breast and/or ovarian carcinoma families (Lahti-Domenici et al., 2001). The mutation
frequencies reported here illustrate the burden of the previously identified Finnish
BRCA1/BRCA2 mutations on Finnish ovarian carcinoma patients and families. Moreover, we
believe that our results provide good estimates of the actual impact of BRCA1/BRCA2
mutations in these patient groups since founder mutations appear to account for most
BRCA1/BRCA2 mutation in Finland (Vehmanen et al., 1997a, 1997b; Huusko et al., 1998;
unpublished data). Among unselected ovarian carcinoma patients, the real mutation
frequencies might, however, be somewhat higher, as BRCA1/BRCA2 mutation-associated
ovarian carcinomas are typically of advanced stage, and moderate to high grade (Rubin et al.,
1996; Aida et al., 1998; Boyd et al., 2000; Moslehi et al., 2000; Werness et al., 2000; Risch et
al., 2001; IV), but in our retrospectively collected study cohort, such tumours were slightly
underrepresented. Moreover, mucinous carcinoma were somewhat overrepresented in our
study cohort, while carcinomas of mucinous subtype appear to be very rare in BRCA1/BRCA2
mutation carriers (Boyd et al., 2000; Moslehi et al., 2000; Werness et al., 2000; Risch et al.,
2001; III; IV).
Germline mutations in BRCA1 and BRCA2 have been proposed to be sufficient to
explain the majority of hereditary ovarian carcinoma (Gayther et al., 1999; Antoniou et al.,
2000). Here, a BRCA1 or BRCA2 mutation was found in all three site-specific ovarian
carcinoma families with three affected cases, whereas most families (9/11) with only two
ovarian carcinomas were mutation-negative (Roth et al., 1998; IV). Similarly, among the
unselected ovarian carcinoma patients (III), all 13 patients who reported one first- or second-
degree relative with ovarian carcinoma only were mutation-negative. Our findings are in line
with reports from other populations; in British families with two ovarian carcinoma cases in
first- or second-degree relatives, BRCA1/BRCA2 mutations were found in only 20%, while
mutations were detected in 70% of families with at least three cases of ovarian carcinoma and
no more than one case of breast carcinoma (Gayther et al., 1999). Furthermore, a study
consisting of French, British, and American families has suggested that the vast majority of
site-specific ovarian carcinoma families with at least three affected family members are
carriers of BRCA1 germline mutations (Steichen-Gersdorf et al., 1994). Most ovarian
75
carcinoma families without BRCA1/BRCA2 mutations may be explained by incomplete
sensitivity of mutation detection, chance clustering of sporadic cases, and non-genetic
familial factors (Gayther et al., 1999; Antoniou et al., 2000). Additionally, germline
mutations in the genes involved in the HNPCC syndrome may account for a small proportion
of BRCA1/BRCA2 mutation-negative ovarian carcinoma families (Rubin et al., 1998).
However, in some families, germline mutations in yet unknown ovarian cancer-susceptibility
genes, possibly with lower penetrance, may be present (Sutcliffe et al., 2000).
In contrast to ovarian carcinoma, where germline mutations of BRCA1 and BRCA2
appear to explain the majority of hereditary cases (Gayther et al., 1999; Antoniou et al.,
2000), only a minority of hereditary breast carcinomas seem to be due to BRCA1/BRCA2
germline mutations, and evidence suggests that other still undiscovered breast cancer-
susceptibility genes also exist (Serova et al., 1997; Ford et al., 1998; Kainu et al., 2000;
Antoniou et al., 2001; Cui et al., 2001; Eerola et al., 2001a). In Finnish families with at least
three cases of breast or ovarian carcinoma in first- or second-degree relatives, the frequency
of BRCA1/BRCA2 mutations was only 5% among site-specific breast carcinoma families,
while in families with both breast and ovarian carcinoma, 50% were found to be mutation-
positive (Vahteristo et al., 2001). In line with this, the frequency of BRCA1/BRCA2 mutations
was considerably higher in unselected Finnish ovarian carcinoma patients (5.6%) (III) than in
unselected Finnish breast carcinoma patients (1.8%) (Syrjäkoski et al., 2000).
Most hereditary ovarian carcinomas have been suggested to be due to mutations in
BRCA1, while the contribution of BRCA2 mutations to ovarian carcinoma has been proposed
to be smaller (Ford et al., 1998). Accordingly, we found a significantly higher proportion of
ovarian carcinoma in Finnish families with BRCA1 mutations than in those with BRCA2
mutations (I). Also among unselected ovarian carcinoma patients, BRCA1 mutations were
considerably more frequent than mutations in BRCA2 (4.7% and 0.9%, respectively) (III). In
contrast, among unselected Finnish breast carcinoma patients, the reverse was observed
(BRCA1 and BRCA2 mutation frequencies being 0.4% and 1.4%, respectively) (Syrjäkoski et
al., 2000). The frequencies of BRCA1 and BRCA2 mutations among unselected ovarian
carcinoma patients vary considerably between populations (for BRCA1 between 2% and 27%,
and for BRCA2 between 0% and 14%) (Johannesdottir et al., 1996; Takahashi et al., 1996;
Stratton et al., 1997; Berchuck et al., 1998; Rubin et al., 1998; Janezic et al., 1999; Tonin et
al., 1999; Anton-Culver et al., 2000; Moslehi et al., 2000; Tobias et al., 2000; van der Looij
et al., 2000; Risch et al., 2001; Smith et al., 2001; Khoo et al., 2002; Liede et al., 2002). The
highest BRCA1/BRCA2 mutation frequencies have been described among Ashkenazi Jews
(25–41%) (Moslehi et al., 2000; Tobias et al., 2000) and Pakistanis (16%) (Liede et al.,
2002). In the Icelandic population, approximately 8% of unselected ovarian carcinoma
76
patients are carriers of the BRCA2 999del5 mutation (Johannesdottir et al., 1996).
Frequencies similar to the ones observed here have been reported in admixed British and
American populations (2–9% for BRCA1, and 1–3% for BRCA2) (Stratton et al., 1997;
Berchuck et al., 1998; Rubin et al., 1998; Janezic et al., 1999; Anton-Culver et al., 2000;
Smith et al., 2001).
For genetic couselling purposes, it is important to identify clinical risk factors that
could best predict the presence of a BRCA1/BRCA2 mutation in a family so that diagnostic
mutation screening could be directed to potential mutation carrier families. Among the
unselected Finnish ovarian carcinoma patients, the most significant predictor of a
BRCA1/BRCA2 mutation was the presence of both breast and ovarian carcinoma in the same
patient (III). Furthermore, family history of breast carcinoma was strongly related to mutation
carrier status. We also found that BRCA1/BRCA2 mutation frequencies were higher among
patients who had a history of early onset breast carcinoma (<40 or <50 years) as compared
with those with a history of breast carcinoma diagnosed at a later age (>50 years). The
number of breast cancer cases for which information on the age at onset was available was,
however, small, and no statistically significant association between mutation carrier status
and young age (<50 years) at breast cancer onset was observed (III). In the phenotype analysis
of the 71 Finnish BRCA1/BRCA2 mutation-positive families (I), young age at breast cancer
diagnosis was characteristic for both BRCA1 and BRCA2 mutation carriers; more than 60% of
breast carcinomas in BRCA1 and BRCA2 mutation carriers had been diagnosed before the age
of 50 years, while in the general breast cancer patient population, the corresponding
proportion was only about 25%. Among Finnish breast cancer families with at least three
breast or ovarian cancers in first- or second-degree relatives, the strongest predictors of a
BRCA1 or BRCA2 mutation are age of the youngest breast cancer patient and number of
ovarian cancer cases in the family (Vahteristo et al., 2001). In contrast to the present study
(III), the occurrence of both breast and ovarian carcinoma in the same woman was not an
independent predictor of a BRCA1/BRCA2 mutation in the study of Vahteristo et al. (2001),
probably because it is closely associated with ovarian carcinoma cases overall. In addition to
the number of breast and ovarian cancer cases in a family and the age at breast cancer onset,
the number and relationship of unaffected family members, along with their current ages or
ages at death, are taken into consideration in several models developed to estimate the
probability that a BRCA1 or BRCA2 mutation is present in a family (Berry et al., 1997;
Parmigiani et al., 1998; Schmidt et al., 1998).
In the phenotype analysis of the 71 Finnish BRCA1/BRCA2 mutation-positive families,
the age at ovarian carcinoma onset was significantly younger in families with BRCA1
mutations than in those with BRCA2 mutations (I); in the BRCA1 families, almost 60% of the
77
ovarian carcinomas had been diagnosed before the age of 50 years, whereas in the BRCA2
families, the corresponding proportion was less than 20% (I). The distribution of ages at
diagnosis of ovarian carcinoma was similar in the BRCA2 mutation carriers and in the general
Finnish ovarian carcinoma patient population (I). Several other studies have also reported that
BRCA1 mutation-associated ovarian carcinomas are diagnosed on average at a younger age
than sporadic ones, while for BRCA2 mutation-associated ovarian carcinomas, the mean age
at ovarian cancer onset does not differ from that of sporadic patients (Boyd et al., 2000;
Moslehi et al., 2000; Risch et al., 2001; IV). Furthermore, the proportion of ovarian cancer
has been suggested to vary according to the location of the mutation in BRCA1 and BRCA2
(Gayther et al., 1995, 1997b; Risch et al., 2001; Thompson and Easton, 2001, 2002). Here,
we observed that in the BRCA1 mutation-positive families with mutations in exon 11, the
proportion of ovarian carcinoma was significantly higher than in the families carrying
mutations 3´ of this exon (I). This is supported by other studies that report a tendency for
BRCA1 families with mutations towards the 3´ end of the gene to have a lower than average
proportion of ovarian cancer cases (Gayther et al., 1995; Risch et al., 2001; Thompson and
Easton, 2002). In BRCA2, mutations located in the OCCR have been found to be associated
with a higher ratio of ovarian cancer to breast cancer than mutations located outside this
region (Gayther et al., 1997b; Thompson and Easton, 2001). The relationship between the
location of the mutation within or outside the OCCR and the proportion of ovarian carcinoma
could not be studied here as only a few families carried mutations located within the OCCR
defined by Gayther et al. (1997b).
Studies on cancer risks in BRCA1/BRCA2 mutation-positive families have reported
similar estimates of breast cancer risk for both BRCA1 and BRCA2 mutation carriers (35–
87% and 26–84%, respectively, by the age of 70 years), but higher ovarian carcinoma risks
for BRCA1 mutation carriers as compared with BRCA2 mutation carriers (26–66% for
BRCA1 mutation carriers versus 9–27% for BRCA2 mutation carriers by age 70) (Ford et al.,
1994, 1998; Easton et al., 1995; Narod et al., 1995; Schubert et al., 1997; Thorlacius et al.,
1998; the BCLC, 1999; Warner et al., 1999; ABCSG, 2000; Antoniou et al., 2000, 2002;
Satagopan et al., 2001). Nevertheless, the risk of ovarian carcinoma in BRCA2 mutation
carriers is still considerable. Interestingly, we observed BRCA2 mutations in four and BRCA1
mutations in two of the 23 Finnish ovarian carcinoma families (IV), and none of the BRCA2
mutations were located within the suggested OCCR (Gayther et al., 1997b; Thompson and
Easton, 2001). In Finnish families with at least three cases of breast or ovarian carcinoma in
first- or second-degree relatives, the cumulative risk of subsequent ovarian cancer for breast
cancer patients by the age of 70 years has been reported to be 29% in BRCA1 and 8% in
BRCA2 mutation-positive families (Eerola et al., 2001a). However, by the age of 80 years, the
78
risk reaches approximately 30% for both BRCA1 and BRCA2 mutation carriers, and the
higher relative risk of subsequent ovarian cancer for BRCA1 mutation carriers [standardized
incidence ratio (SIR) 61 vs. 38] has been attributed to the earlier onset of ovarian cancer in
BRCA1 mutation-associated families (Eerola et al., 2001a).
Apart from age, no significant differences in clinicopathological characteristics have
been reported between BRCA1 and BRCA2 mutation-associated ovarian carcinomas (Boyd et
al., 2000; Moslehi et al., 2000; Ramus et al., 2001; Risch et al., 2001). In our studies (III, IV),
all BRCA1 mutation-associated ovarian carcinomas, except for one, were of serous or
undifferentiated histology, while BRCA2 mutation-associated ovarian carcinomas were of
various histological subtypes. The number of BRCA1/BRCA2 mutation-associated ovarian
carcinomas was, however, small, and in larger studies, ovarian carcinomas of both BRCA1
and BRCA2 mutation carriers have been reported to typically be of serous histology (Rubin et
al., 1996; Aida et al., 1998; Boyd et al., 2000; Moslehi et al., 2000; Werness et al., 2000;
Risch et al., 2001). No carcinomas of mucinous subtype were seen among mutation carriers
(III, IV), and almost all BRCA1/BRCA2 mutation-associated carcinomas were of advanced
stage, and moderate to high grade (IV), which is in line with other reports (Rubin et al., 1996;
Aida et al., 1998; Boyd et al., 2000; Moslehi et al., 2000; Werness et al., 2000; Risch et al.,
2001). Aneuploid tumours were more frequent in BRCA1/BRCA2 mutation carriers than in
non-carriers (IV). Also in the study of Jóhannsson et al. (1997), four out of five BRCA1
mutation-associated ovarian carcinomas were aneuploid. In contrast to breast carcinoma,
where the histopathological phenotype has been suggested to be useful in predicting BRCA1
mutation carrier status (Eisinger et al., 1999; Cortesi et al., 2000; Lidereau et al., 2000), no
ovarian tumour characteristics have been found to be significant predictors of BRCA1/BRCA2
mutations.
79
SUMMARY AND CONCLUSIONS
We examined ancestral origins and geographical distribution of Finnish families with
recurrent BRCA1/BRCA2 mutations. In addition, we studied breast and ovarian carcinoma
phenotypes of Finnish BRCA1 and BRCA2 mutation carriers and evaluated the prevalence of
BRCA1 and BRCA2 founder mutations in Finnish ovarian carcinoma patients and ovarian
carcinoma families. Our conclusions are as follows:
1) Carriers of the same recurrent BRCA1/BRCA2 mutation, except for those with the BRCA2
999del5 mutation, shared an identical core haplotype, indicating a common ancestor for
the families. In families with the 999del5 mutation, two distinct core haplotypes were
seen. Geographical clustering of the 999del5 mutation-positive families as well as the
population history of Finland support the hypothesis that the two distinct haplotypes are
due to gene conversion. The lengths of the shared core haplotypes varied widely (1.6 to
26 cM) between families with different mutations, and the estimates of time elapsed from
a common ancestor varied accordingly (6 to 32 generations). Finnish families with one of
the 999del5 mutation-associated haplotypes shared a 0.5 cM haplotype with Icelandic
families with the same mutation, which may indicate a common ancient origin for the
Finnish and Icelandic 999del5 mutation-positive families. However, distinct mutational
events cannot be ruled out.
2) In families with the founder mutations, birthplaces of the parents, grandparents, or great-
grandparents of index patients were found to be clustered within distinct geographical
areas of Finland. This facilitates BRCA1/BRCA2 mutation testing in certain regions where
the mutation spectra are still narrow.
3) The proportion of ovarian carcinoma was significantly higher in BRCA1 than BRCA2
mutation-positive families. Moreover, within BRCA1 families, the proportion of ovarian
carcinoma was significantly higher in families carrying mutations in exon 11 as compared
with those carrying mutations 3´ of this exon. The mean age at ovarian carcinoma onset
was significantly younger in families with BRCA1 mutations than in those with BRCA2
mutations (51 years vs. 61 years). For breast carcinoma, the distribution of ages at
diagnosis was similar in BRCA1 and BRCA2 mutation-positive families, and mutation
carriers were characterized by early age at diagnosis (mean, 46 years and 48 years).
Phenotypic characteristics associated with BRCA1 and BRCA2 germline mutations are
80
important when deciding clinical management of mutation carriers.
4) Among unselected ovarian carcinoma patients, germline mutations of BRCA1 and BRCA2
were found in 4.7% and 0.9% of patients, respectively. The most significant predictor of a
BRCA1/BRCA2 mutation was the presence of both breast and ovarian carcinoma in the
same patient. In addition, family history of breast carcinoma in first- and second-degree
relatives was strongly related to mutation carrier status.
5) Germline mutations of BRCA1 and BRCA2 may account for most Finnish site-specific
ovarian carcinoma families with at least three affected cases. However, a minority of
families with only two ovarian carcinomas can be linked to BRCA1/BRCA2 germline
mutations. A significant fraction of the minor familial aggregation of ovarian carcinoma
may be explained by chance clustering of sporadic cases and by shared environmental
factors. Nevertheless, unidentified ovarian cancer-susceptibility genes, possibly with
lower penetrance, may also exist.
81
ACKNOWLEDGEMENTS
This study was carried out at the Department of Obstetrics and Gynaecology, Helsinki
University Central Hospital, during 1996–2002, and for a six-month period in 1998–1999, at
the Cancer Genetics Branch, National Human Genome Research Institute, National Institutes
of Health (NIH), USA. Professors Markku Seppälä and Olavi Ylikorkala, the former and
present heads of the Department of Obstetrics and Gynaecology, Docent Maija Haukkamaa,
the administrative head of the Department, and Dr. Jeffrey Trent, the chief of the Cancer
Genetics Branch, are acknowledged for providing excellent research facilities.
I am thankful to all the people I have worked with over these years. The support and
encouragement I received from numerous colleagues, friends, and family members have been
of great value. I am indebted to all patients and their family members for volunteering to
participate in these studies. I especially wish to acknowledge:
Docent Heli Nevanlinna, PhD, my supervisor, for introducing me to the world of
hereditary breast and ovarian cancer, for providing the facilities required, and for guidance
throughout this project. Heli has patiently reminded me not to forget to look for the forest
through the trees.
Professor Päivi Peltomäki, MD, PhD, and Docent Ulla Puistola, MD, PhD, the official
reviewers of this thesis, for their thorough review and valuable comments.
All my co-authors in Helsinki, Kuopio, Tampere, and Oulu, as well as in Iceland, in
Sweden, and at the NIH, for their contribution to this work. In particular, I owe my thanks to
Professor Juha Kere, MD, PhD, for his expertise in handling haplotype data, for helpful
discussions about population genetics, and for his friendly attitude; to Docent Ralf Bützow,
MD, PhD, for providing the samples and clinical information for the study of unselected
ovarian carcinoma patients and for his valuable contribution; to Docent Annika Auranen,
MD, PhD, for providing the material for the study of ovarian carcinoma families and for
pleasant collaboration; to Docent Robert Winqvist, MD, PhD, and Pia Huusko, MD, PhD, for
their substantial contribution to the study of Finnish founder mutations; to Dr. Rósa Björk
Barkardóttir for her kind help and her essential role in the study of the 999del5 mutation; to
Professor Olli-Pekka Kallioniemi, MD, PhD, for the opportunity to work in his laboratory at
the NIH and for his support and interest towards my work; to Elizabeth Gillanders for her
patient guidance in the laboratory and for the times we shared outside the lab during my visit
at the NIH. Special thanks are due to Pia Vahteristo, MSc, Anitta Tamminen, MSc,
Hannaleena Eerola, MD, PhD, and Paula Vehmanen, PhD, the other ”girls” from Heli’s lab,
for smooth collaboration and for sharing the ups and downs of scientific work. Their
82
friendship throughout the years and the many discussions about work and life in general have
been invaluable; they have made the load seem lighter. Pia is also warmly thanked for
companionship at a number of congresses.
Minna Merikivi, Merja Lindfors, and Gynel Arifdshan for sample collection, expert
technical assistance, and friendship.
My colleagues and friends in the research laboratory of the Department of Obstetrics
and Gynaecology and the Department of Clinical Chemistry: Heini Lassus, Johanna Tapper,
Annukka Paju, Outi Kilpivaara, Can Hekim, Erik Mandelin, Susanna Lintula, Kristina
Hotakainen, Patrik Finne, Hannu Koistinen, Marianne Niemelä, Jakob Stenman, Liisa Airas,
Ping Wu, Piia Vuorela, Oso Rissanen, Taina Grönholm, Anne Ahmanheimo, Maarit
Leinimaa, Sirpa Stick, and all the others, for the friendly working atmosphere. Help and
advice when in trouble with work, and companionship during lunch and coffee breaks have
always been available. Activities outside the lab have also been refreshing. I express my
thanks to senior researchers Professor Ulf-Håkan Stenman, MD, PhD, for excellent working
facilities, and Docent Riitta Koistinen, PhD, for support and interest towards my work.
My friends outside the laboratory for support and encouragement and for many joyful
and relaxing times together. I especially wish to thank Karin Haraldsson for warm
companionship during our stay in the US and for ongoing friendship.
My parents, Unnukka and Rauno, for their constant love, care, and support throughout
my life. My brother Antti for love and for sharing so much. My sister Sara for many joyful
moments together. My grandmother Sirkka-Liisa for her love and support. Esko for generous
help and companionship on many nice trips. Anitta for her encouragement, support, and
interest in my work. Salli and Lauri for support and many relaxing times in Ristiina. Emmi
for her patience and understanding and her positive attitude and ready smile.
My warmest thanks are due to Jari for his patience, constant love, and support. Thank
you for sharing all kinds of days with me and for teaching me to adapt a positive, not-so-
serious attitude towards life. I am also thankful to our unborn child for reminding me of the
real meaning of life and for making me work efficiently to get this project finished.
Financial support of the Biomedicum Helsinki Foundation, the Cancer Society of
Finland, the Clinical Research Fund of the Helsinki University Central Hospital, the Ella and
Georg Ehrnrooth Foundation, the Ida Montin Foundation, the Paulo Foundation, and the
University of Helsinki is gratefully acknowledged.
Helsinki, November 2002
83
REFERENCES
Abbott DW, Freeman ML, Holt JT. Double-strand break repair deficiency and radiation sensitivity in BRCA2
mutant cancer cells. J Natl Cancer Inst 90: 978-85, 1998
Aida H, Takakuwa K, Nagata H, Tsuneki I, Takano M, Tsuji S, Takahashi T, Sonoda T, Hatae M, Takahashi K,
Hasegawa K, Mizunuma H, Toyoda N, Kamata H, Torii Y, Saito N, Tanaka K, Yakushiji M, Araki T,
Tanaka K. Clinical features of ovarian cancer in Japanese women with germ-line mutations of BRCA1. Clin
Cancer Res 4: 235-40, 1998
Albertsen HM, Smith SA, Mazoyer S, Fujimoto E, Stevens J, Williams B, Rodriguez P, Cropp CS, Slijepcevic
P, Carlson M, Robertson M, Bradley P, Lawrence E, Harrington T, Sheng ZM, Hoopes R, Sternberg N,
Brothman A, Callahan R, Ponder BAJ, White R. A physical map and candidate genes in the BRCA1 region
on chromosome 17q12-21. Nat Genet 7: 472-9, 1994
Altmüller J, Palmer LJ, Fischer G, Scherb H, Wjst M. Genomewide scans of complex human diseases: true
linkage is hard to find. Am J Hum Genet 69: 936-50, 2001
The American Society of Clinical Oncology. Statement of the American Society of Clinical Oncology: genetic
testing for cancer susceptibility, Adopted on February 20, 1996. J Clin Oncol 14: 1730-6; discussion 1737-
40, 1996
Anglian Breast Cancer Study Group. Prevalence and penetrance of BRCA1 and BRCA2 mutations in a
population-based series of breast cancer cases. Br J Cancer 83: 1301-8, 2000
Anton-Culver H, Cohen PF, Gildea ME, Ziogas A. Characteristics of BRCA1 mutations in a population-based
case series of breast and ovarian cancer. Eur J Cancer 36: 1200-8, 2000
Antoniou AC, Gayther SA, Stratton JF, Ponder BA, Easton DF. Risk models for familial ovarian and breast
cancer. Genet Epidemiol 18: 173-90, 2000
Antoniou AC, Pharoah PD, McMullan G, Day NE, Ponder BA, Easton D. Evidence for further breast cancer
susceptibility genes in addition to BRCA1 and BRCA2 in a population-based study. Genet Epidemiol 21:
1-18, 2001
Antoniou AC, Pharoah PD, McMullan G, Day NE, Stratton MR, Peto J, Ponder BJ, Easton DF. A
comprehensive model for familial breast cancer incorporating BRCA1, BRCA2 and other genes. Br J
Cancer 86: 76-83, 2002
Arason A, Barkardottir RB, Egilsson V. Linkage analysis of chromosome 17q markers and breast-ovarian
cancer in Icelandic families, and possible relationship to prostatic cancer. Am J Hum Genet 52: 711-7,
1993
Armes JE, Trute L, White D, Southey MC, Hammet F, Tesoriero A, Hutchins AM, Dite GS, McCredie MR,
Giles GG, Hopper JL, Venter DJ. Distinct molecular pathogeneses of early-onset breast cancers in BRCA1
and BRCA2 mutation carriers: a population-based study. Cancer Res 59: 2011-7, 1999
Arver B, Du Q, Chen J, Luo L, Lindblom A. Hereditary breast cancer: a review. Semin Cancer Biol 10: 271-88,
2000
Auranen A, Grénman S, Klemi PJ. Immunohistochemically detected p53 and HER-2/neu expression and nuclear
DNA content in familial epithelial ovarian carcinomas. Cancer 79: 2147-53, 1997
84
Auranen A, Grénman S, Mäkinen J, Pukkala E, Sankila R, Salmi T. Borderline ovarian tumors in Finland:
epidemiology and familial occurrence. Am J Epidemiol 144: 548-53, 1996a
Auranen A, Iselius L. Segregation analysis of epithelial ovarian cancer in Finland. Br J Cancer 77: 1537-41,
1998
Auranen A, Pukkala E, Mäkinen J, Sankila R, Grénman S, Salmi T. Cancer incidence in the first-degree relatives
of ovarian cancer patients. Br J Cancer 74: 280-4, 1996b
Barkardottir RB, Arason A, Egilsson V, Gudmundsson J, Jonasdottir A, Johannesdottir G. Chromosome 17q-
linkage seems to be infrequent in Icelandic families at risk of breast cancer. Acta Oncol 34: 657-62, 1995
Ben David Y, Chetrit A, Hirsh-Yechezkel G, Friedman E, Beck BD, Beller U, Ben-Baruch G, Fishman A,
Levavi H, Lubin F, Menczer J, Piura B, Struewing JP, Modan B. Effect of BRCA mutations on the length
of survival in epithelial ovarian tumors. J Clin Oncol 20: 463-6, 2002
Berchuck A, Heron KA, Carney ME, Lancaster JM, Fraser EG, Vinson VL, Deffenbaugh AM, Miron A, Marks
JR, Futreal PA, Frank TS. Frequency of germline and somatic BRCA1 mutations in ovarian cancer. Clin
Cancer Res 4: 2433-7, 1998
Berg JW, Hutter RV. Breast cancer. Cancer 75: 257-69, 1995
Bergman A, Einbeigi Z, Olofsson U, Taib Z, Wallgren A, Karlsson P, Wahlström J, Martinsson T, Nordling M.
The western Swedish BRCA1 founder mutation 3171ins5; a 3.7 cM conserved haplotype of today is a
reminiscence of a 1500-year-old mutation. Eur J Hum Genet 9: 787-93, 2001
Bergthorsson JT, Jonasdottir A, Johannesdottir G, Arason A, Egilsson V, Gayther S, Borg A, Hakanson S,
Ingvarsson S, Barkardottir RB. Identification of a novel splice-site mutation of the BRCA1 gene in two
breast cancer families: screening reveals low frequency in Icelandic breast cancer patients. Hum Mutat
Suppl: S195-7, 1998
Berman DB, Wagner-Costalas J, Schultz DC, Lynch HT, Daly M, Godwin AK. Two distinct origins of a
common BRCA1 mutation in breast-ovarian cancer families: a genetic study of 15 185delAG-mutation
kindreds. Cancer Res 56: 2539-45, 1996
Berry DA, Parmigiani G, Sanchez J, Schildkraut J, Winer E. Probability of carrying a mutation of breast-ovarian
cancer gene BRCA1 based on family history. J Natl Cancer Inst 89: 227-38, 1997
Borg Å, Sandberg T, Nilsson K, Johannsson O, Klinker M, Måsbäck A, Westerdahl J, Olsson H, Ingvar C. High
frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive
melanoma families. J Natl Cancer Inst 92: 1260-6, 2000
Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction
fragment length polymorphisms. Am J Hum Genet 32: 314-31, 1980
Boyd J. Molecular genetics of hereditary ovarian cancer. Oncology (Huntingt) 12: 399-406, 1998
Boyd J, Sonoda Y, Federici MG, Bogomolniy F, Rhei E, Maresco DL, Saigo PE, Almadrones LA, Barakat RR,
Brown CL, Chi DS, Curtin JP, Poynor EA, Hoskins WJ. Clinicopathologic features of BRCA-linked and
sporadic ovarian cancer. JAMA 283: 2260-5, 2000
The Breast Cancer Information Core database. http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/
Bic/
The Breast Cancer Linkage Consortium. Pathology of familial breast cancer: differences between breast cancers
in carriers of BRCA1 or BRCA2 mutations and sporadic cases. Lancet 349: 1505-10, 1997
85
The Breast Cancer Linkage Consortium. Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst 91: 1310-
6, 1999
Brunet JS, Ghadirian P, Rebbeck TR, Lerman C, Garber JE, Tonin PN, Abrahamson J, Foulkes WD, Daly M,
Wagner-Costalas J, Godwin A, Olopade OI, Moslehi R, Liede A, Futreal PA, Weber BL, Lenoir GM,
Lynch HT, Narod SA. Effect of smoking on breast cancer in carriers of mutant BRCA1 or BRCA2 genes. J
Natl Cancer Inst 90: 761-6, 1998
Brzovic PS, Meza JE, King M-C, Klevit RE. BRCA1 RING domain cancer-predisposing mutations. Structural
consequences and effects on protein-protein interactions. J Biol Chem 276: 41399-406, 2001
Buchholz TA, Wu X, Hussain A, Tucker SL, Mills GB, Haffty B, Bergh S, Story M, Geara FB, Brock WA.
Evidence of haplotype insufficiency in human cells containing a germline mutation in BRCA1 or BRCA2.
Int J Cancer 97: 557-61, 2002
Buller RE, Lallas TA, Shahin MS, Sood AK, Hatterman-Zogg M, Anderson B, Sorosky JI, Kirby PA. The p53
mutational spectrum associated with BRCA1 mutant ovarian cancer. Clin Cancer Res 7: 831-8, 2001
Callen DF, Thompson AD, Shen Y, Phillips HA, Richards RI, Mulley JC, Sutherland GR. Incidence and origin
of "null" alleles in the (AC)n microsatellite markers. Am J Hum Genet 52: 922-7, 1993
Castilla LH, Couch FJ, Erdos MR, Hoskins KF, Calzone K, Garber JE, Boyd J, Lubin MB, Deshano ML, Brody
LC, Collins FS, Weber BL. Mutations in the BRCA1 gene in families with early-onset breast and ovarian
cancer. Nat Genet 8: 387-91, 1994
Cavalieri E, Frenkel K, Liehr JG, Rogan E, Roy D. Estrogens as endogenous genotoxic agents - DNA adducts
and mutations. J Natl Cancer Inst Monogr: 75-93, 2000
Chen J, Silver DP, Walpita D, Cantor SB, Gazdar AF, Tomlinson G, Couch FJ, Weber BL, Ashley T,
Livingston DM, Scully R. Stable interaction between the products of the BRCA1 and BRCA2 tumor
suppressor genes in mitotic and meiotic cells. Mol Cell 2: 317-28, 1998
Claus EB, Risch N, Thompson WD. Genetic analysis of breast cancer in the cancer and steroid hormone study.
Am J Hum Genet 48: 232-42, 1991
Claus EB, Schildkraut JM, Thompson WD, Risch NJ. The genetic attributable risk of breast and ovarian cancer.
Cancer 77: 2318-24, 1996
Compagni A, Christofori G. Recent advances in research on multistage tumorigenesis. Br J Cancer 83: 1-5, 2000
Cortesi L, Turchetti D, Bertoni C, Bellei R, Mangone L, Vinceti M, Federico M, Silingardi V, Ferrari S.
Comparison between genotype and phenotype identifies a high-risk population carrying BRCA1 mutations.
Genes Chromosomes Cancer 27: 130-5, 2000
Couch FJ, DeShano ML, Blackwood MA, Calzone K, Stopfer J, Campeau L, Ganguly A, Rebbeck T, Weber
BL. BRCA1 mutations in women attending clinics that evaluate the risk of breast cancer. N Engl J Med
336: 1409-15, 1997
Couch FJ, Rommens JM, Neuhausen SL, Bélanger C, Dumont M, Abel K, Bell R, Berry S, Bogden R, Cannon-
Albright L, Farid L, Frye C, Hattier T, Janecki T, Jiang P, Kehrer R, Leblanc JF, McArthur-Morrison J,
McSweeney D, Miki Y, Peng Y, Samson C, Schroeder M, Snyder SC, Stringfellow M, Stroup C, Swedlund
B, Swensen J, Teng D, Thakur S, Tran T, Tranchant M, Welver-Feldhaus J, Wong AKC, Shizuya H,
Labrie F, Skolnick MH, Goldgar DE, Kamb A, Weber BL, Tavtigian SV, Simard J. Generation of an
integrated transcription map of the BRCA2 region on chromosome 13q12-q13. Genomics 36: 86-99, 1996
86
Crook T, Brooks LA, Crossland S, Osin P, Barker KT, Waller J, Philp E, Smith PD, Yulug I, Peto J, Parker G,
Allday MJ, Crompton MR, Gusterson BA. p53 mutation with frequent novel codons but not a mutator
phenotype in BRCA1- and BRCA2-associated breast tumours. Oncogene 17: 1681-9, 1998
Cui J, Antoniou AC, Dite GS, Southey MC, Venter DJ, Easton DF, Giles GG, McCredie MR, Hopper JL. After
BRCA1 and BRCA2-what next? Multifactorial segregation analyses of three-generation, population-based
Australian families affected by female breast cancer. Am J Hum Genet 68: 420-31, 2001
Daly MJ, Rioux JD, Schaffner SF, Hudson TJ, Lander ES. High-resolution haplotype structure in the human
genome. Nat Genet 29: 229-32, 2001
de la Chapelle A, Wright FA. Linkage disequilibrium mapping in isolated populations: the example of Finland
revisited. Proc Natl Acad Sci USA 95: 12416-23, 1998
de La Hoya M, Osorio A, Godino J, Sulleiro S, Tosar A, Perez-Segura P, Fernandez C, Rodríguez R, Díaz-
Rubio E, Benítez J, Devilee P, Caldés T. Association between BRCA1 and BRCA2 mutations and cancer
phenotype in Spanish breast/ovarian cancer families: implications for genetic testing. Int J Cancer 97: 466-
71, 2002
Deffenbaugh AM, Frank TS, Hoffman M, Cannon-Albright L, Neuhausen SL. Characterization of common
BRCA1 and BRCA2 variants. Genet Test 6: 119-21, 2002
Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau P, Marc S, Hazan J, Seboun E, Lathrop
M, Gyapay G, Morissette J, Weissenbach J. A comprehensive genetic map of the human genome based on
5,264 microsatellites. Nature 380: 152-4, 1996
Dickman PW, Hakulinen T, Luostarinen T, Pukkala E, Sankila R, Söderman B, Teppo L. Survival of cancer
patients in Finland 1955-1994. Acta Oncol 38: 1-103, 1999
Duffy SW, Nixon RM. Estimates of the likely prophylactic effect of tamoxifen in women with high risk BRCA1
and BRCA2 mutations. Br J Cancer 86: 218-21, 2002
Dunning AM, Chiano M, Smith NR, Dearden J, Gore M, Oakes S, Wilson C, Stratton M, Peto J, Easton D,
Clayton D, Ponder BAJ. Common BRCA1 variants and susceptibility to breast and ovarian cancer in the
general population. Hum Mol Genet 6: 285-9, 1997
Durocher F, Shattuck-Eidens D, McClure M, Labrie F, Skolnick MH, Goldgar DE, Simard J. Comparison of
BRCA1 polymorphisms, rare sequence variants and/or missense mutations in unaffected and breast/ovarian
cancer populations. Hum Mol Genet 5: 835-42, 1996
Easton DF, Bishop DT, Ford D, Crockford GP, the Breast Cancer Linkage Consortium. Genetic linkage analysis
in familial breast and ovarian cancer: results from 214 families. Am J Hum Genet 52: 678-701, 1993
Easton DF, Ford D, Bishop DT, the Breast Cancer Linkage Consortium. Breast and ovarian cancer incidence in
BRCA1-mutation carriers. Am J Hum Genet 56: 265-71, 1995
Easton DF, Steele L, Fields P, Ormiston W, Averill D, Daly PA, McManus R, Neuhausen SL, Ford D, Wooster
R, Cannon-Albright LA, Stratton MR, Goldgar DE. Cancer risks in two large breast cancer families linked
to BRCA2 on chromosome 13q12-13. Am J Hum Genet 61: 120-8, 1997
Eaves IA, Merriman TR, Barber RA, Nutland S, Tuomilehto-Wolf E, Tuomilehto J, Cucca F, Todd JA. The
genetically isolated populations of Finland and Sardinia may not be a panacea for linkage disequilibrium
mapping of common disease genes. Nat Genet 25: 320-3, 2000
Edmondson RJ, Monaghan JM. The epidemiology of ovarian cancer. Int J Gynecol Cancer 11: 423-9, 2001
87
Eerola H, Pukkala E, Pyrhönen S, Blomqvist C, Sankila R, Nevanlinna H. Risk of cancer in BRCA1 and
BRCA2 mutation-positive and -negative breast cancer families (Finland). Cancer Causes Control 12: 739-
46, 2001a
Eerola H, Vahteristo P, Sarantaus L, Kyyrönen P, Pyrhönen S, Blomqvist C, Pukkala E, Nevanlinna H, Sankila
R. Survival of breast cancer patients in BRCA1, BRCA2, and non-BRCA1/2 breast cancer families: a
relative survival analysis from Finland. Int J Cancer 93: 368-72, 2001b
Eisinger F, Alby N, Bremond A, Dauplat J, Espié M, Janiaud P, Kuttenn F, Lebrun JP, Lefranc JP, Pierret J,
Sobol H, Stoppa-Lyonnet D, Thouvenin D, Tristant H, Feingold J. Recommendations for medical
management of hereditary breast and ovarian cancer: the French National Ad Hoc Committee. Ann Oncol
9: 939-50, 1998
Eisinger F, Nogues C, Guinebretiere JM, Peyrat JP, Bardou VJ, Noguchi T, Vennin P, Sauvan R, Lidereau R,
Birnbaum D, Jacquemier J, Sobol H. Novel indications for BRCA1 screening using individual clinical and
morphological features. Int J Cancer 84: 263-7, 1999
Ekelund J, Lichtermann D, Hovatta I, Ellonen P, Suvisaari J, Terwilliger JD, Juvonen H, Varilo T, Arajarvi R,
Kokko-Sahin ML, Lönnqvist J, Peltonen L. Genome-wide scan for schizophrenia in the Finnish population:
evidence for a locus on chromosome 7q22. Hum Mol Genet 9: 1049-57, 2000
Ellisen LW, Haber DA. Hereditary breast cancer. Annu Rev Med 49: 425-36, 1998
Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature 411: 342-8, 2001
Fackenthal JD, Cartegni L, Krainer AR, Olopade OI. BRCA2 T2722R is a deleterious allele that causes exon
skipping. Am J Hum Genet 71: 625-31, 2002
Fan S, Wang J, Yuan R, Ma Y, Meng Q, Erdos MR, Pestell RG, Yuan F, Auborn KJ, Goldberg ID, Rosen EM.
BRCA1 inhibition of estrogen receptor signaling in transfected cells. Science 284: 1354-6, 1999
Fearon ER. Human cancer syndromes: clues to the origin and nature of cancer. Science 278: 1043-50, 1997
Fero ML, Randel E, Gurley KE, Roberts JM, Kemp CJ. The murine gene p27Kip1 is haplo-insufficient for
tumour suppression. Nature 396: 177-80, 1998
Feunteun J, Narod SA, Lynch HT, Watson P, Conway T, Lynch J, Parboosingh J, O'Connell P, White R, Lenoir
GM. A breast-ovarian cancer susceptibility gene maps to chromosome 17q21. Am J Hum Genet 52: 736-
42, 1993
The Finnish Cancer Registy. Cancer incidence in Finland in 1995. Cancer Statistics of the National Research
and Development Centre for Welfare and Health. Cancer Society of Finland Publication No. 58. Helsinki,
1997
The Finnish Cancer Registy. Cancer incidence in Finland in 1999. Cancer Statistics of the National Research
and Development Centre for Welfare and Health. Cancer Society of Finland Publication at
http://www.cancerregistry.fi/v99/v9900conts.html, 2002
Fodor FH, Weston A, Bleiweiss IJ, McCurdy LD, Walsh MM, Tartter PI, Brower ST, Eng CM. Frequency and
carrier risk associated with common BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer
patients. Am J Hum Genet 63: 45-51, 1998
Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE, the Breast Cancer Linkage Consortium. Risks of
cancer in BRCA1-mutation carriers. Cancer Res 54: 1791-4, 1994
88
Ford D, Easton DF, Peto J. Estimates of the gene frequency of BRCA1 and its contribution to breast and ovarian
cancer incidence. Am J Hum Genet 57: 1457-62, 1995
Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, Bishop DT, Weber B, Lenoir G, Chang-Claude
J, Sobol H, Teare MD, Struewing J, Arason A, Scherneck S, Peto J, Rebbeck TR, Tonin P, Neuhausen S,
Barkardottir R, Eyfjord J, Lynch H, Ponder BAJ, Gayther SA, Birch JM, Lindblom A, Stoppa-Lyonnet D,
Bignon YJ, Borg A, Hamann U, Haites N, Scott RJ, Maugard CM, Vasen H, Seitz S, Cannon-Albright L,
Schofield A, Zelada-Hedman M, the Breast Cancer Linkage Consortium. Genetic heterogeneity and
penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. Am J Hum Genet 62: 676-
89, 1998
Frank TS, Manley SA, Olopade OI, Cummings S, Garber JE, Bernhardt B, Antman K, Russo D, Wood ME,
Mullineau L, Isaacs C, Peshkin B, Buys S, Venne V, Rowley PT, Loader S, Offit K, Robson M, Hampel H,
Brener D, Winer EP, Clark S, Weber B, Strong LC, Rieger P, McClure M, Ward BE, Shattuch-Eidens D,
Oliphant A, Skolnick MH, Thomas A. Sequence analysis of BRCA1 and BRCA2: correlation of mutations
with family history and ovarian cancer risk. J Clin Oncol 16: 2417-25, 1998
Friedlander ML. Prognostic factors in ovarian cancer. Semin Oncol 25: 305-14, 1998
Friedman LS, Gayther SA, Kurosaki T, Gordon D, Noble B, Casey G, Ponder BA, Anton-Culver H. Mutation
analysis of BRCA1 and BRCA2 in a male breast cancer population. Am J Hum Genet 60: 313-9, 1997
Friedman LS, Szabo CI, Ostermeyer EA, Dowd P, Butler L, Park T, Lee MK, Goode EL, Rowell SE, King MC.
Novel inherited mutations and variable expressivity of BRCA1 alleles, including the founder mutation
185delAG in Ashkenazi Jewish families. Am J Hum Genet 57: 1284-97, 1995
Frisse L, Hudson RR, Bartoszewicz A, Wall JD, Donfack J, Di Rienzo A. Gene conversion and different
population histories may explain the contrast between polymorphism and linkage disequilibrium levels. Am
J Hum Genet 69: 831-43, 2001
Futreal PA, Barrett JC, Wiseman RW. Dinucleotide repeat polymorphism in the THRA1 gene. Hum Mol Genet
1: 66, 1992
Futreal PA, Kasprzyk A, Birney E, Mullikin JC, Wooster R, Stratton MR. Cancer and genomics. Nature 409:
850-2, 2001
Gaffney DK, Brohet RM, Lewis CM, Holden JA, Buys SS, Neuhausen SL, Steele L, Avizonis V, Stewart JR,
Cannon-Albright LA. Response to radiation therapy and prognosis in breast cancer patients with BRCA1
and BRCA2 mutations. Radiother Oncol 47: 129-36, 1998
Gayther SA, Harrington P, Russell P, Kharkevich G, Garkavtseva RF, Ponder BAJ. Frequently occurring germ-
line mutations of the BRCA1 gene in ovarian cancer families from Russia. Am J Hum Genet 60: 1239-42,
1997a
Gayther SA, Mangion J, Russell P, Seal S, Barfoot R, Ponder BA, Stratton MR, Easton D. Variation of risks of
breast and ovarian cancer associated with different germline mutations of the BRCA2 gene. Nat Genet 15:
103-5, 1997b
Gayther SA, Russell P, Harrington P, Antoniou AC, Easton DF, Ponder BAJ. The contribution of germline
BRCA1 and BRCA2 mutations to familial ovarian cancer: no evidence for other ovarian cancer-
susceptibility genes. Am J Hum Genet 65: 1021-9, 1999
89
Gayther SA, Warren W, Mazoyer S, Russell PA, Harrington PA, Chiano M, Seal S, Hamoudi R, van Rensburg
EJ, Dunning AM, Love R, Evans G, Easton D, Clayton D, Stratton MR, Ponder BA. Germline mutations of
the BRCA1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype
correlation. Nat Genet 11: 428-33, 1995
Généthon. ftp://ftp.genethon.fr/pub/Gmap/Nature-1995/
The Genome Database. http://gdbwww.gdb.org/
Goelen G, Teugels E, Bonduelle M, Neyns B, De Greve J. High frequency of BRCA1/2 germline mutations in
42 Belgian families with a small number of symptomatic subjects. J Med Genet 36: 304-8, 1999
Górski B, Byrski T, Huzarski T, Jakubowska A, Menkiszak J, Gronwald J, Pluzánska A, Bebenek M, Fischer-
Maliszewska L, Grzybowska E, Narod SA, Lubinski J. Founder mutations in the BRCA1 gene in Polish
families with breast-ovarian cancer. Am J Hum Genet 66: 1963-8, 2000
Gotlieb WH, Friedman E, Bar-Sade RB, Kruglikova A, Hirsh-Yechezkel G, Modan B, Inbar M, Davidson B,
Kopolovic J, Novikov I, Ben-Baruch G. Rates of Jewish ancestral mutations in BRCA1 and BRCA2 in
borderline ovarian tumors. J Natl Cancer Inst 90: 995-1000, 1998
Gowen LC, Avrutskaya AV, Latour AM, Koller BH, Leadon SA. BRCA1 required for transcription-coupled
repair of oxidative DNA damage. Science 281: 1009-12, 1998
Greenblatt MS, Chappuis PO, Bond JP, Hamel N, Foulkes WD. TP53 mutations in breast cancer associated with
BRCA1 or BRCA2 germ-line mutations: distinctive spectrum and structural distribution. Cancer Res 61:
4092-7, 2001
Gudmundsson J, Johannesdottir G, Arason A, Bergthorsson JT, Ingvarsson S, Egilsson V, Barkardottir RB.
Frequent occurrence of BRCA2 linkage in Icelandic breast cancer families and segregation of a common
BRCA2 haplotype. Am J Hum Genet 58: 749-56, 1996
Håkansson S, Johannsson O, Johansson U, Sellberg G, Loman N, Gerdes A-M, Holmberg E, Dahl N, Pandis N,
Kristoffersson U, Olsson H, Borg Å. Moderate frequency of BRCA1 and BRCA2 germ-line mutations in
Scandinavian familial breast cancer. Am J Hum Genet 60: 1068-78, 1997
Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, King M-C. Linkage of early-onset familial
breast cancer to chromosome 17q21. Science 250: 1684-9, 1990
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 100: 57-70, 2000
Hartmann LC, Schaid DJ, Woods JE, Crotty TP, Myers JL, Arnold PG, Petty PM, Sellers TA, Johnson JL,
McDonnell SK, Frost MH, Jenkins RB. Efficacy of bilateral prophylactic mastectomy in women with a
family history of breast cancer. N Engl J Med 340: 77-84, 1999
Hästbacka J, de la Chapelle A, Kaitila I, Sistonen P, Weaver A, Lander E. Linkage disequilibrium mapping in
isolated founder populations: diastrophic dysplasia in Finland. Nat Genet 2: 204-11, 1992
Hästbacka J, de la Chapelle A, Mahtani MM, Clines G, Reeve-Daly MP, Daly M, Hamilton BA, Kusumi K,
Trivedi B, Weaver A, Coloma A, Lovett M, Buckler A, Kaitila I, Lander ES. The diastrophic dysplasia
gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium
mapping. Cell 78: 1073-87, 1994Healey CS, Dunning AM, Teare MD, Chase D, Parker L, Burn J, Chang-
Claude J, Mannermaa A, Kataja V, Huntsman DG, Pharoah PDP, Luben RN, Easton DF, Ponder BAJ. A
common variant in BRCA2 is associated with both breast cancer risk and prenatal viability. Nat Genet 26:
362-4, 2000
90
Hedenfalk I, Duggan D, Chen Y, Radmacher M, Bittner M, Simon R, Meltzer P, Gusterson B, Esteller M,
Raffeld M, Yakhini Z, Ben-Dor A, Dougherty E, Kononen J, Bubendorf L, Fehrle W, Pittaluga S,
Gruvberger S, Loman N, Johannsson O, Olsson H, Wilfond B, Sauter G, Kallioniemi O-P, Trent J. Gene-
expression profiles in hereditary breast cancer. N Engl J Med 344: 539-48, 2001
Heintz AP, Odicino F, Maisonneuve P, Beller U, Benedet JL, Creasman WT, Ngan HY, Sideri M, Pecorelli S.
Carcinoma of the ovary. J Epidemiol Biostat 6: 107-38, 2001
Henderson BE, Feigelson HS. Epidemiology and screening. In: Textbook of breast cancer. A clinical guide to
therapy (ed. Bonadonna G, Hortobagyi GN, Gianni AM), 1-16. Martin Dunitz, London, 1998
Hilakivi-Clarke L. Estrogens, BRCA1, and breast cancer. Cancer Res 60: 4993-5001, 2000
Hodgson SV, Heap E, Cameron J, Ellis D, Mathew CG, Eeles RA, Solomon E, Lewis CM. Risk factors for
detecting germline BRCA1 and BRCA2 founder mutations in Ashkenazi Jewish women with breast or
ovarian cancer. J Med Genet 36: 369-73, 1999
Hogervorst FBL, Cornelis RS, Bout M, van Vliet M, Oosterwijk JC, Olmer R, Bakker B, Klijn JGM, Vasen
HFA, Meijers-Heijboer H, Menko FH, Cornelisse CJ, den Dunnen JT, Devilee P, van Ommen G-JB. Rapid
detection of BRCA1 mutations by the protein truncation test. Nat Genet 10: 208-12, 1995
Höglund P, Haila S, Socha J, Tomaszewski L, Saarialho-Kere U, Karjalainen-Lindsberg M-L, Airola K,
Holmberg C, de la Chapelle A, Kere J. Mutations of the Down-regulated in adenoma (DRA) gene cause
congenital chloride diarrhoea. Nat Genet 14: 316-9, 1996
Höglund P, Sistonen P, Norio R, Holmberg C, Dimberg A, Gustavson K-H, de la Chapelle A, Kere J. Fine
mapping of the congenital chloride diarrhea gene by linkage disequilibrium. Am J Hum Genet 57: 95-102,
1995
Holmberg M, Kristo P, Chadwicks RB, Mecklin JP, Järvinen H, de la Chapelle A, Nyström-Lahti M, Peltomäki
P. Mutation sharing, predominant involvement of the MLH1 gene and description of four novel mutations
in hereditary nonpolyposis colorectal cancer. Mutations in brief no. 144. Online. Hum Mutat 11: 482, 1998
Holschneider CH, Berek JS. Ovarian cancer: epidemiology, biology, and prognostic factors. Semin Surg Oncol
19: 3-10, 2000
Holt JT, Thompson ME, Szabo C, Robinson-Benion C, Arteaga CL, King M-C, Jensen RA. Growth retardation
and tumour inhibition by BRCA1. Nat Genet 12: 298-302, 1996
Hopper JL, Southey MC, Dite GS, Jolley DJ, Giles GG, McCredie MR, Easton DF, Venter DJ, the Australian
Breast Cancer Family Study. Population-based estimate of the average age-specific cumulative risk of
breast cancer for a defined set of protein-truncating mutations in BRCA1 and BRCA2. Cancer Epidemiol
Biomarkers Prev 8: 741-7, 1999
Hovatta I, Varilo T, Suvisaari J, Terwilliger JD, Ollikainen V, Arajärvi R, Juvonen H, Kokko-Sahin M-L,
Väisänen L, Mannila H, Lönnqvist J, Peltonen L. A genomewide screen for schizophrenia genes in an
isolated Finnish subpopulation, suggesting multiple susceptibility loci. Am J Hum Genet 65: 1114-24,
1999
Hulka BS, Moorman PG. Breast cancer: hormones and other risk factors. Maturitas 38: 103-13, 2001
Huusko P, Pääkkönen K, Launonen V, Pöyhönen M, Blanco G, Kauppila A, Puistola U, Kiviniemi H, Kujala M,
Leisti J, Winqvist R. Evidence of founder mutations in Finnish BRCA1 and BRCA2 families. Am J Hum
Genet 62: 1544-8, 1998
91
Ikeda N, Miyoshi Y, Yoneda K, Shiba E, Sekihara Y, Kinoshita M, Noguchi S. Frequency of BRCA1 and
BRCA2 germline mutations in Japanese breast cancer families. Int J Cancer 91: 83-8, 2001
Isola J, DeVries S, Chu L, Ghazvini S, Waldman F. Analysis of changes in DNA sequence copy number by
comparative genomic hybridization in archival paraffin-embedded tumor samples. Am J Pathol 145: 1301-
8, 1994
Janezic SA, Ziogas A, Krumroy LM, Krasner M, Plummer SJ, Cohen P, Gildea M, Barker D, Haile R, Casey G,
Anton-Culver H. Germline BRCA1 alterations in a population-based series of ovarian cancer cases. Hum
Mol Genet 8: 889-97, 1999
Jazaeri AA, Yee CJ, Sotiriou C, Brantley KR, Boyd J, Liu ET. Gene expression profiles of BRCA1-linked,
BRCA2-linked, and sporadic ovarian cancers. J Natl Cancer Inst 94: 990-1000, 2002
Jeffreys AJ, Kauppi L, Neumann R. Intensely punctate meiotic recombination in the class II region of the major
histocompatibility complex. Nat Genet 29: 217-22, 2001
Jernström H, Lerman C, Ghadirian P, Lynch HT, Weber B, Garber J, Daly M, Olopade OI, Foulkes WD,
Warner E, Brunet JS, Narod SA. Pregnancy and risk of early breast cancer in carriers of BRCA1 and
BRCA2. Lancet 354: 1846-50, 1999
Johannesdottir G, Gudmundsson J, Bergthorsson JT, Arason A, Agnarsson BA, Eiriksdottir G, Johannsson OT,
Borg A, Ingvarsson S, Easton DF, Egilsson V, Barkardottir RB. High prevalence of the 999del5 mutation
in Icelandic breast and ovarian cancer patients. Cancer Res 56: 3663-5, 1996
Johannsson O, Loman N, Borg Å, Olsson H. Pregnancy-associated breast cancer in BRCA1 and BRCA2
germline mutation carriers. Lancet 352: 1359-60, 1998
Johannsson O, Loman N, Möller T, Kristoffersson U, Borg Å, Olsson H. Incidence of malignant tumours in
relatives of BRCA1 and BRCA2 germline mutation carriers. Eur J Cancer 35: 1248-57, 1999
Jóhannsson ÓT, Idvall I, Anderson C, Borg Å, Barkardóttir RB, Egilsson V, Olsson H. Tumour biological
features of BRCA1-induced breast and ovarian cancer. Eur J Cancer 33: 362-71, 1997
Jóhannsson ÓT, Ranstam J, Borg Å, Olsson H. Survival of BRCA1 breast and ovarian cancer patients: a
population-based study from southern Sweden. J Clin Oncol 16: 397-404, 1998
Jorde LB. Linkage disequilibrium as a gene-mapping tool. Am J Hum Genet 56: 11-4, 1995
Jorde LB. Linkage disequilibrium and the search for complex disease genes. Genome Res 10: 1435-44, 2000
Jutikkala E, Pirinen K. A history of Finland (5th ed). WSOY, Juva, 1996
Kainu T, Juo S-HH, Desper R, Schaffer AA, Gillanders E, Rozenblum E, Freas-Lutz D, Weaver D, Stephan D,
Bailey-Wilson J, Kallioniemi O-P, Tirkkonen M, Syrjäkoski K, Kuukasjärvi T, Koivisto P, Karhu R, Holli
K, Arason A, Johannesdottir G, Bergthorsson JT, Johannsdottir H, Egilsson V, Barkardottir RB,
Johannsson O, Haraldsson K, Sandberg T, Holmberg E, Grönberg H, Olsson H, Borg Å, Vehmanen P,
Eerola H, Heikkilä P, Pyrhönen S, Nevanlinna H. Somatic deletions in hereditary breast cancers implicate
13q21 as a putative novel breast cancer susceptibility locus. Proc Natl Acad Sci USA 97: 9603-8, 2000
Kamb A, Shattuck-Eidens D, Eeles R, Liu Q, Gruis NA, Ding W, Hussey C, Tran T, Miki Y, Weaver-Feldhaus
J, McClure M, Aitken JF, E. AD, Bergman W, Frants R, Goldgar DE, Green A, MacLennan R, Martin NG,
Meyer LJ, Youl P, Zone JJ, H. SM, Cannon-Albright LA. Analysis of the p16 gene (CDKN2) as a
candidate for the chromosome 9p melanoma susceptibility locus. Nat Genet 8: 22-6, 1994
Kere J. Human population genetics: lessons from Finland. Annu Rev Genomics Hum Genet 2: 103-28, 2001
92
Khoo US, Chan KY, Cheung AN, Xue WC, Shen DH, Fung KY, Ngan HY, Choy KW, Pang CP, Poon CS,
Poon AY, Ozcelik H. Recurrent BRCA1 and BRCA2 germline mutations in ovarian cancer: a founder
mutation of BRCA1 identified in the Chinese population. Hum Mutat 19: 307-8, 2002
Khoo US, Ngan HY, Cheung AN, Chan KY, Lu J, Chan VW, Lau S, Andrulis IL, Ozcelik H. Mutational
analysis of BRCA1 and BRCA2 genes in Chinese ovarian cancer identifies 6 novel germline mutations.
Hum Mutat 16: 88-9, 2000
King M-C, Wieand S, Hale K, Lee M, Walsh T, Owens K, Tait J, Ford L, Dunn BK, Costantino J, Wickerham
L, Wolmark N, Fisher B. Tamoxifen and breast cancer incidence among women with inherited mutations in
BRCA1 and BRCA2: National Surgical Adjuvant Breast and Bowel Project (NSABP-P1) Breast Cancer
Prevention Trial. JAMA 286: 2251-6, 2001
Kinzler KW, Vogelstein B. Cancer-susceptibility genes. Gatekeepers and caretakers. Nature 386: 761-3, 1997
Kinzler KW, Vogelstein B. Landscaping the cancer terrain. Science 280: 1036-7, 1998
Kittles RA, Perola M, Peltonen L, Bergen AW, Aragon RA, Virkkunen M, Linnoila M, Goldman D, Long JC.
Dual origins of Finns revealed by Y chromosome haplotype variation. Am J Hum Genet 62: 1171-9, 1998
Koivisto UM, Turtola H, Aalto-Setälä K, Top B, Frants RR, Kovanen PT, Syvänen AC, Kontula K. The familial
hypercholesterolemia (FH)-North Karelia mutation of the low density lipoprotein receptor gene deletes
seven nucleotides of exon 6 and is a common cause of FH in Finland. J Clin Invest 90: 219-28, 1992
Koivisto UM, Viikari JS, Kontula K. Molecular characterization of minor gene rearrangements in Finnish
patients with heterozygous familial hypercholesterolemia: identification of two common missense
mutations (Gly823-->Asp and Leu380-->His) and eight rare mutations of the LDL receptor gene. Am J
Hum Genet 57: 789-97, 1995
Krainer M, Silva-Arrieta S, FitzGerald MG, Shimada A, Ishioka C, Kanamaru R, MacDonald DJ, Unsal H,
Finkelstein DM, Bowcock A, Isselbacher KJ, Haber DA. Differential contributions of BRCA1 and BRCA2
to early-onset breast cancer. N Engl J Med 336: 1416-21, 1997
Kruglyak L. Genetic isolates: separate but equal? Proc Natl Acad Sci USA 96: 1170-2, 1999
Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified
multipoint approach. Am J Hum Genet 58: 1347-63, 1996
Kuokkanen S, Gschwend M, Rioux JD, Daly MJ, Terwilliger JD, Tienari PJ, Wikström J, Palo J, Stein LD,
Hudson TJ, Lander ES, Peltonen L. Genomewide scan of multiple sclerosis in Finnish multiplex families.
Am J Hum Genet 61: 1379-87, 1997
Kwabi-Addo B, Giri D, Schmidt K, Podsypanina K, Parsons R, Greenberg N, Ittmann M. Haploinsufficiency of
the Pten tumor suppressor gene promotes prostate cancer progression. Proc Natl Acad Sci USA 98: 11563-
8, 2001
Laan M, Pääbo S. Demographic history and linkage disequilibrium in human populations. Nat Genet 17: 435-8,
1997
Lahti-Domenici J, Rapakko K, Pääkkönen K, Allinen M, Nevanlinna H, Kujala M, Huusko P, Winqvist R.
Exclusion of large deletions and other rearrangements in BRCA1 and BRCA2 in Finnish breast and ovarian
cancer families. Cancer Genet Cytogenet 129: 120-3, 2001
93
Laiho E, Ignatius J, Mikkola H, Yee VC, Teller DC, Niemi KM, Saarialho-Kere U, Kere J, Palotie A.
Transglutaminase 1 mutations in autosomal recessive congenital ichthyosis: private and recurrent mutations
in an isolated population. Am J Hum Genet 61: 529-38, 1997
Laitinen T, Daly MJ, Rioux JD, Kauppi P, Laprise C, Petays T, Green T, Cargill M, Haahtela T, Lander ES,
Laitinen LA, Hudson TJ, Kere J. A susceptibility locus for asthma-related traits on chromosome 7 revealed
by genome-wide scan in a founder population. Nat Genet 28: 87-91, 2001
Lakhani SR, Jacquemier J, Sloane JP, Gusterson BA, Anderson TJ, van de Vijver MJ, Farid LM, Venter D,
Antoniou A, Storfer-Isser A, Smyth E, Steel CM, Haites N, Scott RJ, Goldgar D, Neuhausen S, Daly PA,
Ormiston W, McManus R, Scherneck S, Ponder BA, Ford D, Peto J, Stoppa-Lyonnet D, Bignon Y-J,
Struewing JP, Spurr NK, Bishop DT, Klijn JGM, Devilee P, Cornelisse CJ, Lasset C, Lenoir G,
Barkardottir RB, Egilsson V, Hamann U, Chang-Claude J, Sobol H, Weber B, Stratton MR, Easton DF.
Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and
BRCA2 mutations. J Natl Cancer Inst 90: 1138-45, 1998
Langston AA, Stanford JL, Wicklund KG, Thompson JD, Blazej RG, Ostrander EA. Germ-line BRCA1
mutations in selected men with prostate cancer. Am J Hum Genet 58: 881-4, 1996
Lee H, Trainer AH, Friedman LS, Thistlethwaite FC, Evans MJ, Ponder BA, Venkitaraman AR. Mitotic
checkpoint inactivation fosters transformation in cells lacking the breast cancer susceptibility gene, Brca2.
Mol Cell 4: 1-10, 1999
Lehesjoki AE, Koskiniemi M, Norio R, Tirrito S, Sistonen P, Lander E, de la Chapelle A. Localization of the
EPM1 gene for progressive myoclonus epilepsy on chromosome 21: linkage disequilibrium allows high
resolution mapping. Hum Mol Genet 2: 1229-34, 1993
Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J, Koskenvuo M, Pukkala E, Skytthe A, Hemminki
K. Environmental and heritable factors in the causation of cancer--analyses of cohorts of twins from
Sweden, Denmark, and Finland. N Engl J Med 343: 78-85, 2000
Lidereau R, Eisinger F, Champeme MH, Nogues C, Bieche I, Birnbaum D, Pallud C, Jacquemier J, Sobol H.
Major improvement in the efficacy of BRCA1 mutation screening using morphoclinical features of breast
cancer. Cancer Res 60: 1206-10, 2000
Liede A, Malik IA, Aziz Z, de los Rios P, Kwan E, Narod SA. Contribution of BRCA1 and BRCA2 mutations to
breast and ovarian cancer in Pakistan. Am J Hum Genet 71: 595-606, 2002
Liehr JG. Is estradiol a genotoxic mutagenic carcinogen? Endocr Rev 21: 40-54, 2000
Ligtenberg MJL, Hogervorst FBL, Willems HW, Arts PJW, Brink G, Hageman S, Bosgoed EAJ, Van der Looij
E, Rookus MA, Devilee P, Vos EMAW, Wigbout G, Struycken PM, Menko FH, Rutgers EJT, Hoefsloot
EH, Mariman ECM, Brunner HG, Van't Veer LJ. Characteristics of small breast and/or ovarian cancer
families with germline mutations in BRCA1 and BRCA2. Br J Cancer 79: 1475-78, 1999
Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature 411: 375-9, 2001
Liu H-X, Cartegni L, Zhang MQ, Krainer AR. A mechanism for exon skipping caused by nonsense or missense
mutations in BRCA1 and other genes. Nature 27: 55-8, 2001
Liu X, Barker DF. Evidence for effective suppression of recombination in the chromosome 17q21 segment
spanning RNU2-BRCA1. Am J Hum Genet 64: 1427-39, 1999
94
Loman N, Johannsson O, Bendahl PO, Borg Å, Fernö M, Olsson H. Steroid receptors in hereditary breast
carcinomas associated with BRCA1 or BRCA2 mutations or unknown susceptibility genes. Cancer 83:
310-9, 1998
Loman N, Johannsson O, Kristoffersson U, Olsson H, Borg Å. Family history of breast and ovarian cancers and
BRCA1 and BRCA2 mutations in a population-based series of early-onset breast cancer. J Natl Cancer Inst
93: 1215-23, 2001
Lu M, Conzen SD, Cole CN, Arrick BA. Characterization of functional messenger RNA splice variants of
BRCA1 expressed in nonmalignant and tumor-derived breast cells. Cancer Res 56: 4578-81, 1996
Luria S-E, Delbrück M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28: 491-511,
1943
Lynch HT, Albano W, Black L, Lynch JF, Recabaren J, Pierson R. Familial excess of cancer of the ovary and
other anatomic sites. JAMA 245: 261-4, 1981
Lynch HT, Harris RE, Guirgis HA, Maloney K, Carmody LL, Lynch JF. Familial association of breast/ovarian
carcinoma. Cancer 41: 1543-9, 1978
Lynch HT, Krush AJ, Lemon HM, Kaplan AR, Condit PT, Bottomley RH. Tumor variation in families with
breast cancer. JAMA 222: 1631-5, 1972
Macleod K. Tumor suppressor genes. Curr Opin Genet Dev 10: 81-93, 2000
Madigan MP, Ziegler RG, Benichou J, Byrne C, Hoover RN. Proportion of breast cancer cases in the United
States explained by well-established risk factors. J Natl Cancer Inst 87: 1681-5, 1995
Malone KE, Daling JR, Thompson JD, O'Brien CA, Francisco LV, Ostrander EA. BRCA1 mutations and breast
cancer in the general population: analyses in women before age 35 years and in women before age 45 years
with first-degree family history. JAMA 279: 922-9, 1998
Marcus JN, Watson P, Page DL, Narod SA, Lenoir GM, Tonin P, Linder-Stephenson L, Salerno G, Conway
TA, Lynch HT. Hereditary breast cancer: pathobiology, prognosis, and BRCA1 and BRCA2 gene linkage.
Cancer 77: 697-709, 1996
Marks JR, Huper G, Vaughn JP, Davis PL, Norris J, McDonnell DP, Wiseman RW, Futreal PA, Iglehart JD.
BRCA1 expression is not directly responsive to estrogen. Oncogene 14: 115-21, 1997
Marquis ST, Rajan JV, Wynshaw-Boris A, Xu J, Yin G-Y, Abel KJ, Weber BL, Chodosh LA. The
developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues.
Nat Genet 11: 17-26, 1995
Martin A-M, Blackwood MA, Antin-Ozerkis D, Shih HA, Calzone K, Colligon TA, Seal S, Collins N, Stratton
MR, Weber BL, Nathanson KL. Germline mutations in BRCA1 and BRCA2 in breast-ovarian families from
a breast cancer risk evaluation clinic. J Clin Oncol 19: 2247-53, 2001
Mavraki E, Gray IC, Bishop DT, Spurr NK. Germline BRCA2 mutations in men with breast cancer. Br J Cancer
76: 1428-31, 1997
Mazoyer S, Dunning AM, Serova O, Dearden J, Puget N, Healey CS, Gayther SA, Mangion J, Stratton MR,
Lynch HT, Goldgar DE, Ponder BAJ, Lenoir GM. A polymorphic stop codon in BRCA2. Nat Genet 14:
253-4, 1996
McPherson K, Steel CM, Dixon JM. ABC of breast diseases. Breast cancer-epidemiology, risk factors, and
genetics. BMJ 321: 624-8, 2000
95
Meindl A, German Consortium for Hereditary Breast and Ovarian Cancer. Comprehensive analysis of 989
patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for
the German population. Int J Cancer 97: 472-80, 2002
Miki Y, Katagiri T, Kasumi F, Yoshimoto T, Nakamura Y. Mutation analysis in the BRCA2 gene in primary
breast cancers. Am J Hum Genet 58: 1166-76, 1996
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM,
Ding W, Bell R, Rosenthal J, Hussey C, Tran T, McClure M, Frye C, Hattier T, Phelps R, Haugen-Strano
A, Katcher H, Yakumo K, Gholami Z, Shaffer D, Stone S, Bayer S, Wray C, Bogden R, Dayananth P,
Ward J, Tonin P, Narod S, Bristow PK, Norris FH, Helvering L, Morrison P, Rosteck P, Lai M, Barrett JC,
Lewis C, Neuhausen S, Cannon-Albright L, Goldgar D, Wiseman R, Kamb A, Skolnick MH. A strong
candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266: 66-71, 1994
Miyoshi H, Nakau M, Ishikawa TO, Seldin MF, Oshima M, Taketo MM. Gastrointestinal hamartomatous
polyposis in Lkb1 heterozygous knockout mice. Cancer Res 62: 2261-6, 2002
Modan B, Hartge P, Hirsh-Yechezkel G, Chetrit A, Lubin F, Beller U, Ben-Baruch G, Fishman A, Menczer J,
Ebbers SM, Tucker MA, Wacholder S, Struewing JP, Friedman E, Piura B. Parity, oral contraceptives, and
the risk of ovarian cancer among carriers and noncarriers of a BRCA1 or BRCA2 mutation. N Engl J Med
345: 235-40, 2001
Moisio A-L, Sistonen P, Weissenbach J, de la Chapelle A, Peltomäki P. Age and origin of two common MLH1
mutations predisposing to hereditary colon cancer. Am J Hum Genet 59: 1243-51, 1996
Moslehi R, Chu W, Karlan B, Fishman D, Risch H, Fields A, Smotkin D, Ben-David Y, Rosenblatt J, Russo D,
Schwartz P, Tung N, Warner E, Rosen B, Friedman J, Brunet J-S, Narod SA. BRCA1 and BRCA2 mutation
analysis of 208 Ashkenazi Jewish women with ovarian cancer. Am J Hum Genet 66: 1259-72, 2000
Moynahan ME, Pierce AJ, Jasin M. BRCA2 is required for homology-directed repair of chromosomal breaks.
Mol Cell 7: 263-72, 2001
Murrell JR, Koller D, Foroud T, Goedert M, Spillantini MG, Edenberg HJ, Farlow MR, Ghetti B. Familial
multiple-system tauopathy with presenile dementia is localized to chromosome 17. Am J Hum Genet 61:
1131-8, 1997
Narod SA, Brunet J-S, Ghadirian P, Robson M, Heimdal K, Neuhausen SL, Stoppa-Lyonnet D, Lerman C,
Pasini B, de los Rios P, Weber B, Lynch H. Tamoxifen and risk of contralateral breast cancer in BRCA1
and BRCA2 mutation carriers: a case-control study. Lancet 356: 1876-81, 2000
Narod SA, Feunteun J, Lynch HT, Watson P, Conway T, Lynch J, Lenoir GM. Familial breast-ovarian cancer
locus on chromosome 17q12-q23. Lancet 338: 82-3, 1991
Narod SA, Ford D, Devilee P, Barkardottir RB, Lynch HT, Smith SA, Ponder BAJ, Weber BL, Garber JE,
Birch JM, Cornelis RS, Kelsell DP, Spurr NK, Smyth E, Haites N, Sobol H, Bignon Y-J, Chang-Claude J,
Hamann U, A. L, Borg A, Piver MS, Gallion HH, Struewing JP, Whittemore A, Tonin P, Goldgar DE,
Easton DF, the Breast Cancer Linkage Consortium. An evaluation of genetic heterogeneity in 145 breast-
ovarian cancer families. Am J Hum Genet 56: 254-64, 1995
Nathanson KL, Weber BL. "Other" breast cancer susceptibility genes: searching for more holy grail. Hum Mol
Genet 10: 715-20, 2001
National Center for Biotechnology Information. http://www.ncbi.nih.gov/
96
Ness RB, Cramer DW, Goodman MT, Kjaer SK, Mallin K, Mosgaard BJ, Purdie DM, Risch HA, Vergona R,
Wu AH. Infertility, fertility drugs, and ovarian cancer: a pooled analysis of case-control studies. Am J
Epidemiol 155: 217-24, 2002
Neuhausen SL. Ethnic differences in cancer risk resulting from genetic variation. Cancer 86: 2575-82, 1999
Neuhausen SL, Godwin AK, Gershoni-Baruch R, Schubert E, Garber J, Stoppa-Lyonnet D, Olah E, Csokay B,
Serova O, Lalloo F, Osorio A, Stratton M, Offit K, Boyd J, Caligo MA, Scott RJ, Schofield A, Teugels E,
Schwab M, Cannon-Albright L, Bishop T, Easton D, Benitez J, King M-C, Ponder BAJ, Weber B, Devilee
P, Borg Å, Narod SA, Goldgar D. Haplotype and phenotype analysis of nine recurrent BRCA2 mutations in
111 families: results of an international study. Am J Hum Genet 62: 1381-8, 1998
Neuhausen SL, Mazoyer S, Friedman L, Stratton M, Offit K, Caligo A, Tomlinson G, Cannon-Albright L,
Bishop T, Kelsell D, Solomon E, Weber B, Couch F, Struewing J, Tonin P, Durocher F, Narod S, Skolnick
MH, Lenoir G, Serova O, Ponder B, Stoppa-Lyonnet D, Easton D, King M-C, Goldgar DE. Haplotype and
phenotype analysis of six recurrent BRCA1 mutations in 61 families: results of an international study. Am J
Hum Genet 58: 271-80, 1996
Nevanlinna HR. The Finnish population structure. A genetic and genealogical study. Hereditas 71: 195-236,
1972
Newman B, Austin MA, Lee M, King MC. Inheritance of human breast cancer: evidence for autosomal
dominant transmission in high-risk families. Proc Natl Acad Sci USA 85: 3044-8, 1988
Nguyen HN, Averette HE, Hoskins W, Sevin B-U, Penalver M, Steren A. National survey of ovarian carcinoma.
VI. Critical assessment of current International Federation of Gynecology and Obstetrics staging system.
Cancer 72: 3007-11, 1993
Noguchi S, Kasugai T, Miki Y, Fukutomi T, Emi M, Nomizu T. Clinicopathologic analysis of BRCA1- or
BRCA2-associated hereditary breast carcinoma in Japanese women. Cancer 85: 2200-5, 1999
Nordling M, Karlsson P, Wahlstrom J, Engwall Y, Wallgren A, Martinsson T. A large deletion disrupts the exon
3 transcription activation domain of the BRCA2 gene in a breast/ovarian cancer family. Cancer Res 58:
1372-5, 1998
Norio R. Suomineidon geenit. Tautiperinnön takana juurillemme johtamassa (In Finnish). Otavan Kirjapaino
Oy, Keuruu, 2000
Norio R, Nevanlinna HR, Perheentupa J. Hereditary diseases in Finland; rare flora in rare soil. Ann Clin Res 5:
109-41, 1973
Nowell PC. The clonal evolution of tumor cell populations. Science 194: 23-8, 1976
Nyström-Lahti M, Kristo P, Nicolaides NC, Chang SY, Aaltonen LA, Moisio AL, Järvinen HJ, Mecklin JP,
Kinzler KW, Vogelstein B, de la Chapelle A, Peltomäki P. Founding mutations and Alu-mediated
recombination in hereditary colon cancer. Nat Med 1: 1203-6, 1995
Nyström-Lahti M, Sistonen P, Mecklin JP, Pylkkänen L, Aaltonen LA, Järvinen H, Weissenbach J, de la
Chapelle A, Peltomäki P. Close linkage to chromosome 3p and conservation of ancestral founding
haplotype in hereditary nonpolyposis colorectal cancer families. Proc Natl Acad Sci USA 91: 6054-8, 1994
Nyström-Lahti M, Wu Y, Moisio AL, Hofstra RM, Osinga J, Mecklin JP, Järvinen HJ, Leisti J, Buys CH, de la
Chapelle A, Peltomäki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative
hereditary non-polyposis colorectal cancer. Hum Mol Genet 5: 763-9, 1996
97
Ozelius LJ, Kramer PL, de Leon D, Risch N, Bressman SB, Schuback DE, Brin MF, Kwiatkowski DJ, Burke
RE, Gusella JF, Fahn S, Breakefield XO. Strong allelic association between the torsion dystonia gene
(DYT1) and loci on chromosome 9q34 in Ashkenazi Jews. Am J Hum Genet 50: 619-28, 1992
Parazzini F, Negri E, La Vecchia C, Restelli C, Franceschi S. Family history of reproductive cancers and
ovarian cancer risk: an Italian case-control study. Am J Epidemiol 135: 35-40, 1992
Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer
80: 827-41, 1999
Parmigiani G, Berry DA, Aguilar O. Determining carrier probabilities for breast cancer-susceptibility genes
BRCA1 and BRCA2. Am J Hum Genet 62: 145-58, 1998
Patil N, Berno AJ, Hinds DA, Barrett WA, Doshi JM, Hacker CR, Kautzer CR, Lee DH, Marjoribanks C,
McDonough DP, Nguyen BT, Norris MC, Sheehan JB, Shen N, Stern D, Stokowski RP, Thomas DJ,
Trulson MO, Vyas KR, Frazer KA, Fodor SP, Cox DR. Blocks of limited haplotype diversity revealed by
high-resolution scanning of human chromosome 21. Science 294: 1719-23, 2001
Paunio T, Ekelund J, Varilo T, Parker A, Hovatta I, Turunen JA, Rinard K, Foti A, Terwilliger JD, Juvonen H,
Suvisaari J, Arajärvi R, Suokas J, Partonen T, Lönnqvist J, Meyer J, Peltonen L. Genome-wide scan in a
nationwide study sample of schizophrenia families in Finland reveals susceptibility loci on chromosomes
2q and 5q. Hum Mol Genet 10: 3037-48, 2001
Peelen T, van Vliet M, Petrij-Bosch A, Mieremet R, Szabo C, van den Ouweland AMW, Hogervorst F, Brohet
R, Ligtenberg MJL, Teugels E, van der Luijt R, van der Hout AH, Gille JJP, Pals G, Jedema I, Olmer R,
van Leeuwen I, Newman B, Plandsoen M, van der Est M, Brink G, Hageman S, Arts PJW, Bakker MM,
Willems HW, van der Looij E, Neyns B, Bonduelle M, Jansen R, Oosterwijk JC, Sijmons R, Smeets HJM,
van Asperen CJ, Meijers-Heijboer M, Klijn JGM, de Greve J, King M-C, Menko FH, Brunner HG, Halley
D, van Ommen G-JB, Vasen HFA, Cornelisse CJ, van't Veer LJ, de Knijff P, Bakker E, Devilee P. A high
proportion of novel mutations in BRCA1 with strong founder effects among Dutch and Belgian hereditary
breast and ovarian cancer families. Am J Hum Genet 60: 1041-49, 1997
Peltonen L. Positional cloning of disease genes: advantages of genetic isolates. Hum Hered 50: 66-75, 2000a
Peltonen L, Jalanko A, Varilo T. Molecular genetics of the Finnish disease heritage. Hum Mol Genet 8: 1913-
23, 1999
Peltonen L, Palotie A, Lange K. Use of population isolates for mapping complex traits. Nat Rev Genet 1: 182-
90, 2000b
Peltonen L, Pekkarinen P, Aaltonen J. Messages from an isolate: lessons from the Finnish gene pool. Biol Chem
Hoppe-Seyler 376: 697-704, 1995
Peto J, Collins N, Barfoot R, Seal S, Warren W, Rahman N, Easton DF, Evans C, Deacon J, Stratton MR.
Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer. J Natl Cancer
Inst 91: 943-9, 1999
Petrij-Bosch A, Peelen T, van Vliet M, van Eijk R, Olmer R, Drüsedau M, Hogervorst FBL, Hageman S, Arts
PJW, Ligtenberg MJL, Meijers-Heijboer H, Klijn JGM, Vasen HF, Cornelisse CJ, van't Veer LJ, Bakker
E, van Ommen G-JB, Devilee P. BRCA1 genomic deletions are major founder mutations in Dutch breast
cancer patients. Nat Genet 17: 341-5, 1997
98
Pharoah PD, Day NE, Duffy S, Easton DF, Ponder BA. Family history and the risk of breast cancer: a
systematic review and meta-analysis. Int J Cancer 71: 800-9, 1997
Pharoah PDP, Easton DF, Stockton DL, Gayther S, Ponder BAJ, the United Kingdom Coordinating Committee
for Cancer Research (UKCCCR) Familial Ovarian Cancer Study Group. Survival in familial, BRCA1-
associated, and BRCA2-associated epithelial ovarian cancer. Cancer Res 59: 868-71, 1999
Phillips KA. Immunophenotypic and pathologic differences between BRCA1 and BRCA2 hereditary breast
cancers. J Clin Oncol 18: 107S-12S, 2000
Pike MC, Krailo MD, Henderson BE, Casagrande JT, Hoel DG. 'Hormonal' risk factors, 'breast tissue age' and
the age-incidence of breast cancer. Nature 303: 767-70, 1983
Ponder BA. Cancer genetics. Nature 411: 336-41, 2001
Pritchard JK, Przeworski M. Linkage disequilibrium in humans: models and data. Am J Hum Genet 69: 1-14,
2001
Przeworski M, Wall JD. Why is there so little intragenic linkage disequilibrium in humans? Genet Res 77: 143-
51, 2001
Puget N, Stoppa-Lyonnet D, Sinilnikova OM, Pages S, Lynch HT, Lenoir GM, Mazoyer S. Screening for germ-
line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions.
Cancer Res 59: 455-61, 1999
Puget N, Torchard D, Serova-Sinilnikova OM, Lynch HT, Feunteun J, Lenoir GM, Mazoyer S. A 1-kb Alu-
mediated germ-line deletion removing BRCA1 exon 17. Cancer Res 57: 828-31, 1997
Rafnsson S. The Atlantic islands. In: The Oxford illustrated history of the Vikings (ed. Sawyer P), 110-33.
Oxford University Press, New York, 1999
Rajan JV, Marquis ST, Gardner HP, Chodosh LA. Developmental expression of Brca2 colocalizes with Brca1
and is associated with proliferation and differentiation in multiple tissues. Dev Biol 184: 385-401, 1997
Rajan JV, Wang M, Marquis ST, Chodosh LA. Brca2 is coordinately regulated with Brca1 during proliferation
and differentiation in mammary epithelial cells. Proc Natl Acad Sci USA 93: 13078-83, 1996
Ramus SJ, Bobrow LG, Pharoah PD, Finnigan DS, Fishman A, Altaras M, Harrington PA, Gayther SA, Ponder
BA, Friedman LS. Increased frequency of TP53 mutations in BRCA1 and BRCA2 ovarian tumours. Genes
Chromosomes Cancer 25: 91-6, 1999
Ramus SJ, Fishman A, Pharoah PD, Yarkoni S, Altaras M, Ponder BA. Ovarian cancer survival in Ashkenazi
Jewish patients with BRCA1 and BRCA2 mutations. Eur J Surg Oncol 27: 278-81, 2001
Ramus SJ, Kote-Jarai Z, Friedman LS, van der Looij M, Gayther SA, Csokay B, Ponder BA, Olah E. Analysis
of BRCA1 and BRCA2 mutations in Hungarian families with breast or breast-ovarian cancer. Am J Hum
Genet 60: 1242-6, 1997
Randrianarison V, Marot D, Foray N, Cabannes J, Meret V, Connault E, Vitrat N, Opolon P, Perricaudet M,
Feunteun J. BRCA1 carries tumor suppressor activity distinct from that of p53 and p21. Cancer Gene Ther
8: 759-70, 2001
Rannala B, Bertorelle G. Using linked markers to infer the age of a mutation. Hum Mutat 18: 87-100, 2001
Ravid A, Barschack I, Hirsh-Yechezkel G, Goldberg I, Bar-Sade RB, Chetrit A, Reder I, Ben-Baruch G, Gotlieb
WH, Koplovic J, Friedman E. Immunohistochemical analyses of sporadic and familial (185delAG carriers)
ovarian cancer in Israel. Eur J Cancer 36: 1120-4, 2000
99
Rebbeck TR, Couch FJ, Kant J, Calzone K, DeShano M, Peng Y, Chen K, Garber JE, Weber BL. Genetic
heterogeneity in hereditary breast cancer: role of BRCA1 and BRCA2. Am J Obstet Gynecol 175: 738-46,
1996
Rebbeck TR, Kantoff PW, Krithivas K, Neuhausen S, Blackwood MA, Godwin AK, Daly MB, Narod SA,
Garber JE, Lynch HT, Weber BL, Brown M. Modification of BRCA1-associated breast cancer risk by the
polymorphic androgen-receptor CAG repeat. Am J Hum Genet 64: 1371-7, 1999a
Rebbeck TR, Levin AM, Eisen A, Snyder C, Watson P, Cannon-Albright L, Isaacs C, Olopade O, Garber JE,
Godwin AK, Daly MB, Narod SA, Neuhausen SL, Lynch HT, Weber BL. Breast cancer risk after bilateral
prophylactic oophorectomy in BRCA1 mutation carriers. J Natl Cancer Inst 91: 1475-9, 1999b
Rebbeck TR, Lynch HT, Neuhausen SL, Narod SA, Van't Veer L, Garber JE, Evans G, Isaacs C, Daly MB,
Matloff E, Olopade OI, Weber BL. Prophylactic oophorectomy in carriers of BRCA1 or BRCA2
mutations. N Engl J Med 346: 1616-22, 2002
Rebbeck TR, Wang Y, Kantoff PW, Krithivas K, Neuhausen SL, Godwin AK, Daly MB, Narod SA, Brunet JS,
Vesprini D, Garber JE, Lynch HT, Weber BL, Brown M. Modification of BRCA1- and BRCA2-associated
breast cancer risk by AIB1 genotype and reproductive history. Cancer Res 61: 5420-4, 2001
Reich DE, Cargill M, Bolk S, Ireland J, Sabeti PC, Richter DJ, Lavery T, Kouyoumjian R, Farhadian SF, Ward
R, Lander ES. Linkage disequilibrium in the human genome. Nature 411: 199-204, 2001
Rhei E, Bogomolniy F, Federici MG, Maresco DL, Offit K, Robson ME, Saigo PE, Boyd J. Molecular genetic
characterization of BRCA1- and BRCA2-linked hereditary ovarian cancers. Cancer Res 58: 3193-6, 1998
Risch HA, McLaughlin JR, Cole DEC, Rosen B, Bradley L, Kwan E, Jack E, Vesprini DJ, Kuperstein G,
Abrahamson JL, Fan I, Wong B, Narod SA. Prevalence and penetrance of germline BRCA1 and BRCA2
mutations in a population series of 649 women with ovarian cancer. Am J Hum Genet 68: 700-10, 2001
Roa BB, Boyd AA, Volcik K, Richards CS. Ashkenazi Jewish population frequencies for common mutations in
BRCA1 and BRCA2. Nat Genet 14: 185-7, 1996
Roest PA, Roberts RG, Sugino S, van Ommen GJ, den Dunnen JT. Protein truncation test (PTT) for rapid
detection of translation-terminating mutations. Hum Mol Genet 2: 1719-21, 1993
Rohlfs EM, Puget N, Graham ML, Weber BL, Garber JE, Skrzynia C, Halperin JL, Lenoir GM, Silverman LM,
Mazoyer S. An Alu-mediated 7.1 kb deletion of BRCA1 exons 8 and 9 in breast and ovarian cancer
families that results in alternative splicing of exon 10. Genes Chromosomes Cancer 28: 300-7, 2000
Roth S, Kristo P, Auranen A, Shayehgi M, Seal S, Collins N, Barfoot R, Rahman N, Klemi PJ, Grénman S,
Sarantaus L, Nevanlinna H, Butzow R, Ashworth A, Stratton MR, Aaltonen LA. A missense mutation in
the BRCA2 gene in three siblings with ovarian cancer. Br J Cancer 77: 1199-202, 1998
Rubin SC, Benjamin I, Behbakht K, Takahashi H, Morgan MA, LiVolsi VA, Berchuck A, Muto MG, Garber JE,
Weber BL, Lynch HT, Boyd J. Clinical and pathological features of ovarian cancer in women with germ-
line mutations of BRCA1. N Engl J Med 335: 1455-6, 1996
Rubin SC, Blackwood MA, Bandera C, Behbakht K, Benjamin I, Rebbeck TR, Boyd J. BRCA1, BRCA2, and
hereditary nonpolyposis colorectal cancer gene mutations in an unselected ovarian cancer population:
relationship to family history and implications for genetic testing. Am J Obstet Gynecol 178: 670-7, 1998
Runnebaum IB, Wang-Gohrke S, Vesprini D, Kreienberg R, Lynch H, Moslehi R, Ghadirian P, Weber B,
Godwin AK, Risch H, Garber J, Lerman C, Olopade OI, Foulkes WD, Karlan B, Warner E, Rosen B,
100
Rebbeck T, Tonin P, Dube MP, Kieback DG, Narod SA. Progesterone receptor variant increases ovarian
cancer risk in BRCA1 and BRCA2 mutation carriers who were never exposed to oral contraceptives.
Pharmacogenetics 11: 635-8, 2001
Russell P. Surface epithelial-stromal tumors of the ovary. In: Blaustein's pathology of the female genital tract
(ed. Kurman RJ, 4. ed ), 709-47. Springer-Verlag, New York, 1994
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified beta-globin and
HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324: 163-6, 1986
Sajantila A, Salem A-H, Savolainen P, Bauer K, Gierig C, Pääbo S. Paternal and maternal DNA lineages reveal
a bottleneck in the founding of the Finnish population. Proc Natl Acad Sci USA 93: 12035-9, 1996
Salovaara R, Loukola A, Kristo P, Kääriäinen H, Ahtola H, Eskelinen M, Härkönen N, Julkunen R, Kangas E,
Ojala S, Tulikoura J, Valkamo E, Järvinen H, Mecklin JP, Aaltonen LA, de la Chapelle A. Population-
based molecular detection of hereditary nonpolyposis colorectal cancer. J Clin Oncol 18: 2193-200, 2000
The Sanger Centre. ftp://ftp.sanger.ac.uk/pub/human/sequences/Chr_13/
Satagopan JM, Offit K, Foulkes W, Robson ME, Wacholder S, Eng CM, Karp SE, Begg CB. The lifetime risks
of breast cancer in Ashkenazi Jewish carriers of BRCA1 and BRCA2 mutations. Cancer Epidemiol
Biomarkers Prev 10: 467-73, 2001
Sattin RW, Rubin GL, Webster LA, Huezo CM, Wingo PA, Ory HW, Layde PM. Family history and the risk of
breast cancer. JAMA 253: 1908-13, 1985
Sawyer P. The age of Vikings, and before. In: The Oxford illustrated history of the Vikings (ed. Sawyer P), 1-
18. Oxford University Press, New York, 1999
Schildkraut JM, Risch N, Thompson WD. Evaluating genetic association among ovarian, breast, and
endometrial cancer: evidence for a breast/ovarian cancer relationship. Am J Hum Genet 45: 521-9, 1989
Schleutker J, Laine A-P, Haataja L, Renlund M, Weissenbach J, Aula P, Peltonen L. Linkage disequilibrium
utilized to establish a refined genetic position of the Salla disease locus on 6q14-q15. Genomics 27: 286-
92, 1995
Schmidt S, Becher H, Chang-Claude J. Breast cancer risk assessment: use of complete pedigree information and
the effect of misspecified ages at diagnosis of affected relatives. Hum Genet 102: 348-56, 1998
Schmucker B, Krawczak M. Meiotic microdeletion breakpoints in the BRCA1 gene are significantly associated
with symmetric DNA-sequence elements. Am J Hum Genet 61: 1454-6, 1997
Schubert EL, Lee MK, Mefford HC, Argonza RH, Morrow JE, Hull J, Dann JL, King M-C. BRCA2 in
American families with four or more cases of breast or ovarian cancer: recurrent and novel mutations,
variable expression, penetrance, and the possibility of families whose cancer is not attributable to BRCA1
or BRCA2. Am J Hum Genet 60: 1031-40, 1997
Scully R, Chen J, Ochs RL, Keegan K, Hoekstra M, Feunteun J, Livingston DM. Dynamic changes of BRCA1
subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90: 425-35, 1997
Scully R, Ganesan S, Vlasakova K, Chen J, Socolovsky M, Livingston DM. Genetic analysis of BRCA1
function in a defined tumor cell line. Mol Cell 4: 1093-9, 1999
Sekine M, Nagata H, Tsuji S, Hirai Y, Fujimoto S, Hatae M, Kobayashi I, Fujii T, Nagata I, Ushijima K, Obata
K, Suzuki M, Yoshinaga M, Umesaki N, Satoh S, Enomoto T, Motoyama S, Tanaka K. Localization of a
101
novel susceptibility gene for familial ovarian cancer to chromosome 3p22-p25. Hum Mol Genet 10: 1421-
9, 2001a
Sekine M, Nagata H, Tsuji S, Hirai Y, Fujimoto S, Hatae M, Kobayashi I, Fujii T, Nagata I, Ushijima K, Obata
K, Suzuki M, Yoshinaga M, Umesaki N, Satoh S, Enomoto T, Motoyama S, Tanaka K. Mutational
analysis of BRCA1 and BRCA2 and clinicopathologic analysis of ovarian cancer in 82 ovarian cancer
families: two common founder mutations of BRCA1 in Japanese population. Clin Cancer Res 7: 3144-50,
2001b
Serova OM, Mazoyer S, Puget N, Dubois V, Tonin P, Shugart YY, Goldgar D, Narod SA, Lynch HT, Lenoir
GM. Mutations in BRCA1 and BRCA2 in breast cancer families: are there more breast cancer-
susceptibility genes? Am J Hum Genet 60: 486-95, 1997
Sharan SK, Morimatsu M, Albrecht U, Lim DS, Regel E, Dinh C, Sands A, Eichele G, Hasty P, Bradley A.
Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386:
804-10, 1997
Shattuck-Eidens D, Oliphant A, McClure M, McBride C, Gupte J, Rubano T, Pruss D, Tavtigian SV, Teng DH,
Adey N, Staebell M, Gumpper K, Lundstrom R, Hulick M, Kelly M, Holmen J, Lingenfelter B, Manley S,
Fujimura F, Luce M, Ward B, Cannon-Albright L, Steele L, Offit K, Gilewski T, Norton L, Brown K,
Schulz C, Hampel H, Schluger A, Giulotto E, Zoli W, Ravaioli A, Nevanlinna H, Pyrhonen S, Rowley P,
Loader S, Osborne M, Daly M, Tepler I, Weinstein P, Scalia J, Michaelson R, Scott R, Radice P, Pierotti
M, Garber J, Isaacs C, Peshkin B, Lippman M, Dosik M, Caligo M, Greenstein R, Pilarski R, Weber B,
Burgemeister R, Frank T, Skolnick M, Thomas A. BRCA1 sequence analysis in women at high risk for
susceptibility mutations: risk factor analysis and implications for genetic testing. JAMA 278: 1242-50,
1997
Simard J, Tonin P, Durocher F, Morgan K, Rommens J, Gingras S, Samson C, Leblanc J-F, Bélanger C, Dion F,
Liu Q, Skolnick M, Goldgar D, Shattuck-Eidens D, Labrie F, Narod SA. Common origins of BRCA1
mutations in Canadian breast and ovarian cancer families. Nat Genet 8: 392-8, 1994
Sinclair CS, Berry R, Schaid D, Thibodeau SN, Couch FJ. BRCA1 and BRCA2 have a limited role in familial
prostate cancer. Cancer Res 60: 1371-5, 2000
Slatkin M, Rannala B. Estimating allele age. Annu Rev Genomics Hum Genet 1: 225-49, 2000
Slattery ML, Kerber RA. A comprehensive evaluation of family history and breast cancer risk. The Utah
Population Database. JAMA 270: 1563-8, 1993
Smith SA, Richards WE, Caito K, Hanjani P, Markman M, DeGeest K, Gallion HH. BRCA1 germline
mutations and polymorphisms in a clinic-based series of ovarian cancer cases: a Gynecologic Oncology
Group study. Gynecol Oncol 83: 586-92, 2001
Smith TM, Lee MK, Szabo CI, Jerome N, McEuen M, Taylor M, Hood L, King MC. Complete genomic
sequence and analysis of 117 kb of human DNA containing the gene BRCA1. J Med Genet 33: 889-98,
1996
Somasundaram K, Zhang H, Zeng YX, Houvras Y, Peng Y, Zhang H, Wu GS, Licht JD, Weber BL, El-Deiry
WS. Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor
p21WAF1/CiP1. Nature 389: 187-90, 1997
102
Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol
Biol 98: 503-17, 1975
Spain BH, Larson CJ, Shihabuddin LS, Gage FH, Verma IM. Truncated BRCA2 is cytoplasmic: implications
for cancer-linked mutations. Proc Natl Acad Sci USA 96: 13920-5, 1999
Spillman MA, Bowcock AM. BRCA1 and BRCA2 mRNA levels are coordinately elevated in human breast
cancer cells in response to estrogen. Oncogene 13: 1639-45, 1996
Steichen-Gersdorf E, Gallion HH, Ford D, Girodet C, Easton DF, DiCioccio RA, Evans G, Ponder MA, Pye C,
Mazoyer S, Noguchi T, Karengueven F, Sobol H, Hardouin A, Bignon Y-J, Piver MS, Smith SA, Ponder
BAJ. Familial site-specific ovarian cancer is linked to BRCA1 on 17q12-21. Am J Hum Genet 55: 870-5,
1994
Stoppa-Lyonnet D, Laurent-Puig P, Essioux L, Pagés S, Ithier G, Ligot L, Fourquet A, Salmon RJ, Clough KB,
Pouillart P, the Institut Curie Breast Cancer Group, Bonaiti-Pellié C, Thomas G. BRCA1 sequence
variations in 160 individuals referred to a breast/ovarian family cancer clinic. Am J Hum Genet 60: 1021-
30, 1997
Stratton JF, Gayther SA, Russell P, Dearden J, Gore M, Blake P, Easton D, Ponder BA. Contribution of BRCA1
mutations to ovarian cancer. N Engl J Med 336: 1125-30, 1997
Stratton JF, Pharoah P, Smith SK, Easton D, Ponder BA. A systematic review and meta-analysis of family
history and risk of ovarian cancer. Br J Obstet Gynaecol 105: 493-9, 1998
Stratton JF, Thompson D, Bobrow L, Dalal N, Gore M, Bishop DT, Scott I, Evans G, Daly P, Easton DF,
Ponder BA. The genetic epidemiology of early-onset epithelial ovarian cancer: a population-based study.
Am J Hum Genet 65: 1725-32, 1999
Struewing JP, Hartge P, Wacholder S, Baker SM, Berlin M, McAdams M, Timmerman MM, Brody LC, Tucker
MA. The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews.
N Engl J Med 336: 1401-8, 1997
Sutcliffe S, Pharoah PDP, Easton DF, Ponder BAJ, the UKCCCR Familial Ovarian Cancer Study Group.
Ovarian and breast cancer risks to women in families with two or more cases of ovarian cancer. Int J
Cancer 87: 110-7, 2000
Swensen J, Hoffman M, Skolnick MH, Neuhausen SL. Identification of a 14 kb deletion involving the promoter
region of BRCA1 in a breast cancer family. Hum Mol Genet 6: 1513-7, 1997
Syrjäkoski K, Vahteristo P, Eerola H, Tamminen A, Kivinummi K, Sarantaus L, Holli K, Blomqvist C,
Kallioniemi O-P, Kainu T, Nevanlinna H. Population-based study of BRCA1 and BRCA2 mutations in
1035 unselected Finnish breast cancer patients. J Natl Cancer Inst 92: 1529-31, 2000
Szabo CI, King M-C. Population genetics of BRCA1 and BRCA2. Am J Hum Genet 60: 1013-20, 1997
Takahashi H, Chiu HC, Bandera CA, Behbakht K, Liu PC, Couch FJ, Weber BL, LiVolsi VA, Furusato M,
Rebane BA, Cardonick A, Benjamin I, Morgan MA, King SA, Mikuta JJ, Rubin SC, Boyd J. Mutations of
the BRCA2 gene in ovarian carcinomas. Cancer Res 56: 2738-41, 1996
Tang NL, Pang CP, Yeo W, Choy KW, Lam PK, Suen M, Law LK, King WW, Johnson P, Hjelm M. Prevalence
of mutations in the BRCA1 gene among Chinese patients with breast cancer. J Natl Cancer Inst 91: 882-5,
1999
103
Tanner MM, Grenman S, Koul A, Johannsson O, Meltzer P, Pejovic T, Borg Å, Isola JJ. Frequent amplification
of chromosomal region 20q12-q13 in ovarian cancer. Clin Cancer Res 6: 1833-9, 2000
Tapper J, Sarantaus L, Vahteristo P, Nevanlinna H, Hemmer S, Seppälä M, Knuutila S, Butzow R. Genetic
changes in inherited and sporadic ovarian carcinomas by comparative genomic hybridization: extensive
similarity except for a difference at chromosome 2q24-q32. Cancer Res 58: 2715-9, 1998
Tavtigian SV, Simard J, Rommens J, Couch F, Shattuck-Eidens D, Neuhausen S, Merajver S, Thorlacius S,
Offit K, Stoppa-Lyonnet D, Belanger C, Bell R, Berry S, Bogden R, Chen Q, Davis T, Dumont M, Frye C,
Hattier T, Jammulapati S, Janecki T, Jiang P, Kehrer R, Leblanc J-F, Mitchell JY, McArthur-Morrison J,
Nguyen K, Peng Y, Samson C, Schroeder M, Snyder SC, Steele L, Stringfellow M, Stroup C, Swedlund B,
Swensen J, Teng D, Thomas A, Tran T, Tranchant M, Weaver-Feldhaus J, Wong AKC, Shizuya H,
Eyfjord JE, Cannon-Albright L, Labrie F, Skolnick MH, Weber B, Kamb A, Goldgar DE. The complete
BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat Genet 12: 333-7, 1996
Thomas JE, Smith M, Tonkinson JL, Rubinfeld B, Polakis P. Induction of phosphorylation on BRCA1 during
the cell cycle and after DNA damage. Cell Growth Differ 8: 801-9, 1997
Thompson D, Easton D. Variation in cancer risks, by mutation position, in BRCA2 mutation carriers. Am J Hum
Genet 68: 410-9, 2001
Thompson D, Easton D. Variation in BRCA1 cancer risks by mutation position. Cancer Epidemiol Biomarkers
Prev 11: 329-36, 2002
Thompson ME, Jensen RA, Obermiller PS, Page DL, Holt JT. Decreased expression of BRCA1 accelerates
growth and is often present during sporadic breast cancer progression. Nat Genet 9: 444-50, 1995
Thorlacius S, Olafsdottir G, Tryggvadottir L, Neuhausen S, Jonasson JG, Tavtigian SV, Tulinius H,
Ögmundsdottir HM, Eyfjörd JE. A single BRCA2 mutation in male and female breast cancer families from
Iceland with varied cancer phenotypes. Nat Genet 13: 117-9, 1996
Thorlacius S, Sigurdsson S, Bjarnadottir H, Olafsdottir G, Jonasson JG, Tryggvadottir L, Tulinius H, Eyfjörd
JE. Study of a single BRCA2 mutation with high carrier frequency in a small population. Am J Hum Genet
60: 1079-84, 1997
Thorlacius S, Struewing JP, Hartge P, Olafsdottir GH, Sigvaldason H, Tryggvadottir L, Wacholder S, Tulinius
H, Eyfjörd JE. Population-based study of risk of breast cancer in carriers of BRCA2 mutation. Lancet 352:
1337-9, 1998
Thorlacius S, Tryggvadottir L, Olafsdottir GH, Jonasson JG, Ogmundsdottir HM, Tulinius H, Eyfjord JE.
Linkage to BRCA2 region in hereditary male breast cancer. Lancet 346: 544-5, 1995
Tirkkonen M, Johannsson O, Agnarsson BA, Olsson H, Ingvarsson S, Karhu R, Tanner M, Isola J, Barkardottir
RB, Borg Å, Kallioniemi O-P. Distinct somatic genetic changes associated with tumor progression in
carriers of BRCA1 and BRCA2 germ-line mutations. Cancer Res 57: 1222-7, 1997
Tobias DH, Eng C, McCurdy LD, Kalir T, Mandelli J, Dottino PR, Cohen CJ. Founder BRCA 1 and 2
mutations among a consecutive series of Ashkenazi Jewish ovarian cancer patients. Gynecol Oncol 78:
148-51, 2000
Tonin PM, Mes-Masson AM, Narod SA, Ghadirian P, Provencher D. Founder BRCA1 and BRCA2 mutations in
French Canadian ovarian cancer cases unselected for family history. Clin Genet 55: 318-24, 1999
104
Tonin PN, Mes-Masson A-M, Futreal PA, Morgan K, Mahon M, Foulkes WD, Cole DEC, Provencher D,
Ghadirian P, Narod SA. Founder BRCA1 and BRCA2 mutations in French Canadian breast and ovarian
cancer families. Am J Hum Genet 63: 1341-51, 1998
Tonin PN, Perret C, Lambert JA, Paradis AJ, Kantemiroff T, Benoit MH, Martin G, Foulkes WD, Ghadirian P.
Founder BRCA1 and BRCA2 mutations in early-onset French Canadian breast cancer cases unselected for
family history. Int J Cancer 95: 189-93, 2001
Unger MA, Nathanson KL, Calzone K, Antin-Ozerkis D, Shih HA, Martin AM, Lenoir GM, Mazoyer S, Weber
BL. Screening for genomic rearrangements in families with breast and ovarian cancer identifies BRCA1
mutations previously missed by conformation-sensitive gel electrophoresis or sequencing. Am J Hum
Genet 67: 841-50, 2000
Vahteristo P, Eerola H, Tamminen A, Blomqvist C, Nevanlinna H. A probability model for predicting BRCA1
and BRCA2 mutations in breast and breast-ovarian cancer families. Br J Cancer 84: 704-8, 2001
Vallon-Christersson J, Cayanan C, Haraldsson K, Loman N, Bergthorsson JT, Brondum-Nielsen K, Gerdes A-
M, Moller P, Kristoffersson U, Olsson H, Borg Å, Monteiro AN. Functional analysis of BRCA1 C-terminal
missense mutations identified in breast and ovarian cancer families. Hum Mol Genet 10: 353-60, 2001
van der Looij M, Szabo C, Besznyak I, Liszka G, Csokay B, Pulay T, Toth J, Devilee P, King M-C, Olah E.
Prevalence of founder BRCA1 and BRCA2 mutations among breast and ovarian cancer patients in Hungary.
Int J Cancer 86: 737-40, 2000
Varilo T, Savukoski M, Norio R, Santavuori P, Peltonen L, Järvelä I. The age of human mutation: genealogical
and linkage disequilibrium analysis of the CLN5 mutation in the Finnish population. Am J Hum Genet 58:
506-12, 1996
Vehmanen P, Friedman LS, Eerola H, McClure M, Ward B, Sarantaus L, Kainu T, Syrjäkoski K, Pyrhönen S,
Kallioniemi O-P, Muhonen T, Luce M, Frank TS, Nevanlinna H. Low proportion of BRCA1 and BRCA2
mutations in Finnish breast cancer families: evidence for additional susceptibility genes. Hum Mol Genet 6:
2309-15, 1997a
Vehmanen P, Friedman LS, Eerola H, Sarantaus L, Pyrhönen S, Ponder BA, Muhonen T, Nevanlinna H. A low
proportion of BRCA2 mutations in Finnish breast cancer families. Am J Hum Genet 60: 1050-8, 1997b
Venesmaa P. Epithelial ovarian cancer: impact of surgery and chemotherapy on survival during 1977-1990.
Obstet Gynecol 84: 8-11, 1994
Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 108: 171-82, 2002
Verhoog LC, Berns EM, Brekelmans CT, Seynaeve C, Meijers-Heijboer EJ, Klijn JG. Prognostic significance
of germline BRCA2 mutations in hereditary breast cancer patients. J Clin Oncol 18: 119S-24S, 2000
Verhoog LC, Brekelmans CT, Seynaeve C, Dahmen G, van Geel AN, Bartels CC, Tilanus-Linthorst MM,
Wagner A, Devilee P, Halley DJ, van den Ouweland AM, Meijers-Heijboer EJ, Klijn JG. Survival in
hereditary breast cancer associated with germline mutations of BRCA2. J Clin Oncol 17: 3396-402, 1999
Verhoog LC, Brekelmans CT, Seynaeve C, van den Bosch LM, Dahmen G, van Geel AN, Tilanus-Linthorst
MM, Bartels CC, Wagner A, van den Ouweland A, Devilee P, Meijers-Heijboer EJ, Klijn JG. Survival and
tumour characteristics of breast-cancer patients with germline mutations of BRCA1. Lancet 351: 316-21,
1998
105
Verhoog LC, van den Ouweland AM, Berns E, van Veghel-Plandsoen MM, van Staveren IL, Wagner A, Bartels
CC, Tilanus-Linthorst MM, Devilee P, Seynaeve C, Halley DJ, Niermeijer MF, Klijn JG, Meijers-Heijboer
H. Large regional differences in the frequency of distinct BRCA1/BRCA2 mutations in 517 Dutch breast
and/or ovarian cancer families. Eur J Cancer 37: 2082-90, 2001
Wagner TMU, Hirtenlehner K, Shen P, Moeslinger R, Muhr D, Fleischmann E, Concin H, Doeller W, Haid A,
Lang AH, Mayer P, Petru E, Ropp E, Langbauer G, Kubista E, Scheiner O, Underhill P, Mountain J,
Stierer M, Zielinski C, Oefner P. Global sequence diversity of BRCA2: analysis of 71 breast cancer
families and 95 control individuals of worldwide populations. Hum Mol Genet 8: 413-23, 1999
Wagner TMU, Moslinger RA, Muhr D, Langbauer G, Hirtenlehner K, Concin H, Doeller W, Haid A, Lang AH,
Mayer P, Ropp E, Kubista E, Amirimani B, Helbich T, Becherer A, Scheiner O, Breiteneder H, Borg A,
Devilee P, Oefner P, Zielinski C. BRCA1-related breast cancer in Austrian breast and ovarian cancer
families: specific BRCA1 mutations and pathological characteristics. Int J Cancer 77: 354-60, 1998
Wang SC, Shao R, Pao AY, Zhang S, Hung MC, Su LK. Inhibition of cancer cell growth by BRCA2. Cancer
Res 62: 1311-4, 2002
Warner E, Foulkes W, Goodwin P, Meschino W, Blondal J, Paterson C, Ozcelik H, Goss P, Allingham-Hawkins
D, Hamel N, Di Prospero L, Contiga V, Serruya C, Klein M, Moslehi R, Honeyford J, Liede A, Glendon
G, Brunet JS, Narod S. Prevalence and penetrance of BRCA1 and BRCA2 gene mutations in unselected
Ashkenazi Jewish women with breast cancer. J Natl Cancer Inst 91: 1241-7, 1999
Watkins WS, Zenger R, O'Brien E, Nyman D, Eriksson AW, Renlund M, Jorde LB. Linkage disequilibrium
patterns vary with chromosomal location: a case study from the von Willebrand factor region. Am J Hum
Genet 55: 348-55, 1994
Weber JL, Kwitek AE, May PE, Wallace MR, Collins FS, Ledbetter DH. Dinucleotide repeat polymorphisms at
the D17S250 and D17S261 loci. Nucleic Acids Res 18: 4640, 1990
Weber JL, Wong C. Mutation of human short tandem repeats. Hum Mol Genet 2: 1123-8, 1993
Weinberg RA. Oncogenes, antioncogenes, and the molecular bases of multistep carcinogenesis. Cancer Res 49:
3713-21, 1989
Welcsh PL, King MC. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet 10:
705-13, 2001
Werness BA, Ramus SJ, Whittemore AS, Garlinghouse-Jones K, Oakley-Girvan I, Dicioccio RA, Tsukada Y,
Ponder BA, Piver MS. Histopathology of familial ovarian tumors in women from families with and without
germline BRCA1 mutations. Hum Pathol 31: 1420-4, 2000
Whittemore AS, Gong G, Itnyre J. Prevalence and contribution of BRCA1 mutations in breast cancer and
ovarian cancer: results from three U.S. population-based case-control studies of ovarian cancer. Am J Hum
Genet 60: 496-504, 1997
Whittemore AS, Harris R, Itnyre J. Characteristics relating to ovarian cancer risk: collaborative analysis of 12
US case-control studies. II. Invasive epithelial ovarian cancers in white women. Collaborative Ovarian
Cancer Group. Am J Epidemiol 136: 1184-203, 1992
Wilson CA, Payton MN, Elliott GS, Buaas FW, Cajulis EE, Grosshans D, Ramos L, Reese DM, Slamon DJ,
Calzone FJ. Differential subcellular localization, expression and biological toxicity of BRCA1 and the
splice variant BRCA1-delta11b. Oncogene 14: 1-16, 1997
106
Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G,
Barfoot R, Hamoudi R, Patel S, Rice C, Biggs P, Hashim Y, Smith A, Connor F, Arason A, Gudmundsson
J, Ficenec D, Kelsell D, Ford D, Tonin P, Bishop DT, Spurr NK, Ponder BAJ, Eeles R, Peto J, Devilee P,
Cornelisse C, Lynch H, Narod S, Lenoir G, Egilsson V, Barkardottir RB, Easton DF, Bentley DR, Futreal
PA, Ashworth A, Stratton MR. Identification of the breast cancer susceptibility gene BRCA2. Nature 378:
789-92, 1995
Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Nguyen K, Seal S, Tran T, Averill D, Fields
P, Marshall G, Narod S, Lenoir GM, Lynch H, Feunteun J, Devilee P, Cornelisse CJ, Menko FH, Daly PA,
Ormiston W, McManus R, Pye C, Lewis CM, Cannon-Albright LA, Peto J, Ponder BAJ, Skolnick MH,
Easton DF, Goldgar DE, Stratton MR. Localization of a breast cancer susceptibility gene, BRCA2, to
chromosome 13q12-13. Science 265: 2088-90, 1994
Wright AF, Carothers AD, Pirastu M. Population choice in mapping genes for complex diseases. Nat Genet 23:
397-404, 1999
Wu LC, Wang ZW, Tsan JT, Spillman MA, Phung A, Xu XL, Yang MC, Hwang LY, Bowcock AM, Baer R.
Identification of a RING protein that can interact in vivo with the BRCA1 gene product. Nat Genet 14:
430-40, 1996
Xu X, Weaver Z, Linke SP, Li C, Gotay J, Wang XW, Harris CC, Ried T, Deng CX. Centrosome amplification
and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient
cells. Mol Cell 3: 389-95, 1999
Yarden RI, Pardo-Reoyo S, Sgagias M, Cowan KH, Brody LC. BRCA1 regulates the G2/M checkpoint by
activating Chk1 kinase upon DNA damage. Nat Genet 30: 285-9, 2002
Zavattari P, Deidda E, Whalen M, Lampis R, Mulargia A, Loddo M, Eaves I, Mastio G, Todd JA, Cucca F.
Major factors influencing linkage disequilibrium by analysis of different chromosome regions in distinct
populations: demography, chromosome recombination frequency and selection. Hum Mol Genet 9: 2947-
57, 2000
Zelada-Hedman M, Wasteson Arver B, Claro A, Chen J, Werelius B, Kok H, Sandelin K, Håkansson S,
Andersen TI, Borg Å, Borresen Dale A-L, Lindblom A. A screening for BRCA1 mutations in breast and
breast-ovarian cancer families from the Stockholm region. Cancer Res 57: 2474-7, 1997
Zheng L, Li S, Boyer TG, Lee W-H. Lessons learned from BRCA1 and BRCA2. Oncogene 19: 6159-75, 2000
Zou JP, Hirose Y, Siddique H, Rao VN, Reddy ES. Structure and expression of variant BRCA2a lacking the
transactivation domain. Oncol Rep 6: 437-40, 1999
Zweemer RP, Shaw PA, Verheijen RM, Ryan A, Berchuck A, Ponder BA, Risch H, McLaughlin JR, Narod SA,
Menko FH, Kenemans P, Jacobs IJ. Accumulation of p53 protein is frequent in ovarian cancers associated
with BRCA1 and BRCA2 germline mutations. J Clin Pathol 52: 372-5, 1999