GERMLINE MUTATIONS OF BRCA1 AND BRCA2 GENES

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


Helsinki University Central Hospital


REVIEWED BY

Professor Päivi Peltomäki, MD, PhD

Department of Medical Genetics


Docent Ulla Puistola, MD, PhD


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

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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.

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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

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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

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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,

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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

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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.

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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).

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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


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


26.5 13.0 39.5

Ikeda et al.,2001

Japanese >2 bc (in 1st-degreerelatives), no ovarian cancer


7.9 20.8 28.7

Meindl andGermanConsortiumfor HBOC,2002

German >2 bc, with >2 diagnosed atage <50 y, no ovarian cancer


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)


35.7 7.1 42.9

Gayther etal., 1999

British 2 oc (in 1st/2nd-degreerelatives), no breast cancer


16.0 4.0 20.0

Sekine etal., 2001b

Japanese >2 oc (in 1st/2nd-degreerelatives), no breast cancer


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)


107 W, BRCA1 1.9

Rubin et al.,1998

American(PA)



8.6 0.9 9.5

Berchuck etal., 1998

American(NC)


103 W, BRCA1 3.9

Risch et al.,2001

Canadian(Ontario)


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


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)


92 F, 2 in BRCA1and 1in BRCA2

17.4 7.6 25.0

Moslehi etal., 2000

AshkenaziJewish(Israel and N.America)


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, 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 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.

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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

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GERMLINE MUTATIONS OF BRCA1 AND BRCA2 GENES