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OULU 2007

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Katri Pylkäs

ATM, ATR AND MRE11 COMPLEX GENESIN HEREDITARY SUSCEPTIBILITY TOBREAST CANCER

FACULTY OF MEDICINE,DEPARTMENT OF CLINICAL GENETICS,UNIVERSITY OF OULU

D 914

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TA K

atri Pylkäs

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 9 1 4

KATRI PYLKÄS

ATM, ATR AND MRE11 COMPLEX GENES IN HEREDITARY SUSCEPTIBILITY TOBREAST CANCER

Academic dissertation to be presented, with the assent ofthe Faculty of Medicine of the University of Oulu, forpublic defence in Auditorium 5 of Oulu UniversityHospital, on April 20th, 2007, at 12 noon

OULUN YLIOPISTO, OULU 2007

Copyright © 2007Acta Univ. Oul. D 914, 2007

Supervised byDocent Robert Winqvist

Reviewed byProfessor Marikki LaihoProfessor Annika Lindblom

ISBN 978-951-42-8382-6 (Paperback)ISBN 978-951-42-8383-3 (PDF)http://herkules.oulu.fi/isbn9789514283833/ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)http://herkules.oulu.fi/issn03553221/

Cover designRaimo Ahonen

OULU UNIVERSITY PRESSOULU 2007

Pylkäs, Katri, ATM, ATR and Mre11 complex genes in hereditary susceptibility tobreast cancerFaculty of Medicine, Department of Clinical Genetics, University of Oulu, P.O.Box 5000, FI-90014University of Oulu, Finland Acta Univ. Oul. D 914, 2007Oulu, Finland

AbstractMutations in BRCA1 and BRCA2 explain only about 20% of familial aggregation of breast cancer,suggesting involvement of additional susceptibility genes. In this study five DNA damage responsegenes, ATM, ATR, MRE11, NBS1 and RAD50, were considered as putative candidates to explain someof the remaining familial breast cancer risk, and were screened for germline mutations in familiesdisplaying genetic predisposition.

Analysis of ATM indicated that clearly pathogenic mutations seem to be restricted to thosereported in ataxia-telangiectasia (A-T). However, a cancer risk modifying effect was suggested for acombination of two ATM polymorphisms, 5557G>A and IVS38-8T>C, as this allele seemed toassociate with bilateral breast cancer (OR 10.2, 95% CI 3.1–33.8, p = 0.001).

The relevance of ATM mutations, originally identified in Finnish A-T patients, in breast cancersusceptibility was evaluated by a large case-control study. Two such alleles, 6903insA and 7570G>C,in addition to 8734A>G previously associated with breast cancer susceptibility, were observed. Theoverall mutation frequency in unselected cases (7/1124) was higher than in controls (1/1107), but asignificantly elevated frequency was observed only in familial cases (6/541, p = 0.006, OR 12.4, 95%CI 1.5–103.3). These three mutations showed founder effects in their geographical occurrence, andhad different functional consequences at protein level.

In ATR no disease-related mutations were observed, suggesting that it is not a breast cancersusceptibility gene.

The mutation screening of the Mre11 complex genes, MRE11, NBS1 and RAD50, revealed twonovel potentially breast cancer associated alleles: NBS1 Leu150Phe and RAD50 687delT wereobserved in 2.0% (3/151) of the studied families. The subsequent study of newly diagnosed,unselected breast cancer cases indicated that RAD50 687delT is a relatively common low-penetrancesusceptibility allele in Northern Finland (cases 8/317 vs. controls 6/1000, OR 4.3, 95% CI 1.5–12.5,p = 0.008). NBS1 Leu150Phe (2/317) together with a novel RAD50 IVS3-1G>A mutation (1/317)was also observed, both being absent from controls. Loss of the wild-type allele was not observed inthe tumors of the studied mutation carriers, but they all showed an increase in chromosomalinstability of peripheral T-lymphocytes. This suggests an effect for RAD50 and NBS1haploinsufficiency on genomic integrity and susceptibility to cancer.

Keywords: ATM, ATR, breast neoplasms, DNA double-strand break response, geneticpredisposition to disease, MRE11, NBS1, RAD50

Acknowledgements

This study was carried out at the Department of Clinical Genetics, Oulu University Hospital and University of Oulu, during the years 2002-2007. I wish to express my sincere gratitude to all those who participated in this project:

Professor Jaakko Ignatius and Professor Jaakko Leisti for giving me the opportunity to work in the Department of Clinical Genetics.

Docent Robert Winqvist, my supervisor, for giving me the opportunity to join his research group and to participate in such an interesting project. He is most warmly thanked for giving me the possibility to pursue my own ideas as well, and his guidance and support has been invaluable throughout this project.

Professor Marikki Laiho and Professor Annika Lindblom for their invaluable comments on the manuscript of this thesis. I also want to thank Anna Vuolteenaho for the careful revision of the language in this thesis.

All the co-authors and collaborators who made this study possible, Professor Kristiina Aittomäki, Professor Rosa Barkardóttir, Docent Guillermo Blanco, Professor Carl Blomqvist, Professor Åke Borg, Professor Anne-Lise Børresen-Dale, Dr Magtouf Gatei,

Dr Mervi Grip, Professor Kaija Holli, Professor Olli-Pekka Kallioniemi, Professor Juha Kere, Professor Kum Kum Khanna, Professor Sakari Knuutila, Professor Helena Kääriäinen, Tuija Lundán, M.Sc., Dr Arto Mannermaa, Dr Aki Mustonen, Dr Markus Mäkinen, Docent Heli Nevanlinna, Dr Elina Nieminen, Docent Pentti Nieminen, Professor John Petrini, Docent Ulla Puistola, Docent Ylermi Soini, Dr Kirsi Syrjäkoski, Johanna Tommiska, M.Sc., and Dr Nicola Waddell.

My colleagues Sanna-Maria Karppinen and Katrin Rapakko for their support both inside and outside the laboratory. Especially Sanna is most warmly thanked for the intensive teamwork throughout the project, and Katrin for her invaluable help in chromosomal analysis.

All the past and present group members, especially Minna Allinen, Hannele Erkko and Virpi Mansikka, and the staff at the department of Clinical Genetics for their expertise in our studies and for creating pleasant working conditions. The technical assistance of Kati Outila and Arja Tapio is also greatly appreciated.

My family and friends. I especially want to thank my parents, Mirja and Esa, for their encouragement and support throughout the years. Most of all I am grateful to Tuomo for

his care and unfailing support, and to Emma for bringing all the love and happiness into our lives.

Finally, I want to express my sincere gratitude to all the patients and their family members who volunteered to participate in this study.

The financial support of the University of Oulu, Oulu University Hospital, the Academy of Finland, Nordic Cancer Union, Oulu University Scholarship foundation, Yliopiston apteekin rahasto of University of Oulu, the Finnish Cancer Society, the Cancer Society of Northern Finland, the Finnish Cultural Foundation, the Maud Kuistila Memorial Foundation, the Finnish Breast Cancer Group and the Ida Montin Foundation is gratefully acknowledged.

Oulu, April 2007 Katri Pylkäs

Abbreviations

AI allelic imbalance A-T ataxia-telangiectasia ATLD ataxia-telangiectasia-like disorder ATM ataxia-telangiectasia mutated ATR ataxia-telangiectasia- and Rad3-related BRCA1/2 breast cancer gene 1/2 BRCT BRCA1 carboxy-terminal CHK1/2 checkpoint kinase 1/2 gene CI confidence interval CSGE conformation sensitive gel electrophoresis DSB double-strand break ESE exonic splicing enhancer FA Fanconi anemia FAT FRAP/ATM/TRRAP FATC FRAP/ATM/TRRAP carboxy-terminal HR homologous recombination IR ionizing radiation kDa kilodalton LCL lymphoblast cell line LOH loss of heterozygosity MRE11 meiotic recombination 11, S.cerevisiae homolog, gene NBS Nijmegen breakage syndrome NBS1 Nijmegen breakage syndrome gene NHEJ non-homologous end-joining OR odds ratio p short arm of the chromosome p53 tumor protein 53 q long arm of the chromosome SNP single nucleotide polymorphism ssDNA single-stranded DNA TP53 gene for tumor protein 53

List of original publications

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Heikkinen K‡, Rapakko K, Karppinen S-M, Erkko H, Nieminen P & Winqvist R (2005) Association of common ATM polymorphism with bilateral breast cancer. Int J Cancer 116: 69-72.

II Pylkäs K*, Tommiska J*, Syrjäkoski K, Kere J, Gatei M, Waddell N, Allinen M, Karppinen S-M, Rapakko K, Kääriäinen H, Aittomäki K, Blomqvist C, Mustonen A, Holli K, Khanna KK, Kallioniemi O-P, Nevanlinna H & Winqvist R (2006) Evaluation of the role of Finnish ataxia-telangiectasia mutations in hereditary predisposition to breast cancer. Carcinogenesis, Dec 13 [epub ahead of print].

III Heikkinen K‡, Mansikka V, Karppinen S-M, Rapakko K & Winqvist R (2005) Mutation analysis of the ATR gene in breast and ovarian cancer families. Breast Cancer Res 7: R495-R501.

IV Heikkinen K‡, Karppinen S-M, Soini Y, Mäkinen M & Winqvist R (2003) Mutation screening of Mre11 complex genes: indication of RAD50 involvement in breast and ovarian cancer susceptibility. J Med Genet 40: e131.

V Heikkinen K‡, Rapakko K, Karppinen S-M, Erkko H, Knuutila S, Lundán T, Mannermaa A, Børresen-Dale A-L, Borg Å, Barkardóttir RB, Petrini J & Winqvist R (2006) RAD50 and NBS1 are breast cancer susceptibility genes associated with genomic instability. Carcinogenesis 27: 1593-1599.

*These authors contributed equally to the study ‡ Pylkäs K née Heikkinen K

Contents

Abstract Acknowledgements Abbreviations List of original publications Contents 1 Introduction ................................................................................................................... 13 2 Review of the literature ................................................................................................. 15

2.1 Development of cancer...........................................................................................15 2.2 General features of breast cancer............................................................................17 2.3 Hereditary predisposition to breast cancer..............................................................17

2.3.1 Major breast cancer susceptibility genes .......................................................17 2.3.2 Search for additional susceptibility genes .....................................................18 2.3.3 Selection of candidate genes .........................................................................20

2.4 DNA double-strand break response ........................................................................21 2.4.1 DNA double-strand break response pathway as part of the DNA

repair network .............................................................................................21 2.4.2 DNA double-strand break signaling leading to cell cycle checkpoints .........23

2.5 DNA double-strand break response genes associated with inherited genomic instability syndromes ..............................................................................26 2.5.1 ATM mutations and ataxia-telangiectasia ......................................................26 2.5.1.1 ATM structure and function......................................................................26 2.5.1.2 Ataxia-telangiectasia.................................................................................27 2.5.1.3 Heterozygous ATM mutations and breast cancer susceptibility................28

2.5.2 ATR mutations and Seckel syndrome ............................................................30 2.5.2.1 Structure and function of ATR..................................................................30 2.5.2.2 ATR mutations in Seckel syndrome ..........................................................31

2.5.3 Mutations in the Mre11 complex genes.........................................................32 2.5.3.1 Structure and function of the Mre11 complex ..........................................32 2.5.3.2 Ataxia-telangiectasia-like disorder and Nijmegen breakage

syndrome..................................................................................................34 3 Aims of the study........................................................................................................... 36

4 Material and Methods.................................................................................................... 37 4.1 Subjects ..................................................................................................................37

4.1.1 Studies I, III, IV.............................................................................................37 4.1.2 Study II ..........................................................................................................38 4.1.3 Study V..........................................................................................................38

4.2 DNA extraction (I-V)..............................................................................................39 4.3 mRNA extraction and cDNA synthesis (I, II, IV, V) ..............................................39 4.4 Mutation detection methods ...................................................................................40

4.4.1 Conformation sensitive gel electrophoresis (I-V)..........................................40 4.4.2 Minisequencing (II) .......................................................................................40 4.4.3 Direct sequencing (I-V).................................................................................40

4.5 Real-time Quantitative RT-PCR (I) ........................................................................41 4.6 Allelic imbalance analysis (II, IV, V)......................................................................41 4.7 Haplotype analysis (II, V).......................................................................................41 4.8 Cell cultures (I, II, IV, V)........................................................................................42 4.9 Western blot analysis (II)........................................................................................42 4.10 MTT assay (II)......................................................................................................42 4.11 Cytogenetic analysis (V).......................................................................................43 4.12 Statistical analysis (I-III, V)..................................................................................43 4.13 Ethical issues ........................................................................................................43

5 Results ........................................................................................................................... 44 5.1 Mutation screening of the ATM gene (I).................................................................44 5.2 A-T-related ATM mutations in breast cancer patients (II).......................................45 5.3 Mutation screening of the ATR gene (III) ...............................................................48 5.4 Mutation screening of Mre11 complex genes MRE11, NBS1 and

RAD50 (IV) ...........................................................................................................49 5.5 Occurrence of RAD50 and NBS1 mutations in unselected breast cancer

patients (V) ............................................................................................................50 6 Discussion ..................................................................................................................... 54

6.1 ATM mutations and breast cancer susceptibility (I, II) ...........................................54 6.2 ATR mutations and breast cancer susceptibility (III) ..............................................59 6.3 Mutations in the Mre11 complex genes and breast cancer susceptibility

(IV, V)....................................................................................................................60 7 Concluding remarks....................................................................................................... 65 References Original papers

1 Introduction

Breast cancer is the most common malignancy affecting women in Western countries (Parkin et al. 2001). It is a complex disease caused by a combination of both genetic and environmental factors. Most breast cancers arise in women without apparent family history of the disease, but in approximately 7% of all cases an inherited predisposition is proposed (Claus et al. 1996). Genetic linkage analysis in families with high risk of breast cancer has led to the identification of two major susceptibility genes: BRCA1 and BRCA2 (Miki et al. 1994; Wooster et al. 1995). Germline mutations in these genes explain the majority of the families with apparent autosomal dominant inheritance of susceptibility to both breast and ovarian cancer. However, most families with site-specific female breast cancer cannot be explained by mutations in BRCA1 and BRCA2, and population studies have demonstrated that they account for only a minority of the overall familial risk (Ford et al. 1998). This suggests the contribution of additional susceptibility genes. The identification of these genes may help to clarify the genetic background contributing to the etiology of breast cancer and suggest novel pharmaceutical targets, and it could lead to genetic screening in order to identify individuals at increased risk. Hopefully, this will ultimately lead to improved prevention efforts and treatment.

As genetic linkage analyses have largely failed to identify any additional major susceptibility genes, the remaining predisposition to breast cancer has been explained by a polygenic model (Pharoah et al. 2002). According to this model, the genetic susceptibility is due to several loci, each conferring a modest independent risk. These low- to moderate-penetrance susceptibility alleles are difficult to identify through linkage studies (Risch & Merikangas 1996). Therefore, the search has centered on association studies, the power of which can be enhanced by using cases with family history of the disease (Houlston & Peto 2003). The candidate genes for the association studies are often chosen based on their essential biological functions (Nathanson & Weber 2001). As BRCA1 and BRCA2 have an important role in cell response to DNA double-strand breaks (DSB), and also other genes, ATM, TP53 and CHK2, associated with breast cancer susceptibility function in this pathway (Khanna & Jackson 2001), other similarly acting genes may represent new potential candidates.

In this study, we have evaluated the possibility that germline alterations in selected DNA DSB response genes, ATM, ATR, MRE11, NBS1 and RAD50, could explain a

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fraction of the remaining familial breast cancer risk. Biallelic mutations in these genes have also been associated with inherited genomic instability syndromes. Of these candidates, ATM has previously been classified as a breast cancer susceptibility gene (Easton 1994; Swift et al. 1991; Olsen et al. 2001), but its exact role is still controversial (reviewed in Khanna & Chenevix-Trench 2004). Consequently, ATM was included in the study. In contrast, ATR and the Mre11 complex genes, MRE11, NBS1 and RAD50, were chosen based on their essential function in the maintenance of genomic integrity. The possible role of germline defects in these genes in inherited breast cancer susceptibility has not previously been studied by mutation screening. The entire coding region of these five genes was screened for germline mutations in families displaying genetic predisposition to breast cancer, and the role of potentially pathogenic alterations was evaluated by comparing their frequency to that of controls. Additionally, the overall relevance of the specific ATM, RAD50 and NBS1 alleles in breast cancer predisposition was evaluated by additional patient cohorts and functional analysis.

2 Review of the literature

2.1 Development of cancer

Cancer is a genetic disease that arises from accumulation of mutations in genes that regulate cell proliferation, development and differentiation. Cancer cell phenotype can be described by six hallmark features: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, avoidance of programmed cell death, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis (Hanahan & Weinberg 2000). Generally it has been thought that three to seven successive mutations are required for the malignant conversion (Miller 1980; Weinberg 1989; Vogelstein & Kinzler 1993). Various sources of spontaneous DNA damage are estimated to alter about 25 000 bases per human genome per cell per day out of the 3 x 109 base pairs in the genome. However, cells are endowed with specific mechanisms for repairing the damage and for maintaining mutations at reasonable levels. Consequently, the mutational load at any given time in both germline and somatic cells can be viewed as the outcome of dynamic equilibrium between the extent of DNA damage and the efficiency of DNA repair (Friedberg 2001). Given the normal mutation rate, typically 1.4 x 10-10 mutations per base pair per cell generation, the likelihood that a single cell will acquire enough independent mutations needed for malignant transformation is very low (Loeb 1991). However, it has been suggested that there are two major mechanisms that make the series of cancer-promoting mutations more probable. These are mutations that enhance cell proliferation, hence creating an expanded target population of cells for the next mutation (Nowell 1976), and mutations that affect the stability of the entire genome, either at the DNA or the chromosomal level, thereby increasing the overall mutation rate (Loeb 1991; Loeb 1994).

Most of the genes responsible for the development of malignancy fall into two main categories: proto-oncogenes and tumor suppressor genes (reviewed in Weinberg 1996). Under normal conditions, proto-oncogenes stimulate cell proliferation and differentiation. Their protein products are mainly involved in signal transduction pathways and include growth factors, growth factor receptors, signal transducers and transcription factors. Inappropriate activation may turn them into cancerous oncogenes, and this gain of function mutation even in a single allele confers a growth advantage to a cell. Conversely,

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tumor suppressors are genes whose loss of function promotes malignancy (Frank 2001; Cavenee & White 1995). They are usually negative regulators of growth or other functions that may affect invasive and metastatic potential (Osborne et al. 2004). The tumor suppressor genes can further be divided into two more classes according to their function. Gatekeepers act directly to regulate cell proliferation and are rate limiting for tumorigenesis, whereas caretakers maintain the integrity of the genome and promote tumor growth indirectly by causing an increased mutation rate (Kinzler & Vogelstein 1997).

The contribution of tumor suppressors in tumorigenesis was initially proposed by Alfred Knudson (1971) based on epidemiological studies of retinoblastoma. According to Knudson’s “two-hit” model, both copies of the responsible tumor suppressor gene need to be inactivated for cancer formation. In the familial form of the disease, the mutated allele is inherited from one of the parents and the second is somatically inactivated (Knudson 1971). Conventionally, the second hit often involves a large deletion that can be visualized as loss of heterozygosity (LOH) in the tumor. Knudson’s model has later been applied to other forms of familial cancer as well. However, not all loss of function mutations in tumor suppressor genes are truly recessive (Ponder 1988), and recent evidence suggests that inactivation can also occur by haploinsufficiency (Cook & McCaw 2000). This indicates that a gene-dosage effect rather than biallelic inactivation of tumor suppressor genes may be sufficient to exert a cellular phenotype that leads to tumorigenesis. Particularly, haploinsufficiency in caretaker genes is thought to lead to genomic instability, and thus to accelerated conversion of a normal cell into a neoplastic cell (Fodde & Smits 2002).

The vast majority of genetic alterations in cancer are somatic and found only in the cancer cells of an individual. In contrast, in hereditary susceptibility to cancer gene defects are in the germline, thus present in each cell. This can be considered as the first hit required for tumorigenesis (Frank 2001). Even though additional somatic changes are still needed, the presence of constitutional mutations greatly speeds up the accumulation of mutations needed for malignant conversion, and individuals with such mutations are at higher risk of developing cancer. Mutations with strong effects are likely to cause early onset of the disease and development of multiple primary tumors. They are inherited in simple Mendelian fashion and tend to cause familial clustering of disease (Fearon 1997; Frank 2004). Most cancer susceptibility syndromes result from inherited mutations in tumor suppressor genes rather than proto-oncogenes (Frank 2001). Thus far only three proto-oncogenes, RET, MET and CDK4, have been shown to be involved in hereditary cancer syndromes (Mulligan et al. 1993; Zuo et al. 1996; Schmidt et al. 1997). Although the inherited cancer syndromes are rare, they are of vast biological importance. The study of the genes responsible for the syndromes and the cellular pathways disrupted by the mutated proteins provide significant insights into the molecular origin and pathogenesis of both inherited and sporadic forms of cancer (Fearon 1997).

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2.2 General features of breast cancer

Breast cancer is the most common cancer among women in Western countries, comprising 22% of female cancers (Parkin et al. 2001). In Finland, 3903 new female breast cancer cases were reported in the year 2004, and the annual numbers have been growing (Finnish Cancer Registry 2006). However, mortality rates have been declining at the same time, mostly due to diagnosis at earlier stage associated with the widespread administration of adjuvant therapies (Duffy 2001). The incidence of breast cancer increases with age, doubling approximately every ten years until menopause, after which the rate of increase slows down dramatically (McPherson et al. 2000). The causes of breast cancer are multifactorial, and there is a strong interplay between genetic and environmental factors. The most significant single risk factor for breast cancer is family history. Other important risk determinants are several reproductive and hormonal factors, such as early age at menarche, nulliparity, and late age at menopause (Couch & Weber 1998, and refs. therein), which reflect a hormonal pattern with exposure to high levels of endogenous estrogen. Additional risk factors are previous benign breast disease and ionizing radiation (IR), particularly if exposure occurs in the rapid phase of breast formation during puberty, as well as postmenopausal obesity, alcohol consumption, a high-fat diet, prolonged oral contraceptive use and estrogen replacement therapy (McPherson et al. 2000; Hulka & Moorman 2001).

About 95% of malignant breast tumors are carcinomas that arise from the epithelium of the mammary gland. The transformation typically proceeds through several stages. Normal breast epithelium first transforms into atypical hyperplasia and carcinoma in situ. Eventually the malignant cells develop into invasive carcinoma, leading ultimately to cells with metastatic potential (Couch & Weber 1998). The majority of breast cancers are infiltrating ductal (70%) or lobular (about 6%) carcinomas. Rare histological subtypes are medullar, mucinous, papillary and tubular carcinomas, as well as Paget’s disease (Berg & Hutter 1995). The prognosis of breast cancer patients depends largely on the clinical stage at the time of diagnosis. The five-year relative survival rate for patients with localized disease is 93%, for patients with regional metastases 69%, and for patients with distant metastases 22% (Dickman et al. 1999).

2.3 Hereditary predisposition to breast cancer

2.3.1 Major breast cancer susceptibility genes

Most breast cancers arise in women without apparent family history of the disease. However, in approximately 7% of all cases an inherited predisposition is proposed (Claus et al. 1996). BRCA1 (Miki et al. 1994) and BRCA2 (Wooster et al. 1995) are the most important high-penetrance breast cancer susceptibility genes: the average cumulative risk by the age of 70 years is 65% for BRCA1 carriers and 45% for BRCA2 carriers. While the primary tumor type associated with these mutations is breast cancer, both mutations also predispose to ovarian cancer, the cumulative risk being 39% for BRCA1 carriers and 11%

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for BRCA2 carriers (Antoniou et al. 2003). Additionally, BRCA2 mutations have been linked to increased risk for male breast cancer (Thorlacius et al. 1995). Other cancer types associated with mutations in these genes are prostate and pancreatic cancer for BRCA2 (Breast Cancer Linkage Consortium 1999), and prostate and colon for BRCA1 (Ford et al. 1994).

Mutations in BRCA1 or BRCA2 are found essentially in all families with apparent autosomal dominant inheritance of susceptibility to both breast and ovarian cancer (Ford et al. 1998). However, predisposition in the majority of families with less than six cases of female breast cancer and no ovarian cancer is not due to mutations in BRCA1 or BRCA2 (Rebbeck et al. 1996, Serova et al. 1997, Schubert et al. 1997, Vehmanen et al. 1997a, Vehmanen et al. 1997b, Huusko et al. 1998), and large population-based studies indicate that only 15-20% of the overall familial risk of breast cancer is attributable to mutations in these genes (Anglian Breast Cancer Study Group 2000). Although some BRCA1 or BRCA2 mutations might have been missed and some families may represent chance clustering of sporadic cases, the results from twin studies (Lichtenstein et al. 2000) and the pattern of inheritance in families suggest that there are additional susceptibility genes which may account for a substantial proportion of familial aggregation of breast cancer (Balmain et al. 2003).

A small proportion of the remaining familial breast cancer cases are caused by mutations in other known cancer susceptibility genes, including TP53 (Malkin et al. 1990), PTEN (Nelen et al. 1996), LKB1 (Boardman et al. 1998). However, mutations in these genes confer an increased risk mainly in the context of rare, stringently defined autosomal dominant familial cancer syndromes, Li-Fraumeni syndrome, Cowden’s disease and Peutz-Jeghers syndrome, respectively, and are rarely seen in breast cancer patients without other features of these syndromes (Børresen et al. 1992; Tsou et al. 1997; FitzGerald et al. 1998; Bignell et al. 1998; Huusko et al. 1999; Rapakko et al. 2001). There is also evidence that heterozygous carriers of ATM mutations, the gene defected in the autosomal recessive disorder ataxia-telangiectasia (A-T), are at increased risk of breast cancer (Easton 1994; Swift et al. 1991; Olsen et al. 2001). The role of ATM as a breast cancer susceptibility gene is, however, still controversial and will be discussed in more detail (chapter 2.5.1.3). Nevertheless, even if the effects of all above-mentioned cancer susceptibility genes are taken together, they are estimated to explain only 20-25% of the familial relative risk for breast cancer (Easton 1999). Evidently additional breast cancer susceptibility genes do exist.

2.3.2 Search for additional susceptibility genes

The remaining familial breast cancer risk could be due to a few additional, as yet unidentified, high-penetrance mutations, and several groups have searched for susceptibility genes by genetic linkage studies. Positive linkage has been found at chromosome region 8p12-p22 (Seitz et al. 1997) and 13q21 (Kainu et al. 2000), but these findings have not been confirmed by subsequent studies (Rahman et al. 2000; Thompson et al. 2002). More recently, also 2q32 has been suggested to harbor a susceptibility locus in Finnish breast cancer families (Huusko et al. 2004). Despite all these findings, the

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linkage studies reported so far have failed to identify any additional high risk breast cancer susceptibility genes. This suggests that more than one such gene may exist, and that they might also be limited in their geographical occurrence. Indeed, although linkage analyses have been proven powerful in the identification of BRCA1 and BRCA2, the success of this approach depends on the underlying heterogeneity of the remaining breast cancer susceptibility loci. Heterogeneity limits the usefulness of combining data from multiple families, as different genes might be responsible for clustering of breast cancer in different families. In addition, studies are limited by problems of the phenocopies in the families and the variable penetrance of the mutations concerned. The carrier status of a putative disease allele cannot therefore be reliably concluded from disease status (Fearon 1997; Pharoah et al. 2004). As the attempts to identify additional high-penetrance susceptibility genes have failed, it has been suggested that a polygenic model may provide a more plausible alternative explanation for the remaining familial breast cancer risk (Houlston & Peto 2003). According to this model, the inherited susceptibility to breast cancer is due to several loci, each having a modest effect individually (Pharoah et al. 2002). Various possibilities are consistent with the polygenic inheritance. According to the common variant/common disease model, susceptibility results from the joint action of several common variants with unrelated affected individuals sharing a substantial proportion of disease alleles. Alternatively, susceptibility is caused by many different rare genetic variants with a moderate or relatively large effect produced by each allele (Pritchard 2001; Prichard & Cox 2002; Fearnhead et al. 2004).

The so-called low-penetrance genes are defined as genes in which subtle sequence variants or polymorphisms may be associated with a low to moderate increased relative risk for breast cancer. The low-penetrance susceptibility alleles, which confer relative risks of 2.0 or less, rarely cause multiple-case families and are difficult, or even impossible, to identify through linkage studies (Risch & Merikangas 1996). The search has therefore centered on the comparison of the frequency of candidate alleles in these genes in cases against controls. The power of these association studies can be greatly enhanced by using cases selected for family history of the disease (Houlston & Peto 2003). Ideally, the variants tested in association studies should be those that are directly related to the disease predisposition, but generally these are not known. Instead, a set of single nucleotide polymorphisms (SNPs) is used to define haplotypes across the gene of interest, and the frequency of each haplotype is compared between cases and controls. This method relies on the assumption that the disease-causing variants have arisen only once (Balmain et al. 2003). However, a major disadvantage with the usage of candidate SNP approach is that a lack of association does not rule out the presence of other important variants in the same gene. Furthermore, the indirect SNP approach is unlikely to work for rare variants, because a rare allele will be too weakly correlated with any common SNP haplotype. In contrast, identification of rare variants will require re-sequencing, i.e. mutation screening, of candidate genes, perhaps concentrating on individuals from high-risk families where the frequencies might be higher (Pharoah et al. 2004). The re-sequencing approach also identifies mutations of recent origin, but the frequencies of these alleles are likely to vary between populations.

It has been suggested that the low-penetrance alleles may be relatively common at population level. Thus, despite the low relative risk estimates, they may confer a higher attributable risk in general population than rare mutations in high-penetrance cancer

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susceptibility genes. Consequently, the eventual identification and functional characterization of a large number of these genes could have a major impact on many aspects of breast cancer prevention and treatment (Risch & Merikangas 1996; Peto 2002). However, if most cancer susceptibility is related to fundamental processes of cellular control, rare alleles might turn out to be a more important component of cancer susceptibility than common alleles (Pharoah et al. 2004). Although several common polymorphisms have been reported to have a slightly higher frequency in breast cancer patients than among control individuals (Dunning et al. 1999), CHK2 1100delC was the first example of a low-penetrance alteration that contributes to familial clustering of breast cancer at the population level, and was also found in families with a small number of affected relatives (The CHEK2-Breast Cancer Consortium 2002; Vahteristo et al. 2002). Furthermore, CHK2 1100delC was the first illustration that rare low-penetrance susceptibility alleles can be found by simply comparing their prevalence in patients with that in controls (Peto 2002). Interestingly, virtually all susceptibility alleles identified to date have frequencies of less that 1%. These include not only high-penetrance mutations, but also low-penetrance variants in genes such as ATM and CHK2. The rarity of these alleles might be due to the fact that they are of recent origin and have not had time to spread through the population. However, alleles are likely to be rare also if there is any degree of selection against them, for example when homozygotes are non-viable (Pharoah et al. 2004) or have impaired fertility as in the case of A-T patients (Meyn 1999).

2.3.3 Selection of candidate genes

The candidate genes for SNP or re-sequencing studies are usually chosen on the basis of biological plausibility, so that even slight alterations in the function of these genes could have an effect on pathways involved in carcinogenesis. For example, genes involved in detoxification of environmental carcinogens, steroid hormone metabolism, immune surveillance and DNA damage repair have been hypothesized as candidates (Nathanson & Weber 2001).

It has been suggested that particularly those genes that function in DNA damage response pathways, and in which germline mutations cause autosomal recessive genomic instability syndromes with cancer susceptibility as a prominent feature, may be good candidates for low-penetrance breast cancer susceptibility genes (Nathanson and Weber 2001). The best studied example for this is ATM, although its role as a breast cancer susceptibility gene outside the A-T families is still controversial (reviewed in Khanna & Chenevix-Trench 2004). However, convincing evidence for a shared genetic background between genomic instability syndromes and breast cancer susceptibility has emerged from a recent study that identified biallelic BRCA2 mutations in Fanconi anemia (FA) complementation groups FA-B and FA-D1 (Howlett et al. 2002). In addition, another potential breast cancer susceptibility gene, BACH1, has recently been shown to be defected in complementation group FA-J (Levran et al. 2005; Litman et al. 2005). Nevertheless, even though some germline mutations in BACH1 seem to contribute to breast cancer development, these alleles are rare and their overall contribution to familial

21

breast cancer seems marginal (Cantor et al. 2001; Karppinen et al. 2003; Vahteristo et al. 2006; Seal et al. 2006). Altogether the present data suggests that besides being good candidates for low-penetrance susceptibility, other rare variants or even deleterious mutations in the DNA damage response genes causative for recessive genomic instability syndromes might also be associated with increased risk of breast cancer. These genes therefore represent attractive candidates for re-sequencing studies.

2.4 DNA double-strand break response

2.4.1 DNA double-strand break response pathway as part of the DNA repair network

The genomic integrity of the cell is maintained by monitoring and repairing DNA lesions that are caused by both environmental and endogenous DNA damaging agents. An inability to respond properly to DNA damage leads to genetic instability, which is one of the main forces driving the onset and progression of tumorigenesis (Khanna & Jackson 2001; Hoejimakers 2001). In general, inherited defects in DNA damage response and repair pathways predispose to malignancy. Examples of familial cancer syndromes caused by defects in these processes are presented in Table 1. These either dominantly or recessively inherited syndromes represent cancer predisposition caused by major single gene defects.

No single repair pathway can cope with all kinds of damage; multiple interwoven DNA repair systems are thus required. At least four main DNA damage repair pathways operate in mammals. These are nucleotide-excision repair (NER) including global genome repair (GGR) and transcription-coupled repair (TCR), base-excision repair (BER), mismatch repair (MMR) and recombinational repair including homologous recombination (HR) and nonhomologous end joining (NHEJ) (reviewed in Hoejimakers 2001; Pierce et al. 2001). However, instead of regarding the different DNA repair mechanisms as separate processes, they should be considered as temporary associations made from a larger pool of DNA interacting proteins. These particular complexes associate according to the specific challenges set by damaged DNA or unusual DNA conformations (Cleaver et al. 2001). The formation of the complexes probably changes dynamically during cell cycle, and different multiprotein complexes may be created and disassembled in response to different stimuli (Abraham 2001). One such complex is BASC (BRCA1 associated genome surveillance complex), where BRCA1 has been found to associate with several tumor suppressor proteins, DNA damage sensors and signal transducers including ATM, ATR, Mre11 complex, replication factor C, BLM, and MSH2, MSH6 and MLH1. The members of BASC have roles in recognition of abnormal DNA structures or damaged DNA, and several of these proteins participate in DNA replication-associated repair. Thus, BRCA1 coordinates multiple activities required for the maintenance of genomic integrity and point to a central role in DNA repair (Wang et al. 2000).

22

Table 1. Familial cancer syndromes caused by defects in DNA damage response

Gene Main defected pathway

Syndrome Predominant tumors

A. Dominantly inherited cancer syndromes BRCA1 DSB, HR Hereditary breast and ovarian

cancer (HBOC) Breast, ovarian

BRCA2=FANCD1 DSB, HR HBOC Breast (female/male), ovarian MLH1, MSH2, MSH6

MMR Hereditary nonpolyposis colorectal cancer (HNPCC)

Colorectal

p16/CDKN2A CC Familial melanoma Malignant melanoma, pancreatic TP53 CC Li-Fraumeni syndrome Sarcoma, breast, brain, leukemia RB CC Retinoblastoma Retinoblastoma, osteosarcoma

B. Recessively inherited cancer syndromes

ATM DSB Ataxia-telangiectasia Lymphoma, leukemia BLM HR Bloom syndrome Leukemia, lymphoma, solid

tumors FANCA-FANCG FANCJ, FANCL

HR Fanconi anemia Acute myeloid leukemia

LIG4 NHEJ Ligase IV syndrome Leukemia MUTYH BER Colorectal adenomatous polyposis Colorectal NBS1 DSB Nijmegen breakage syndrome Lymphoma RECQL4 HR, NHEJ Rothmund-Thomson syndrome Osteosarcoma WRN HR Werner syndrome Various cancers XPA-XPG NER, TCR Xeroderma pigmentosum Basal cell carcinoma, melanoma

Abbreviations: BER base excision repair, CC cell cycle checkpoints, DSB double-strand break signaling, HR homologous recombination, MMR mismatch repair, NER nucleotide excision repair, NHEJ nonhomologous end joining, and TCR transcription-coupled repair. (Hoeijmakers 2001; Thompson & Schild 2002; Marsh & Zori 2002; Chow et al. 2004)

Although the repair of different types of DNA lesion relies on different sets of proteins, the various forms of DNA damage trigger common signal transduction pathways. One important feature of this DNA damage response is the slowing down or arrest of cell cycle progression at G1, S and G2 checkpoints (Zhou & Elledge 2000). Another aspect includes changes in chromatin structure at the site of damage, and the transcriptional induction and posttranslational modification of various proteins involved in DNA repair. Alternatively, if the damage is too severe to be repaired, the cell may proceed to apoptosis (reviewed in Rouse & Jackson 2002).

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2.4.2 DNA double-strand break signaling leading to cell cycle checkpoints

DSBs are the most lethal genetic lesions that can result in chromosomal aberrations, disruption of genomic integrity and ultimately cancer (Khanna & Jackson 2001). DSBs arise from many sources, including IR, free radicals and chemicals, but they can also occur during normal DNA processing such as DNA replication and meiosis. In eukaryotes DSB repair is carried out by two major repair pathways: HR and NHEJ. Of these, NHEJ is considered error-prone, as it ligates the exposed DNA ends without any templates, while HR repair uses adjacent homologous sequences as a template for the repair. HR is preferentially used during and after the S-phase, when DNA has replicated and the homologous sister chromatid forms a template for the repair event. In contrast, NHEJ is most relevant in the G1-phase of the cell cycle, when second copy is not available (Haber 2000; Hoeijmakers 2001). In both processes, DSBs have to be detected and processed efficiently to enable subsequent repair steps (Hopfner et al. 2002).

The initial sensing of the DSBs, the amplification of the signal and transduction into appropriate responses is mediated through checkpoint signaling pathways. These signal transduction cascades promote cell cycle delay or arrest in response to DNA damage at G1-, S- and G2-phase checkpoints (Zhou & Elledge 2000). A simplified schematic presentation of the DSB signaling pathway is presented in Figure 1. One of the first processes that is initiated by DSBs is the massive phosphorylation of the histone variant H2AX. This γ-H2AX is critical for mediating the assembly of specific DNA repair complexes on damaged DNA. However, there is a growing notion that the DSB signal might initially be amplified by means of cyclic processes rather than by a series of steps with linear hierarchy (reviewed in Redon et al. 2002; Thompson & Shild 2002).

The Mre11 complex, composed of MRE11, NBS1 and RAD50 proteins, is suggested to be the initial DSB sensor and processor, which determines the timing and magnitude of all downstream processes. The complex possesses a variety of DNA nuclease, helicase, ATPase and annealing activities, and participates in both NHEJ and HR (Futaki & Liu 2001; Connelly & Leach 2002; Lieber et al. 2003; Lukas & Bartek 2004). ATM and ATR protein kinases are the two main signal transducers. In response to DSBs, ATM reacts first and is responsible for the immediate, rapid phase of the response (reviewed in Abraham 2001), whereas ATR along with ATRIP (ATR-interacting protein) joins in later and maintains the phosphorylated state of specific substrates (reviewed in Abraham 2001; Bartek et al. 2004). Downstream of ATM and ATR are two checkpoint kinases, CHK1 and CHK2 (Zhou & Elledge 2000). It has been traditionally thought that CHK1 is devoted to the ATR-mediated signaling pathway, whereas CHK2 functions primarily through ATM. However, recent evidence suggests that ATR-CHK1 and ATM-CHK2 pathways are not parallel branches of the DNA damage response, but that they rather show a high degree of cross-talk and connectivity (Gatei et al. 2003 and refs. therein, Jazayeri et al. 2006). Below the signal transduction level are the effectors that execute the functions of the DNA damage response. These include the substrates of both ATM/ATR and CHK1/CHK2 kinases and proteins involved in DNA repair, transcription regulation and cell cycle control. However, depending on the context, certain molecules may have multiple functions in the signal transduction pathway (reviewed in Zhou & Elledge

24

2000). These so-called mediators represent an emerging class of checkpoint regulators that may function to recruit other factors to the sites of DNA damage, and have a complex function both upstream and downstream of main signal transducers. The proper timing and rapidity of ATM/ATR-controlled response relies on the functional interplay of at least three mediators: MDC1 (Goldberg et al. 2003; Stewart et al. 2003; Lou et al. 2003), 53BP1 (Wang et al. 2002) and BRCA1 (Venkitaraman 2002; Kitagawa et al. 2004).

Fig. 1. A simplified scheme of DSB signaling pathway leading to cell cycle checkpoints. The ATM and ATR kinase phosphorylate (showed with ‘P’) diverse components of the network either directly or through CHK1 and CHK2. The recruitment of MDC1, 53BP1, BRCA1 and Mre11 complex to the sites of DNA damage is independent of ATM and ATR, but their accumulation in the repair foci depends on the ATM/ATR-mediated phosphorylation of H2AX. The grey arrows represent other putative interactions influencing S-phase arrest. Modified from Kastan & Lim (2000), Bartek & Lukas (2001), Falck et al. (2002), Kastan & Bartek (2004).

DSBH2AX

ATM/ATR

CHK2/CHK1 CHK1/CHK2MDM2 p53

MDM2 p53

p21

CDC25A

CDK2/cyclin E CDK2/cyclin E CDC2/cyclin B

BRCA1

SMC1 FANCD2CDC25C

14-3-3σ

14-3-3σ

RAD50

MRE11NBS1

nuclear export

G1 S G2 M

53BP1

BRCA1

MDC1

RAD50

MRE11NBS1

transcriptionalinduction

ubiquitination, proteolysis

CDC25C

P P

PP P

P

P P

PP

P

P

P

P

P P

P

P

P

P

25

The cell cycle arrest at the G1 checkpoint is mediated by two signal transduction pathways. In the dominant, p53-dependent pathway, ATM phosphorylates both p53, on Ser15, and its major binding partner, ubiquitin ligase MDM2. Additionally, ATM activates CHK2 by phosphorylating Thr68, leading to subsequent phosphorylation of p53 on Ser20 by CHK2. Together these events interfere with the binding of p53 to MDM2, which leads to p53 stabilization, allowing it to transcriptionally induce several genes, including p21 (also known as WAF1 and Cip1) (Banin et al. 1998; Canman et al. 1998; Dumaz & Meek 1999; Matsuoka et al. 2000; Melchionna et al. 2000; Maya et al. 2001). The p21 protein binds to and inhibits the activity of cyclin E and its partner CDK2, which is required in progression from G1- to S-phase. This pathway is capable of inducing sustained and sometimes even permanent G1 arrest (Kastan & Lim 2000). The other pathway leading to the G1 checkpoint is ATM-activated but p53-independent, in which the major target is CHK2. In this pathway, phosphorylation of multiple CHK2 Ser/Thr residues, including Thr68, leads to upregulation of CHK2 activity and the phosphorylation of its downstream target CDC25A. This stimulates CDC25A degradation, which blocks the S-phase entry by preventing the CDC25A-dependent dephosphorylation and activation of CDK2 (Bartek & Lukas 2001 and refs. therein). This is a rapid pathway, delaying G1 to S transition only for a few hours, unless the sustained p53-dependent mechanism prolongs the G1 arrest (Kastan & Bartek 2004).

The intra-S-phase checkpoint is manifested by a decreased rate of DNA synthesis after DSBs. There are two parallel, cooperating pathways, which contribute to the rapid and transient inhibition of DNA synthesis (Falck et al. 2002). In the CDC25A branch ATM activates CHK2, which phosphorylates the cell cycle regulator CDC25A, leading to its degradation through the polyubiquitination-mediated proteolysis pathway (Matsuoka et al. 2000; Falck et al. 2001, Falck et al. 2002). The second branch is the ATM-regulated NBS1/BRCA1/SMC1 pathway. In response to DSBs ATM phosphorylates Ser957 and Ser966 of SMC1, and the intra-S-phase checkpoint is activated. The SMC1 phosphorylation requires NBS1 and BRCA1, as both have a role in recruiting activated ATM to the DNA breaks (Yazdi et al. 2002; Kitagawa et al. 2004). In addition, ATM-mediated phosphorylation of NBS1 on Ser343 and Ser278 (Lim et al. 2000; Zhao et al. 2000), and BRCA1 on Ser1387 is required for the checkpoint function (Xu et al. 2002). Recently, the NBS1-dependent phosphorylation of FANCD2 on Ser222 by ATM was also shown to play a role in the activation of this checkpoint (Taniguchi et al. 2002; Nakanishi et al. 2002).

The G2-phase checkpoint is the final checkpoint that blocks the entry of damaged cells into mitosis. The critical target is the mitosis-promoting activity of cyclin B/CDC2 kinase, the activation of which is inhibited by ATM/ATR and CHK1/CHK2. However, it has been indicated that ATM is dispensable for the activation of checkpoint-mediated G2 arrest, unless the DNA damage occurs during G2 itself. CHK2 also appears to play a supporting role in the maintenance rather than initiation of G2 arrest (reviewed in Kastan & Lim 2000). In contrast, the major player in this checkpoint is CHK1, which blocks mitotic entry through phosphorylation of CDC25C on Ser216. This results in its binding to 14-3-3σ proteins and nuclear export. As a result, CDC25C is incapable of removing inhibitory phosphates from CDC2/Cyclin B, and entry into mitosis is prevented (Peng et al. 1997; Sanchez et al. 1997; Liu et al. 2000).

26

Aberrations in the cell cycle checkpoint pathways and the subsequently activated DNA repair machinery lead to accumulation of mutations and an increase in chromosomal instability. Inherited defects in these pathways increase the possibility of developmental malformations as well as the risk of cancer. Five genes operating in DSB response pathways and associated with genomic instability syndromes are presented in the following.

2.5 DNA double-strand break response genes associated with inherited genomic instability syndromes

2.5.1 ATM mutations and ataxia-telangiectasia

2.5.1.1 ATM structure and function

The ATM gene is located in chromosomal region 11q22.3. It comprises 66 exons, 62 of which encode a ubiquitously expressed 370-kDa protein kinase (Savitsky et al. 1995a; Savitsky et al. 1995b; Uziel et al. 1996). ATM is a member of the phosphoinositol 3-kinase family. It has the catalytic kinase domain located near the carboxy-terminus of the protein, flanked by two loosely conserved domains termed FAT (FRAP/ATM/TRRAP) and FATC (C indicating carboxy-terminal) (Figure 2). Although the FAT domain contains no identifiable catalytic sequences, it occurs only in combination with FATC, and it has been suggested that these two domains fold together in a configuration that ensures proper function of the interposed kinase domain (Savitsky et al. 1995a; Bosotti et al. 2000).

Fig. 2. The ATM protein. The main structural domains are indicated above the diagram and other sites important for its function are shown with arrows. Abbreviations: FAT FRAP/ATM/TRRAP, FATC FRAP/ATM/TRRAP carboxy-terminal, NLS nuclear localization signal, PI3K phosphoinositide 3-kinase related catalytic domain. Modified from Lavin et al. (2004), Young et al. (2005).

FAT PI3K FATC

30561

ABL interaction

Ser1981

proline-rich region

leucine zippersubstrate binding

NLSchromatinassociation

ATP binding substrate binding

27

ATM is the major controller of cell response to DNA DSBs. The hallmark of this response is the rapid increase in its kinase activity following DSB formation (Banin et al. 1998; Canman et al. 1998). It has been proposed that ATM molecules are inactive in undamaged cells, being held as homodimers and high-order multimers. Upon DSBs, and possibly other changes in the chromatin structure, ATM autophosphorylates itself at Ser1981 (Bakkenist & Kastan 2003). Recently two other autophosphorylation sites, Ser367 and Ser1893, have also been reported (Kozlov et al. 2006). Autophosphorylation leads to ATM dimer dissociation into active monomers that are recruited to the substrates, some of which localize to sites of DNA damage. This mechanism in ATM regulation permits a rapid and sensitive switch for checkpoint pathways (Bakkenist & Kastan 2003).

In addition to its role in the signaling pathways leading to cell cycle arrests (chapter 2.4.2), ATM activates a separate signal transduction pathway operated through stress-activated protein kinase by interacting with ABL, a nuclear nonreceptor tyrosine kinase (Baskaran et al. 1997; Shafman et al. 1997; Shangary et al. 2000). ATM is also required for the ABL-mediated assembly of the RAD51 repair protein complex following IR (Chen et al. 1999). Another target in this stress signaling pathway is the activation of the NFκB transcription factor, which plays a critical role in cellular protection against a variety of apoptotic stimuli. Through ATM-mediated IκB kinase complex activation, NFκB is released from its inhibitor IκB-α, enabling its relocation in the nucleus and transcriptional activation of a wide variety of stress-responsive genes (Jung et al. 1997; Li et al. 2001). ATM also has other roles essential for the genomic stability, as it participates in the maintenance of telomere length and integrity, a process that is crucial in both aging and cancer (Smilenov 1997; Hande et al. 2001; Pandita 2001). In addition, ATM appears to play a role in apoptosis, during which it is cleaved to generate a kinase-inactive protein that acts through its DNA binding ability in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signaling. The cleavage is dependent on caspases, and ATM is an efficient substrate for caspase 3 (Smith et al. 1999).

Although ATM has a predominantly nuclear localization in most proliferating cells, not induced by DNA damage (Brown et al. 1997), it appears to have cytoplasmic functions as well. Approximately 10% of cell ATM proteins are extranuclear, where it is present in cytoplasmic vesicles, peroxisomes and endosomes (Lim et al. 1998; Watters et al. 1999). No function has been ascribed to ATM in peroxisomes, but it might be part of the cellular response to reactive oxygen intermediates. In endosomes ATM binds to β-adaptin, suggesting a possible role in vesicle and protein transport, too (Rotman & Shiloh 1997; Lim et al. 1998; Barlow et al. 1999; Watters et al. 1999). In Purkinje cells ATM is predominantly cytoplasmic (Oka & Takashima 1998).

2.5.1.2 Ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder resulting from biallelic mutations in the ATM gene (Savitsky et al. 1995a). A-T is characterized by early-onset progressive cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency and predisposition to cancer. Approximately 70% of the malignancies are lymphomas and T-cell leukemias, while the remainder consists of a

28

wide variety of solid tumors, including breast cancer. The ATM defective cells show chromosomal instability, cell cycle checkpoint defects and extreme sensitivity to IR (Lavin & Shiloh 1997). The highly pleiotropic clinical phenotype of A-T patients reflects the numerous cellular processes affected by ATM (Shiloh & Rotman 1996). In addition, it is possible that ATM has different functions in different cell types and at different stages of development (Khanna 2000).

A-T mutations are observed throughout the open reading frame of the ATM transcript, with no apparent “hot-spots”. Interestingly, none of the mutations seem to cause significant instability of mRNA transcript (Lavin & Shiloh 1997). Despite this extensive variation, most of the causative ATM mutations are truncating mutations. The majority of A-T patients are compound heterozygotes (Gilad et al. 1996; Telatar et al. 1996), and there is evidence for heterogeneity in symptoms, with some patients having a milder phenotype. This seems to be related to the nature of the mutation involved and can be explained by certain mutations’ capacity to produce some amount of normal ATM protein (Stankovic et al. 1998; Stewart et al. 2001). In particular, a milder disease phenotype has been suggested for patients homozygous for missense mutations (Dörk et al. 2001; Laake et al. 2000), although some patients carrying such mutations show a classical A-T phenotype (van Belzen et al. 1998; Angèle et al. 2003a).

Even though A-T is an autosomal recessive disease, there is evidence for penetrance in heterozygotes manifested by intermediate sensitivity to IR in cell cultures (Scott et al. 2002 and refs. therein). The carriers are not clinically distinguishable from normal individuals, but heterozygotes have been reported to have an excess risk of cancer (Swift et al. 1987). Interestingly, gene-dosage seems to have an effect on the tumor spectrum: whereas the main cancer types in A-T patients are leukemias and lymphomas (Lavin & Shiloh 1997), A-T heterozygosity predisposes significantly only to breast cancer (Olsen et al. 2001; Thompson et al. 2005).

2.5.1.3 Heterozygous ATM mutations and breast cancer susceptibility

The first suggestion that ATM is a breast cancer susceptibility gene came from studies reporting an increased breast cancer risk among obligate carriers in A-T families (Swift et al. 1987; Swift et al. 1991; Easton 1994; Olsen et al. 2001). This led to the conclusion that A-T heterozygotes are a subpopulation of individuals with increased risk of developing breast cancer (Meyn 1999), the estimated relative risk being 3.9 (Easton 1994). However, this risk appears not to be limited only to young women, but appears even higher at older ages (Athma et al. 1996).

Given that A-T heterozygotes constitute 0.2-1% of the population, the earlier studies suggested that ATM mutations may contribute significantly to the breast cancer incidence at population level, even up to 5% of all breast cancer cases in the general population (Easton 1994; Inskip et al. 1999). However, the evidence for this has been contradictory, as the overall frequency of ATM mutations in breast cancer patients outside the A-T families is low (reviewed in Khanna & Chenevix-trench 2004). There are several explanations for this controversy. First, many of the studies have analyzed breast cancer cases unselected for family history, which might be an inefficient way to detect ATM

29

mutations. Additionally, many of the studies have been too small to have adequate statistical power or have focused only on women with early-onset breast cancer (Vorechovsky et al. 1996; FitzGerald et al. 1997; Chen et al. 1998, Khanna & Chenevix-trench 2004). Second, it may be that only ATM mutations with specific functional consequences predispose to breast cancer. As many of the case-control studies have failed to show an elevated frequency of truncating ATM mutations in breast cancer patients, it has been hypothesized that dominant-negative mutations, particularly missense changes, are the ones mainly responsible for the increased cancer risk. However, these mutations would not necessarily give the full A-T clinical phenotype when paired either with themselves or other mutant ATM alleles (Meyn 1999; Gatti et al. 1999). The dominant-negative mutations give rise to kinase-inactive or non-phosphorylable proteins, which are stable but functionally impaired, interfering with the function of the wild-type protein (Bakkenist & Kastan 2003). In contrast, null or truncating ATM mutations typically produce no detectable protein, despite the apparent stability of the transcript. Consequently, the carriers of these alleles would still have 50% of the wild-type ATM activity, thus a nearly normal phenotype. While the cancer-predisposing effect of these alleles in the heterozygous state cannot be excluded, their relative rarity means that they are not major cancer susceptibility alleles in the general population (Gatti et al. 1999).

A few of the studies concentrating on familial cases have provided evidence for an increased breast cancer risk associated with specific ATM mutations. Thorstenson et al. (2003) identified seven pathogenic ATM mutations in altogether ten of 270 breast and ovarian cancer families. All of these mutations were shown or predicted to cause A-T. However, only two of the changes, 8734A>G (Arg2912Gly) and 9031A>G (Met3011Val), were missense mutations. Both located to the highly conserved carboxy-terminus of the protein that includes the catalytic kinase domain, and have also previously been associated with breast cancer susceptibility (Teraoka et al. 2001). Although these seven mutations were found to be more frequent in cases compared to controls, the difference did not reach statistical significance (Thorstenson et al. 2003). Also two other ATM alleles, 7271T>G (Val2424Gly) and IVS10-6T>G, have been reported in breast cancer families, and both have been suggested to act in a dominant-negative manner (Chenevix-Trench et al. 2002). According to Chenevix-Trench et al. (2002), the risk associated with 7271T>G and IVS10-6T>G appears sufficient to give rise to multiple-case families, but this has been debated by other studies (Bernstein et al. 2003; Szabo et al. 2004). A recent study provided population-based estimates of breast cancer risks associated with these two alleles and suggested that IVS10-6T>G, observed in 0.3% (13/3757) of breast cancer cases and in 0.8% (10/1268) of controls, was not associated with breast cancer. In contrast, 7271T>G, observed in 0.2% (7/3743) cases but not in 1268 controls, was associated with a substantially elevated risk, the estimated cumulative breast cancer risk being 52% by the age of 70 years (Bernstein et al. 2006). Both 7271T>G and IVS10-6T>G have been reported in A-T patients (Stankovic et al. 1998; Dörk et al. 2001). However, despite the one A-T patient homozygous for IVS10-6T>G (Dörk et al. 2001), the association of this allele with A-T remains unclear. It seems to occur too frequently in general population to be pathogenic for A-T and has never been reported at compound heterozygous state (Khanna & Chenevix-Trench 2004).

Some contribution of the A-T-related mutations to familial aggregation of breast cancer has also been suggested in Finland, where the estimated carrier frequency is one in

30

280 (Olsen et al. 2001). In the study by Allinen et al. (2002), 162 families displaying signs of hereditary susceptibility to breast cancer were screened for ATM mutations, originally identified in Finnish A-T patients. Three of the studied families showed heterozygous ATM mutations: two were found positive for 7570G>C (previously marked as 7522G>C) leading to Ala2524Pro substitution and one for 6903insA truncation mutation. Both alleles were absent from the tested 85 sporadic breast cancer cases and 200 healthy controls.

2.5.2 ATR mutations and Seckel syndrome

2.5.2.1 Structure and function of ATR

The ATR gene is located in chromosomal region 3q22-q24, spanning approximately 130 kb and comprising 47 exons (Cimprich et al. 1996). It encodes a protein kinase (Figure 3), which is a member of the phosphoinositol 3-kinase family and a major regulator of the signal transduction cascades induced by DNA lesions (Abraham 2001). In response to IR, ATR acts in parallel and cooperatively with ATM (chapter 2.4.2). Whereas ATM is responsible for the immediate, rapid phase of the response, ATR joins in later and maintains the phosphorylated state of specific substrates (reviewed in Abraham 2001; Bartek et al. 2004).

Fig. 3. The ATR protein. The main structural domains are indicated above the diagram. Abbreviations: FAT FRAP/ATM/TRRAP, FATC FRAP/ATM/TRRAP carboxy-terminal, PI3K phosphoinositide 3-kinase related catalytic domain. Modified from Abraham (2001).

After DNA damage there is no measurable change in ATR kinase activity. Rather, it seems that ATR may be constitutively ready to phosphorylate its substrates, and the cellular functions seem to be largely controlled by its subcellular localization (reviewed in Kastan & Bartek 2004). An important characteristic of ATR is its need for an accessory protein, ATRIP (Cortez et al. 2001). The ATR activation seems to depend on the prior processing of DSBs to single-stranded DNA (ssDNA), and it has been suggested that ATR becomes localized to ssDNA through binding of ATRIP to replication protein A (RPA), a ssDNA-binding protein involved in DNA replication. It seems that both ATM and Mre11 complex are required for the processing of DSBs to generate RPA-coated ssDNA in the S- and G2-phases of the cell cycle. Once the active ATR is localized to the ssDNA region, it can phosphorylate its substrates (reviewed in Kastan & Bartek 2004; Jazayeri et al. 2006). The ATR-controlled checkpoint signaling, at least towards the CHK1, also requires a mediator protein, claspin (Chini & Chen 2004).

FATCFAT PI3K

26441

31

Taking care of the late stage of the DSB response is not, however, the main role of ATR. It also responds to UV-light treatment, hypoxia and stalled replication forks by phosphorylating several substrates, among them p53, BRCA1, NBS1 and FANCD2 (Tibbets et al. 1999; Tibbets et al. 2000; Hammond et al. 2002; Pichierri & Rosselli 2004). Many of the ATR substrates are shared with ATM, and these phosphorylation events represent overlapping pathways partially responding to distinct DNA lesions (reviewed in Shiloh 2003; Zhou & Elledge 2003; Alderton et al. 2004). Thus ATR also links the ATM-mediated web to signals other than DSBs. However, even in the absence of DNA damage and cellular stress ATR is probably required for normal progression through the cell cycle, as it has been suggested to have a critical role in the progression of DNA replication forks (Shechter et al. 2004). Consistent with this, knockout studies have shown that ATR is essential for somatic cell growth and genomic integrity, and that its deletion leads to early embryonic lethality in mice. Furthermore, it has been reported that heterozygous disruption of the ATR gene leads to increased incidence of benign tumors, possibly indicating that deficiency in ATR affects the rate of tumor initiation (Brown & Baltimore 2000).

2.5.2.2 ATR mutations in Seckel syndrome

Seckel syndrome is a clinically and genetically heterogeneous autosomal recessive disorder, in which at least four susceptibility loci have been identified (Goodship et al. 2000; Børglum et al. 2001; Faivre et al. 2002; Kilinc et al. 2003). The syndrome is characterized by dwarfism, developmental delay, severe microcephaly with mental retardation, and a characteristic “bird-like” facial appearance. Although lymphoma has been reported in some Seckel patients, a high incidence of malignancies is not thought to be a prominent part of this syndrome (Butler et al. 1987; Hayani et al. 1994; Chanan-Khan et al. 2003; O’Driscoll & Jeggo 2003). The Seckel syndrome shares an overlap in clinical features with a variety of other syndromes associated with impaired DNA damage response mechanisms including Nijmegen breakage syndrome, Ligase IV (LIG4) syndrome, FA and Cockayne’s syndrome (O’Driscoll et al. 2003).

Recently, a synonymous mutation affecting splicing of the ATR gene was shown to be associated with Seckel syndrome in two related Pakistani families. The causative ATR mutation is hypomorphic: it significantly decreases but crucially does not fully abolish ATR function (O’Driscoll et al. 2003). Correspondingly to the general clinical features of Seckel syndrome, ATR-defective patients do not display cancer. Nevertheless, these patients are very young, and as various cell lines in which ATR has been inactivated show genetic instability, it may anticipate proneness to cancer (O’Driscoll & Jeggo 2003). Recent studies have shown that also several other cell lines derived from Seckel syndrome patients without defects in ATR are unusually sensitive to agents that cause replication stalling. Consequently, it has been suggested that Seckel syndrome might be commonly caused by defects in the ATR-signaling pathway (Alderton et al. 2004).

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2.5.3 Mutations in the Mre11 complex genes

2.5.3.1 Structure and function of the Mre11 complex

The Mre11 complex is composed of MRE11, NBS1 and RAD50 proteins (Figure 4), which are encoded by MRE11 at 11q21 (Petrini et al. 1995), NBS1 at 8q21 (Varon et al. 1998; Carney et al. 1998) and RAD50 at 5q31 (Dolganov et al. 1996), respectively. This protein complex operates in DNA DSB sensing and early processing, cell cycle checkpoints, DNA recombination and in the maintenance of telomeres (Zhu et al. 2000; Wu et al. 2000; Stracker et al. 2004). It integrates DNA repair with the activation of checkpoint signaling through ATM, acting both upstream and downstream of it in the DNA damage response pathway. The downstream role has been demonstrated by the ATM-mediated phosphorylation of at least NBS1 and MRE11 as part of the response to DSBs (Dong et al. 1999; Gatei et al. 2000; Zhao et al. 2000; Lim et al. 2000). Of these the best characterized event is the phosphorylation of NBS1 (chapter 2.4.2), whereas the nature and functional consequences of the MRE11 phosphorylation still remain undefined. Recent studies have also suggested an upstream role for the Mre11 complex, as it seems to be required for the full activation of ATM after low doses of IR (Uziel et al. 2003; Carson et al. 2003; Costanzo et al. 2004; Horejsi et al. 2004). It has been proposed that the Mre11 complex facilitates the recruitment of ATM to damaged sites by tethering DNA, thereby increasing the local concentration of damaged DNA (Dupré et al. 2006). In addition, the Mre11 complex seems to serve as adaptor in the phosphorylation of certain ATM substrates, such as CHK2. For this function, particularly, the presence and phosphorylation of NBS1 seem to be essential (Lee & Paull 2004).

Because of its independent interaction with both NBS1 and RAD50, MRE11 has been viewed as the core of the complex, whereas the interaction between NBS1 and RAD50 is indirect and mediated through MRE11 (Carney et al. 1998; Desai-Mehta et al. 2001; Tauchi et al. 2001). The sites in MRE11 responsible for binding to NBS1 and RAD50 have both been assigned to the amino-terminal half of the protein (Bressan et al. 1998; Desai-Mehta et al. 2001). In NBS1, the MRE11 binding domain has been located to the extreme carboxy-terminus (Desai-Mehta et al. 2001), and the corresponding site in RAD50 has been located to a 40-residue coiled-coil region adjacent to the ABC domain (Hopfner et al. 2000; Hopfner et al. 2001) (Figure 4).

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Fig. 4. Components of the Mre11 complex. Known functional domains and sites important for protein interactions are shown. Abbreviations: FHA forkhead-associated domain, BRCT BRCA1 carboxy-terminal domain. Modified from Hopfner et al. (2001), Hopfner et al. (2002), Stracker et al. (2004) and Falck et al. (2005).

Several important domains for the function of the complex have been identified in each of the proteins (Figure 4). The DNA nuclease activity is restricted to MRE11 and specified by phosphoesterase motifs in the amino-terminal half of the molecule, which also has two DNA-binding motifs (Bressan et al. 1998). The phosphoesterase domain functions as both a single- and double-strand (ds) DNA endonuclease, as well as 3’-5’ dsDNA exonuclease (reviewed in D’Amours & Jackson 2002). Both RAD50 and NBS1 stimulate the enzymatic activity of MRE11, which is also regulated by ATP (Paull & Gellert 1999). The amino-terminus of NBS1 exhibits two distinct domains often found in cell-cycle checkpoint and DNA damage response proteins: a forkhead-associated (FHA) domain, followed by a BRCA1 carboxy-terminal (BRCT) domain (Varon et al. 1998).

These highly conserved domains are involved in protein-protein interactions and are therefore crucial for the functions of the Mre11 complex, including its subcellular localization after DNA damage (Tauchi et al. 2001; Zhao et al. 2002). RAD50 consists of bipartite amino- and carboxy-terminal ATPase segments, which contain Walker A and B motifs that are required for nucleotide binding. The ATPase domains assemble into a single ABC-type cassette at the end of the predicted antiparallel coiled-coil (Hopfner et

MRE11 interaction

nuclease domain

708

DNA binding

phosphoesterase motifs

DNA binding

1

MRE11

FHA BRCT

1 754

ATM interaction

NBS1

ABC-ATPase

1312

ABC-ATPasehook-constructcoiled-coil coiled-coil

MRE11 interaction MRE11 interaction

1

Walker A Walker B

RAD50

34

al. 2000). A functionally important zinc-hook has been identified in the central portion of the coiled-coil domain (Hopfner et al. 2002). This zinc-hook is thought to mediate RAD50 dimerization, which allows the required molecular flexibility of the Mre11 complex and keeps it functionally assembled during the DNA recombination and repair process (reviewed in D’Amours & Jackson 2002).

2.5.3.2 Ataxia-telangiectasia-like disorder and Nijmegen breakage syndrome

The major role of the Mre11 complex in ATM-controlled genome surveillance is illustrated by the fact that deficiency of its components recapitulates some of the key defects observed in A-T (Tauchi et al. 2001). Hypomorphic mutations of MRE11 cause ataxia-telangiectasia-like disorder (ATLD) (Stewart et al. 1999), while hypomorphic NBS1 mutations result in Nijmegen breakage syndrome (NBS) (Varon et al. 1998), both of which are rare recessively inherited disorders phenotypically related to A-T. At cellular level these three disorders exhibit hypersensitivity to IR, radioresistant DNA synthesis, failure to induce stress-activated protein kinases following exposure to IR, and genetic instability (Stewart et al. 1999; Varon et al. 1998, Kastan & Lim 2000).

ATLD patients display most of the hallmarks of A-T, although at later stage and with slower progression (Stewart et al. 1999). However, despite the observed genomic instability predisposition to cancer has not been reported (Stewart et al. 1999; Delia et al. 2004; Fernet et al. 2005). Also NBS patients show a significant overlap in symptoms with those of A-T and ATLD, except that cerebellar degeneration does not occur. In addition, NBS patients exhibit microcephaly and a characteristic “bird-like” facial appearance absent from ATLD and A-T (Weemaes et al. 1994; van der Burgt et al. 1996). While NBS shares overlapping characteristics with A-T, it also has features overlapping with ATR-Seckel syndrome, a subclass of Seckel syndrome with mutations in ATR (chapter 2.5.2.2). This could be related to the integral role of NBS1 in the ATR-dependent signaling, and it has been proposed that the clinical features of NBS are the result of the combined defects in both ATM- and ATR-mediated signaling pathways (Stiff et al. 2005). Besides A-T, ATLD and Seckel syndrome, NBS has clinical overlap with LIG4 syndrome and FA. There are even patients originally diagnosed with FA that were subsequently found to have rare mutations in NBS1 (Gennery et al. 2004; New et al. 2005; Ben-Omran et al. 2005).

A majority of NBS patients are homozygous for the Slavic founder mutation, NBS1 657del5, which leads to a prematurely truncated protein (Matsuura et al. 1998; Varon et al. 1998; Carney et al. 1998). However, the alternative mode of translation permits production of a variant NBS1 protein. Therefore, these NBS cells contain a 70-kD protein lacking the native amino-terminus in addition to the predicted truncated 26-kD amino-terminal protein fragment (Maser et al. 2001). In NBS patients there is an increased incidence of malignancies, particularly lymphomas (Weemaes et al. 1994; van der Burgt et al. 1996). It has been suggested that heterozygous carriers of NBS founder mutation may also be at risk for cancer. Indeed, heterozygosity for NBS1 657del5 has been suggested to associate with an increased susceptibility to prostate, ovarian and breast

35

cancer, malignant melanoma, acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (Plisiecka-Halasa et al. 2002; Dębniak et al. 2003; Górski et al. 2003; Cybulski et al. 2004; Chrzanowska et al. 2005; Steffen et al. 2006), while other germline alterations of NBS1 gene have also been associated with childhood ALL (Varon et al. 2001).

Recently, one patient with a variant form of NBS without immunodeficiency was shown to harbor biallelic mutations in RAD50. The patient is compound heterozygote for nonsense and stop codon mutation. In fibroblast and lymphoblast cell lines of this patient the level of RAD50 protein was reduced less than one tenth of the normal amount, which was associated with a high frequency of spontaneous chromatid changes. However, the patient has not developed any malignancies so far and has no family history of cancer (T. Dörk, personal communication). Consequently, all three components of the Mre11 complex have been implicated in related disorders. However, as the deficiency of the MRE11, NBS1 and RAD50 proteins does not lead to exactly the same clinical picture, it suggests that the components of the complex act separately as well.

3 Aims of the study

Apart from BRCA1 and BRCA2, the lack of convincing evidence of additional major breast cancer susceptibility genes suggests that the remaining familial breast cancer risk could be due to mutations in several other genes, perhaps with lower disease penetrance. As the protein products of the genes so far indicated in hereditary predisposition to breast cancer are involved in pathways responding to DNA DSB, other genes participating in these processes may represent new potential candidates. Here we have evaluated the role of five genes essential for the DNA damage response in breast cancer susceptibility. Mutations in these genes are also associated with genetic instability syndromes. The specific aims of this study were to:

1. Investigate whether ATM mutations apart from those identified in Finnish A-T patients could explain an additional fraction of hereditary predisposition to breast cancer.

2. Determine the significance of seven Finnish A-T-related ATM mutations in breast cancer susceptibility in Finland, and to investigate the functional consequences of the observed breast cancer associated ATM mutations.

3. Screen the entire coding region of new candidate genes, ATR, MRE11, NBS1 and RAD50, for germline mutations that could associate with breast cancer susceptibility.

4. Evaluate the importance of the identified potentially pathogenic RAD50 and NBS1 alleles in hereditary breast cancer susceptibility, and to assess the effect of these mutations on genomic instability.

4 Material and Methods

4.1 Subjects

4.1.1 Studies I, III, IV

Patients from 121-128 breast and/or ovarian cancer families (Table 2) were screened for germline alterations in the exons and flanking intronic sequences of the ATM, ATR MRE11, NBS1 and RAD50 genes. Geographically the studied families derived from the Northern Ostrobothnia Health Care District. Inclusion criteria for the families classified as high risk families were the following: 1) three or more cases of breast and/or ovarian cancer in first- or second-degree relatives, or 2) two cases of breast and/or ovarian cancer in first- or second-degree relatives, of which at least one with early disease onset (≤35 years), bilateral disease or multiple primary tumors. The rest were considered moderate risk families, which displayed two cases of breast and/or ovarian cancer in first- or second-degree relatives. All of the high risk families had previously been screened for germline mutations in BRCA1, BRCA2, CHK2 and TP53 (Huusko et al. 1998; Huusko et al. 1999; Allinen et al. 2001; Rapakko et al. 2001). Ten were found to be BRCA1 or BRCA2 mutation-positive, and three had mutations in TP53 (Huusko et al. 1998; Huusko et al. 1999; Rapakko et al. 2001). All included families had previously also been screened for ATM mutations originally identified in Finnish A-T patients (Allinen et al. 2002), and two families were found to be mutation-positive.

In study IV, besides the high or moderate risk families, 23 families showing a single case of breast or ovarian cancer along with multiple cases (two or more) of other kinds of cancer in first- or second-degree relatives were included. In addition, the occurrence of novel, potentially pathogenic alterations were also investigated in 192 sporadic breast cancer patients deriving from the same geographical area as the studied families.

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Table 2. Summary of the families included in studies I, III and IV

Families Number of families Study I Study III Study IV High risk families 71 75 76 Moderate risk families 50 51 52 Phenotypes

Families with breast cancer 92 a 94 95 Families with breast and ovarian cancer 29 29 29 Families with ovarian cancer - 3 4 Multicancer families showing a single case of breast or ovarian cancer

- - 23

Total number of studied families 121 126 151 a Two families with known pathogenic ATM mutations (Allinen et al. 2002) were excluded.

The geographically matched control material used in studies I, III and IV derived from anonymous Finnish Red Cross blood donors and was collected from May through July 2002. In studies I and III, 306 and 300 age-matched females served as controls, respectively. In study IV, 1000 population controls were used as reference group, and this same control group was utilized in study V.

4.1.2 Study II

Index cases of 541 BRCA1 and BRCA2 mutation-negative families originating from Southern, Central and Northern Finland were screened for seven A-T-related ATM mutations: IVS14+3-4delAT (exon 14 skipped), IVS37+9A>G (insertion of Val, Ser, Stop), 6779-6780delTA (truncation), 6903insA (truncation), 7570G>C (Ala2524Pro), 8710-8715delGAGACA (deletion of Glu and Thr), 9139C>T (Arg3047Stop), and for the occurrence of an additional germline mutation, 8734A>G (Arg2912Gly), observed during the study. Inclusion criteria for the families were 1) three or more affected in the family (285 cases), 2) two affected first-degree relatives (251 cases) or 3) two affected second-degree relatives (5 cases). The frequencies of the observed mutations were compared to those of geographically matched 1124 unselected breast cancer cases and 1107 healthy controls.

4.1.3 Study V

An unselected cohort of 317 consecutive, newly diagnosed breast cancer patients was screened for RAD50 687delT, RAD50 Ile94Leu, RAD50 Arg224His and NBS1 Leu150Phe germline alterations. The patients were operated at Oulu University Hospital at the time period from April 2000 through July 2004. Diagnoses were pathologically confirmed. The patients filled out a questionnaire concerning prevalence of breast, ovarian and other cancer in their family. Only those reporting breast and/or ovarian

39

cancer in first- or second-degree relatives were considered to be familial cases. For the evaluation of the geographical prevalence of RAD50 687delT, additional breast cancer cohorts from Sweden (Lund, 130 cases), Norway (Oslo, 136 cases) and Iceland (Reykjavik, 246 cases) were screened for this mutation.

For the assessment of genomic instability, a cytogenetic analysis of peripheral blood T-lymphocytes of four RAD50 687delT, two RAD50 IVS3-1G>A, and two NBS1 Leu150Phe mutation carriers was performed. The blood samples of these patients were collected at least two years after the initial breast cancer diagnosis. The patients filled out a questionnaire concerning treatment received for cancer and other possible chronic diseases, to exclude cases where recent or ongoing radio- and/or chemotherapy or other medication might have an effect on peripheral lymphocyte chromosomal stability. Seven of the eight cases had received radiotherapy; the time from the last treatment before sample collection for chromosomal analysis was nine months for one patient, and from 2 to 4 years for the rest. Three patients had also received chemotherapy; the time from the last treatment was two years for two cases and one year for one case. Samples from six healthy, age-matched female individuals served as controls.

4.2 DNA extraction (I-V)

DNA from blood lymphocytes of the analyzed patients and control samples was extracted using the standard phenol-chloroform method or the Puregene D-50K purification kit (Gentra). In studies II, IV and V, DNA from paraffin-embedded normal and tumor tissues was extracted according to the standard protocols with slight modifications (Isola et al. 1994). For each specimen, 5x5 μm thick paraffin-embedded dissects were deparaffinized with 1 ml of xylene for 30 min, after which the samples were centrifuged for 10 min and xylene was discarded. The process was repeated until all paraffin had been removed. The paraffin-free tissues were washed two times with 1ml of 100% ethanol, centrifuged and air-dried. Finally, the samples were suspended in 400 μl of DNA extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 0.5% Tween 20) and 15 μl of proteinase K (10 mg/ml), and incubated overnight in a shaker at 55ºC. Twenty-four and forty-eight hours later 10-20 μl of proteinase K was added. After 72 hours of incubation DNA was extracted with the standard phenol-chloroform procedure. DNA was precipitated with 50 μl of 7.5 M sodium acetate and 800 μl of 100% ethanol at -20 ºC overnight and pelleted by centrifugation. After air-drying DNA was resuspended in 50 μl of TE buffer (10 mM Tris, 1 mM EDTA).

4.3 mRNA extraction and cDNA synthesis (I, II, IV, V)

mRNA isolation from lymphoblast cell lines (LCLs) established from the patients studied was done using the FastTrack®2.0 Kit (Invitrogen), and cDNA was synthesized from mRNA with either the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) (study I, II, V) or GeneRacerTM Kit (Invitrogen) (study IV) according to manufacturers’ instructions.

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4.4 Mutation detection methods

4.4.1 Conformation sensitive gel electrophoresis (I-V)

Mutation screening from genomic DNA was done by conformation sensitive gel electrophoresis (CSGE), which has been developed to scan PCR products for single-base and larger mismatches. The assay is based on the assumption that mildly denaturating solvents can emphasize the conformational changes produced by mismatches in double-strand DNA and thereby increase the differential migration of heteroduplexes and homoduplexes (Gangly et al. 1993; Körkkö et al. 1998). In studies I, III and IV, all exons and flanking intronic sequences of the candidate genes were amplified by PCR utilizing AmpliTaqGold polymerase (Applied Biosystems) or Vent DNA polymerase (New England BioLabs). The latter was used in the amplification of fragments containing long poly(T) or poly(A) sequences. In studies II and V, mutation screening was restricted to specific exons. After the PCR amplification the samples were denaturated at 98ºC for 5 min, and kept at 68ºC for 30 min to allow DNA heteroduplex formation. Samples were run on mildly denaturating CSGE gels (25% acrylamide, 10% ethylene glycol, 15% formamide) at 400 V for 20-24 hours. The gels were stained with ethidium bromide, visualized in UV-light and photographed.

4.4.2 Minisequencing (II)

The minisequencing protocol described by Syvänen et al. (1993) was partly used as a mutation detection method in study II.

4.4.3 Direct sequencing (I-V)

All findings in CSGE were confirmed by reamplification of the original DNA sample and direct sequencing. In addition, direct sequencing was used as the primary mutation detection method when analyzing changes in the cDNA sequence or genomic fragments unsuitable for CSGE analysis. The PCR products were purified with QIAquick PCR Purification Kit (Qiagen) or enzymatically with ExoSAP-ITTM (usb). Sequencing reactions were carried out in both forward and reverse orientations with IRD (infrared dye)-labeled primers utilizing the SequiTherm EXELTMII DNA Sequencing Kit-LC (Epicentre Technologies). The products were analyzed with the Li-Cor IR2 4200-S DNA Analysis system (Li-Cor Inc.) according to manufacturer’s instructions.

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4.5 Real-time Quantitative RT-PCR (I)

Real-Time Quantitative RT-PCR analysis was used in study I to analyze the expression level of ATM in heterozygous ATM 5557G>A, IVS38-8T>C compound allele carriers. mRNA was isolated from the seven carrier and six control LCLs and converted into cDNA. The expression level analysis was performed using TaqMan chemistry on an ABI 7700 Sequence Detection System (Applied Biosystems) as described in Majalahti-Palviainen et al. (2000). The ATM expression level results were normalized to that of Glyceraldehyde-3-phosphate dehydrogenase (GADPH), a constitutively expressed gene quantified from the same set of samples. The sequences of forward (F) and reverse (R) primers and dual-labeled fluorogenic probes (P) for mRNA detection were as follows: GADPH (F) 5’-TGTTCGACAGTCAGCCGC-3’, (R) 5’-GGTGTCTGAGCGATGTG-GC-3’, (P) 5’-Fam-TCTTCTTTTGCGTCGCCAGCCG-Tamra-3’ and ATM (F) 5’-GTGGAGTTATTGATGACGTTACATGA-3’, (R) 5’-CCCCTGAAAAGTCACAGA-GGTC-3’, (P) 5’-Fam-CCAGCAAATTCTAGTGCCAGTCAGAGCA-Tamra-3’. Both cDNA synthesis and expression level measurements were performed twice.

4.6 Allelic imbalance analysis (II, IV, V)

LOH in the tumors of mutation carriers was evaluated by analyzing allelic imbalance (AI). Genomic DNA was amplified by PCR utilizing AmpliTaqGold polymerase (Applied Biosystems). The PCR products were analyzed with the Li-Cor IR2 4200-S DNA Analysis system (Li-Cor Inc.) using a IRD800-labeled forward primer. Allele intensity ratios were quantified with the Gene Profiler 4.05 analysis program (Scanalytics, Inc.). AI was calculated from the formula AI = (T2 x N1)/(T1 x N2), where T1/2 represents tumor and N1/2 the corresponding normal alleles. A value >1.67 or <0.60 was considered to indicate AI, meaning that the intensity of one allele had decreased >40% (Tuhkanen et al. 2004). The decrease in the intensity or complete loss of one of the alleles was confirmed by direct sequencing. The microsatellite markers utilized were D11S1819, D11S2179, D11S1778, D11S1294 and D11S1818 for ATM mutation carriers (study II), D11S4176 and D11S1757 for MRE11 mutation carriers (study IV), D8S1800, D8S88, D8S1811 and D8S1724 for NBS1 mutation carriers (study V), and D5S2110, D5S2057, D5S1984, D5S2002 and D5S2117 for RAD50 mutation carriers (studies IV and V).

4.7 Haplotype analysis (II, V)

A haplotype analysis was performed in studies II and V to evaluate the possible founder effect for ATM 6903insA, ATM 7570G>C, ATM 8734A>G, RAD50 687delT and NBS1 Leu150Phe mutations. DNA markers D11S1819, D11S2179, D11S1778, D11S1294 and D11S1818 were used for ATM mutation carriers, markers D5S2057, D5S1984 and D5S2002 for RAD50 687delT carriers, and markers D8S1800, D8S88, D8S1811 and

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D8S1724 for NBS1 Leu150Phe carriers. The genomic DNA was amplified by PCR utilizing AmpliTaqGold polymerase (Applied Biosystems), and the PCR products were analyzed with the Li-Cor (Li-Cor Inc.) using an IRD800-labeled forward primer. Haplotypes were constructed manually.

4.8 Cell cultures (I, II, IV, V)

LCLs were established from mutation carriers and controls in studies I, II, IV and V. Epstein-Barr virus (B95-8) transformed B-lymphocytes were grown in RPMI 1640 medium containing 20% fetal bovine serum, 1% L-glutamine and gentamycin (10 μg/ml) at 37°C in a 5% CO2 atmosphere.

4.9 Western blot analysis (II)

Western blot analysis was used in study II to evaluate the effects of ATM 6903insA, 7570G>C and 8734A>G mutations on ATM expression and kinase activity. Cellular extracts from LCLs were prepared by resuspending the cells in lysis buffer and incubating the mixture on ice for 30 minutes. Supernatants were collected after centrifugation at 14 000g for 15 minutes at 4ºC. ATM was immunoprecipitated with anti-ATM polyclonal antibody against the amino terminus of the protein (residues 250-522). Immunoprecipitates were resolved on SDS-PAGE gels and western blotted with the same anti-ATM antibody.

For assessment of in vivo ATM kinase activity, extracts from mock or irradiated cells (4 Gray, Gy) were analyzed by SDS-PAGE and immunoblotted with appropriate antibody: anti-ATM Ser1981, anti-p53 Ser15 and anti-CHK1 Ser317, with additional anti-p53 and anti-CHK1 antibodies that detects the total pool of these ATM substrates. The phosphorylation of two ATM substrates, p53 (Ser15) and CHK1 (Ser317), and the ATM autophosphorylation site Ser1981, was assessed before and after exposure to IR.

4.10 MTT assay (II)

In study II, the effect of the ATM 6903insA, 7570G>C and 8734A>G mutations on cell survival after IR was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 10 000 cells of each LCL were plated in triplicate into a 96-well plate in 100 µl media, and cell survival was assessed 4 days after exposure to 0.5, 1, 2 and 3 Gy from a 137Cs source, at a dose rate of about 1.1 Gy/min. For assessment of the number of viable cells, 100 µl of MTT solution 1 mg/ml in PBS was added to each well and incubated at 37ºC for 4 hours. The cells were pelleted by centrifugation and resuspended in 150 µl DMSO. The plates were incubated for 1 hour at room temperature and then measured at 570 nm. Cell survival fraction was calculated relative to the number of viable cells in the non-irradiated culture at 96 hrs.

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4.11 Cytogenetic analysis (V)

The cytogenetic analysis of peripheral blood T-lymphocytes was carried out in short-term three-day cultures (Verma & Babu 1989). A minimum of 50 Giemsa-banded metaphases were evaluated for spontaneous chromosomal abnormalities under a light microscope and photographed with an automatic chromosome analyzer (CytoVision version 3.1, Applied Imaging).

4.12 Statistical analysis (I-III, V)

Fisher’s exact test or χ2 test was used to evaluate the differences in allele frequencies between cases and controls (I-III, V). In study I, the comparison of the two expression level measurements performed was assessed by paired-samples t-test, and independent samples t-test was used for the comparison of ATM expression levels between the two subgroups categorized according to their genetic status. In study V, independent samples t-test for normally distributed variables and Mann-Whitney U-test for not normally distributed variables was used to determine the statistical difference in the number of different chromosomal aberrations between the two groups categorized according to their genetic status. All analyses were performed with SPSS (version 12.0 for Windows, SPSS Inc.) and all p-values were two-sided.

4.13 Ethical issues

Approval for the study (I-V) was obtained from the Ethical Board of the Northern Ostrobothnia Health Care District and the Finnish Ministry of Social Affairs, and from the Ethical Boards of the University Hospital Health Care Districts involved (II, V). All patients and family members participating in the study provided an informed consent, and relatives were contacted only with the permission of the index case. Information from the mutation analyses was not given to any patients or family members.

5 Results

5.1 Mutation screening of the ATM gene (I)

The mutation analysis of 185 breast or ovarian cancer patients belonging to 121 families revealed several changes both in the exon and intron region of ATM. However, only one novel amino acid substitution, Arg2691Cys, was observed, but it located outside the known functional domains. All the other amino acid substitutions observed have been described previously. Although a few of the previous studies have proposed a role for some of these alleles in susceptibility to breast cancer (Dörk et al. 2001; Thortenson et al. 2003), the current study did not show any evidence for the co-segregation of these alterations with cancer phenotype, and the comparison of allele frequencies between cases and controls showed no statistical difference. However, it was observed that the combination of 5557G>A (1853Asp>Asn) in cis position with IVS38-8 T>C associated with bilateral breast cancer. Both 5557G>A and IVS38-8T>C are known polymorphisms reported by several studies (Teraoka et al. 2001; Angéle et al. 2003b; Angéle et al. 2004). Although in the current study IVS38-8T>C was always found in cis with 5557A, it has been indicated that IVS38-8T>C also occurs in combination with 5557G (Teraoka et al. 2001). Among altogether 176 breast cancer patients studied 16 had a bilateral disease, five of which (31.3%) were heterozygous for the 5557G>A, IVS38-8T>C compound allele. The frequency was markedly higher compared to 7 carriers in 160 (4.4%) patients with unilateral breast cancer (OR 9.9, 95% CI 2.7-36.5, p=0.002) and to controls (4.2%, 13/306, OR 10.2, 95% CI 3.1-33.8, p=0.001).

As the 5557G>A change has been reported to affect an exonic splicing enhancer (ESE) (Thorstenson et al. 2003), it was hypothesized that the observed compound allele could have some effect on the correct splicing of exon 39. However, no aberrant transcripts were detected by electrophoresis in 1% agarose gel and by direct sequencing, but the results from Real-Time Quantitative RT-PCR analysis showed that ATM expression levels of LCLs from heterozygous carriers of 5557G>A, IVS38-8T>C compound allele (n=7) were lower than in non-carriers (n=6). The mean ATM expression level normalized to GADPH expression was 1.54 (95% CI 0.9-2.2) for carriers, and 2.38 (95% CI 1.4-3.4) for non-carriers. The measurements were performed twice and paired-samples correlations

45

were high (0.971, p<0.001). However, the difference in expression levels did not reach statistical significance (p=0.09), which is at least partially due to the low number of cell lines available for analysis.

5.2 A-T-related ATM mutations in breast cancer patients (II)

Of the seven ATM germline mutations previously identified in Finnish A-T patients, 6903insA and 7570G>C were the only ones observed in the breast cancer cases studied. Additionally, when screening for the A-T related mutation in exon 62 by CSGE another alteration, 8734A>G, was identified. 8734A>G has previously been associated with breast cancer susceptibility (Teraoka et al. 2001; Thorstenson et al. 2003) and has also been suggested to be causative for A-T (Thorstenson et al. 2003). Consequently, it was included in the study.

6903insA (leading to premature translation stop at codon 2372) was observed in the affected index patients of three families (3/541, Table 3) and in five unselected breast cancer cases (5/1124) diagnosed between the age of 41 and 58. Cancer registry inquiries revealed that at least one of the parents of four of these cases had had cancer (lymphoma, uterine, bladder, esophageal, stomach). 6903insA was not observed in the 1107 controls tested. All eight carriers originated form the Tampere region (Figure 5) and shared the same haplotype, further confirming a common origin.

Table 3. Prevalence of ATM mutations in Finnish breast cancer patients

Allele Carrier frequency Family ID Family history of cancer a, b

Effect on protein Familial Unselected Control

6903insA

truncation 3/541 5/1124 0/1107 p088 Br 53(+), Bil Br 48, 75(+), Br 79, Br 49(-), Hp 68(+), Pan, Pro

p1025 Br 81(+), Br 50, Br 70, Pan 79, Col 75 s134 Bil Br 48, 62(+), Br, Br, Lu

7570G>C Ala2524Pro 1/541 2/1124 1/1107 5063 Br 60(+)c, , Br 56, Lu 58+Pro 70, Skin 49

8734A>G d Arg2912Gly 2/541 0/1124 0/1107 p293 Br 58(+), Bil Br 60, 83(+), Br 48(+), Col 52(+), Sto, Lu

p337 Br 44(+), Bil Br 53, 58(+), Br 55(-), Br, Col

a Bil Br, bilateral breast; Br, breast; Col, colorectal; Hp, hypopharyngeal; Lu, lung; Pan, pancreas; Pro, prostate; Sto, stomach b Age at diagnosis is given after the malignancy when known, and the mutation status for the tested individuals is shown in brackets: (+) for carriers and (-) for non-carriers. The + sign indicates more than one primary cancer in the same individual c Heterozygous also for CHK2 1100delC d Not reported so far in A-T families

46

The 7570G>C mutation (Ala2524Pro) was observed in one familial breast cancer case (Table 3) originating maternally from central and paternally from eastern Finland. Two carriers, diagnosed at the age of 44 and 56 years, were also identified in 1124 unselected breast cancer cases. Both of them originated from the Oulu region (Figure 5). One anonymous 7570G>C carrier was also identified among the 1107 healthy controls. The age and the family history of cancer of this carrier are, however, unknown. Because of the seemingly different geographical origins of the currently identified mutation carriers, a common founder for the 7570G>C mutation was confirmed by microsatellite marker analysis. Samples from eight different families, including two A-T and two breast cancer families identified in previous studies (Laake et al. 2000; Allinen et al. 2002), were analyzed. All carriers shared the same haplotype.

8734A>G (Arg2912Gly) was observed in two familial cases, both originating from the Tampere region (Table 3, Figure 5). All carriers shared the same haplotype. In contrast to the 6903insA and 7570G>C mutations, 8734A>G was not found among the 1124 unselected cancer cases or 1107 healthy controls tested.

Fig. 5. Map of Finland showing the geographical origin of ATM 6903insA (grey circle), ATM 7570G>C (black star) and ATM 8734A>G (black triangle) mutation carriers. In addition to the carriers identified in the current study, the origin of two A-T families with 6903insA (FIAT4, FIAT5), two with 7570G>C (FIAT6 and FIAT7), as well as two previously identified 7570G>C and one 6903insA positive breast cancer families are included (Laake et al. 2000; Allinen et al. 2002).

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In the current study, ATM mutations, 6903insA, 7570G>C and 8734A>G, were observed in a total of 6/541 familial cases (p=0.006, OR 12.4, 95% CI 1.5-103.3) and 7/1124 unselected cases (p=0.07, OR 6.9, 95% CI 0.9-56.4) compared to a single 7570G>C carrier in 1107 healthy controls. As the mean age at diagnosis of all the identified carriers was 54 years, the breast cancer risk associated with these mutations appeared not to be restricted to those diagnosed at young age. However, there seemed to be an excess of bilateral cases, as 24% of all mutation carriers with breast cancer had bilateral disease. The study of additional family members in mutation-positive families showed incomplete segregation with the disease, as both unaffected mutation carriers and mutation-negative breast cancer patients were observed.

The AI analysis was performed on available tumor samples of two 7570G>C carriers and one 6903insA carrier, but loss of the wild-type allele was not observed. Subsequently, heterozygous LCLs were used for characterization of the functional consequences of the observed germline defects. LCLs were established from one 7570G>C, one 8734A>G, and two 6903insA carriers (Table 3). LCLs from two healthy individuals and two noncarrier breast cancer cases, together with two A-T LCLs were used as reference. The results showed that the missense mutations had no effect on protein stability, as the amount of ATM protein from 7570G>C and 8734A>G LCLs was comparable to that of the control subject homozygous for wild-type ATM. In 6903insA LCLs no truncated protein was present, and the total amount of endogenous ATM was reduced to approximately half. However, sequencing analysis demonstrated that 6903insA transcripts were still present in the mRNA pool, which indicates that they were not eliminated from the cells by nonsense-mediated decay.

Possible downstream effects of the observed mutations were evaluated by assessing the phosphorylation of two ATM substrates, p53 and CHK1, and the ATM autophosphorylation site Ser1981 for the missense mutations, before and after exposure to IR. In control cell line ATM was activated rapidly, as judged by enhanced phosphorylation of p53 on Ser15, CHK1 on Ser317 and ATM autophosphorylation site Ser1981. In contrast, phosphorylation was barely detectable in the ATM non-expressing A-T cell line. DNA damage-induced phosphorylation of p53, CHK1 and ATM autophosphorylation site seemed also to be dramatically lower in the 7570G>C carrier LCL, whereas the 8734A>G carrier LCL was defective only in CHK1 phosphorylation. For 6903insA LCLs, no significant difference in the phosphorylation was observed when compared to controls.

Since ATM deficiency leads to increase in cellular sensitivity to IR, the possible effects of 7570G>C, 8734A>G and 6903insA on radiosensitivity was evaluated in the carrier LCLs. Two cell lines from A-T patients were used as positive controls. As expected, the A-T cell lines displayed extreme radiosensitivity to IR when compared to control LCLs. The survival of the 7570G>C carrier and 8734A>G carrier LCLs was of the same order as the controls, whereas the survival of the two 6903insA heterozygous LCLs was indistinguishable from that of A-T cell lines at most dose points.

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5.3 Mutation screening of the ATR gene (III)

The mutation analysis of ATR in altogether 126 families revealed several alterations. In total, 23 nucleotide substitutions were observed: seventeen in the exon and six in the intron regions of the gene. Eleven of the exonic changes resulted in amino acid substitutions, eight of which were novel and three were polymorphisms reported in the SNP database (http://www.ncbi.nlm.nih.gov/SNP/). The frequency of all observed germline variants was assessed in either 100 or 300 control individuals. In addition, all nucleotide alterations were assessed for possible effects on splicing consensus sequences by the Splice site prediction program (http://www.fruitfly.org/seq_tools/splice.html). All coding sequence variants were also tested by the ESEfinder program (http://rulai.cshl.edu/tools/ESE/) in order to identify those that reduced the ESE score below the calculated threshold value.

Only four of the observed changes, Glu254Gly, Ser1142Gly, IVS24-48G>A and IVS26+15C>T, were absent from the 300 control individuals tested. However, none of the changes appeared to be clearly pathogenic: none had an effect on splicing consensus sequences, and Glu254Gly and Ser1142Gly did not change ESE sequences. Of the missense changes only Ser1142Gly affected an evolutionarily conserved residue (Xenopus laevis, Drosophila melanogaster) (Perry & Kleckner 2003). Moreover, the segregation with the disease phenotype could not be confirmed in any of the families found positive for these alterations (Table 4).

Table 4. Germline variants of the ATR gene observed in the patients studied but absent from controls

Nt change Effect on protein

Carrier frequency Family ID Family history of cancer a, b

761A>G Glu254Gly 0.8% (1/126) 5 Br 37(+), Bil Br 45, 55(-)

3424A>G Ser1142Gly 1.6% (2/126) 6 Br 64(+), Br 49(-), Br 40(-), Br, Br 7 Br 59(+), Br 45(-), Br, Ut, Pro, Col, Lu, Sto

IVS24-48G>A Unknown 2.4% (3/126) 8 Br 57(+), Br 68 9 Br 46(+), Br 62, Br 68, Br 72, Br+Ut 52(-) 10 Br 71(+), Br 65, Br

IVS26+15C>T Unknown 0.8% (1/126) 11 Br 64(+), Br 49(-), Br 55, Br a Bil Br, bilateral breast; Br, breast; Col, colon; Lu, lung; Pro, prostate; Sto, stomach; Ut, uterine b Age at diagnosis is given after the malignancy when known, and the mutation status for the tested individuals is shown in brackets: (+) for carriers and (-) for non-carriers. The + sign indicates more than one primary cancer in the same individual

49

5.4 Mutation screening of Mre11 complex genes MRE11, NBS1 and RAD50 (IV)

Among the 151 cancer families studied, three novel potentially pathogenic Mre11 complex gene alterations were observed: MRE11 913C>T (Arg305Trp), NBS1 448C>T (Leu150Phe) and RAD50 687delT (stop codon at 234). Of these, MRE11 Arg305Trp was observed in one patient diagnosed with ovarian cancer, and NBS1 Leu150Phe in one breast cancer case (Table 5). Both amino acid changes occurred in the highly conserved protein domains. Although MRE11 Arg305Trp does not affect a residue with specific function, the involved arginine is evolutionarily conserved (e.g. Mus musculus, Gallus gallus, Saccharomyces cerevisiae, Arabidopsis thaliana). Correspondingly, the NBS1 Leu150 residue is conserved between several species (e.g. Mus musculus, Gallus gallus, Drosophila melanogaster), and the substitution occurs in the middle of the functionally important BRCT domain. The pathogenicity of both changes is supported by their absence from 1000 healthy controls. Yet, the segregation of these alleles with the disease phenotype in the mutation positive families could not be confirmed because of the lack of suitable additional DNA samples.

The third potential Mre11 complex mutation, RAD50 687delT, is expected to cause truncation of most of the protein. The analysis of three mutation-carrier LCLs showed that this allele is expressed at least at the mRNA level. RAD50 687delT was observed in two families (1.3%, 2/151), in which altogether four affected carriers were observed (Table 5). Interestingly, the index case of one of the families had also tested positive for the previously identified BRCA1 3745delT mutation (Huusko et al. 1998). In this family there were three healthy individuals carrying RAD50 687delT, but also BRCA1 truncation mutation segregated incompletely with the disease. Surprisingly, RAD50 687delT was observed in healthy controls (0.6%, 6/1000), albeit at a lower frequency. In addition to 687delT, two other novel RAD50 alleles, Ile94Leu (1.3%, 2/151) and Arg224His (2.0%, 3/151), were observed in the current study. Both were classified as unknown variants. Even though these substitutions affected conserved residues, the former in the highly conserved amino-terminal ATPase domain and the latter in the coiled-coil domain (Hopfner et al. 2000; Hopfner et al. 2001), they also occurred in healthy controls, but at lower frequency (0.3%, 3/1000 and 0.9%, 9/1000, respectively). Consequently, their role in breast cancer susceptibility has to be clarified by additional studies.

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Table 5. Putative mutations in the Mre11 complex genes in Finnish families with breast and/or ovarian cancer

Allele Carrier frequency Family ID Family history of cancer a, b Cases Controls MRE11 Arg305Trp 0.7% (1/151) - (0/1000) 1 Ov 37(+), Ut 38, Ut, Ut 28 NBS1 Leu150Phe 0.7% (1/151) - (0/1000) 2 Br 40(+), Ut 47 c, Sto, Sto RAD50 687delT 1.3% (2/151) 0.6% (6/1000) 3 Br 43(+), Br 41(+), Lu, Sto, Sto, Csu, Csu

4 d Ov 50(+) e, Ov 44(+), Br 35(-) a Br, breast; Csu, primary cancer site unknown; Lu, lung; Ov, ovarian; Sto, stomach; Ut, uterine b Age at diagnosis is given after the malignancy when known, and the mutation status for the tested individuals is shown in brackets: (+) for carriers and (-) for non-carriers c Premalignant uterine lesion d BRCA1 mutation positive family e confirmed BRCA1 3745delT carrier

The relatively high frequency of RAD50 687delT at the population level suggested that the allele, if truly pathogenic, exhibits low penetrance and may therefore predispose to cancer without clustering the disease in the pedigrees. Consequently, 192 breast cancer cases without family history of cancer were screened for RAD50 687delT. In parallel, these patients were also screened for the MRE11 and NBS1 mutations, as the segregation of these alterations with the disease phenotype could not be confirmed from the pedigrees. None of these three alterations was present in the sporadic breast cancer cases tested. However, this might be due to coincidence, but also to selection bias, as the frequency of the studied alterations is low (0.7 and 1.3%) in the familial cohort, and it is expected to be even lower in patients without family history.

The AI analysis of the tumors available from one MRE11 Arg305Trp and three RAD50 687delT positive patients showed retention of the wild-type alleles. Although based on a small number of tumors analyzed, it indicates that LOH is unlikely to be involved in disease development in the carriers of these mutations.

5.5 Occurrence of RAD50 and NBS1 mutations in unselected breast cancer patients (V)

The prevalence of the two candidate breast cancer susceptibility alleles, RAD50 687delT and NBS1 Leu150Phe, and the two other novel unknown RAD50 variants, Ile94Leu and Arg224His, identified in study IV was investigated in an unselected cohort consisting of 317 consecutive, newly diagnosed breast cancer patients. Altogether eight RAD50 687delT carriers (8/317) were identified (Table 6), the frequency being significantly higher compared to controls (6/1000, p=0.008, OR 4.3, 95% CI 1.5-12.5). The mean age at diagnosis did not differ between RAD50 687delT carriers (57.9 years) and non-carriers (58.0 years). Of the studied 317 cases 70 were considered familial, two of which were found positive for RAD50 687delT. However, the study of additional affected family members indicated incomplete segregation of the mutation (Table 6). For two RAD50

51

687delT carriers there was family history of other cancers (at least two cases in first- or second-degree relatives), whereas the rest showed no evidence for clustering of the disease in the family. The difference between familial cases (2.9%, 2/70) and cases without family history of breast/ovarian cancer (2.4%, 6/247) was insignificant (p=1.000). Due to the relatively high frequency of RAD50 687delT in the Northern Finnish cohort, altogether 512 breast cancer cases from Sweden, Norway and Iceland were screened to obtain more information about the geographical occurrence of this allele. However, RAD50 687delT was not observed in other Nordic cohorts, suggesting that it is a Finnish founder mutation. The common origin of RAD50 687delT was confirmed by microsatellite marker analysis of fourteen carriers that all shared the same haplotype not present in eight non-carriers analyzed.

Of the other RAD50 alterations studied, Arg224His was observed twice (2/317, 0.6%), which did not differ from the controls (9/1000, 0.9%), whereas Ile94Leu was not detected (0/317), compared to 3/1000 in controls. Consequently, the current results do not support their association with breast cancer susceptibility. However, when screening for the Ile94Leu alteration a novel RAD50 splice site mutation, RAD50 IVS3-1G>A, was observed in one patient. There were several breast cancer cases in the family (Table 6), but only two additional ones were available for mutation testing: the sister of the index case was identified as carrier, whereas a maternal cousin tested negative. RAD50 IVS3-1G>A was not present among 1000 population controls. Subsequently, the effect of RAD50 IVS3-1G>A on mRNA splicing was evaluated by cDNA-specific amplification of RAD50 exon 3. Only one PCR product corresponding to the predicted size was observed. However, sequencing revealed that IVS3-1G>A leads to one base pair deletion between exon 2 and 3 in the mRNA sequence (214delG), thus resulting in a premature translation stop at codon 78. This observation was supported by the Splice site prediction program. Even though IVS3-1G>A abolishes the existing acceptor site (score 0.97) it creates a new one right next to the previous site (score 0.11). The RAD50 214delG transcript was present at lower levels than the wild type, indicating that it is unstable.

The NBS1 Leu150Phe substitution was observed in two patients in the unselected breast cancer cohort (Table 6). One patient had breast cancer at the age of 58, and was considered to be a familial case as her daughter had had ovarian cancer at the age of 17; also the daughter tested positive for the Leu150Phe allele. The other patient had breast cancer at the age of 54. She had a family history of stomach and thyroid cancer, but no additional DNA samples were available for mutation testing. The common origin of the two currently identified NBS1 Leu150Phe carriers and one previously observed carrier (study IV) was verified by microsatellite marker analysis.

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Table 6. Pathogenic germline changesa in the RAD50 and NBS1 genes in unselected Northern Finnish breast cancer patients

Allele Carrier frequency p-value Carrier patient Family history of cancer b, c Cases Controls OR [95% CI] RAD50 687delT 8/317 6/1000 p=0.008 Br 52 Br(-), Br OR 4.3 [1.5-12.5] Br 70 Br 43(-) Br 61 Csu, Csu, He, Lu, Sto, Mel Br 74 Sar 59, Kid 73 Br 46 Sto Br 56 Pan 54 Br 50 None reported Br 54 None reported

RAD50 IVS3-1G>A 1/317 0/1000 p=0.241 Br 56 Br 51(+), Leu, Br 43, Br 71+Ov 56, Br 57(-)

NBS1 Leu150Phe 2/317 0/1000 p=0.058 Br 54 Sto 78, Th 50, Sto Br 58 Ov 17(+), Csu 48 a Based on the effect on the protein and allele prevalence in patients versus controls b Age at diagnosis is given after the malignancy when known, and the mutation status for the tested individuals is shown in brackets: (+) for carriers and (-) for non-carriers. The + sign indicates more than one primary cancer in the same individual c Br, breast; Csu, primary cancer site unknown; He, head; Kid, kidney; Leu, leukemia; Lu, lung; Mel, melanoma; Ov, ovarian; Pan, pancreas; Sar, sarcoma; Sto, stomach; Th, thyroid

Next, the effect of the three potentially pathogenic alterations, RAD50 687delT,

RAD50 IVS3-1G>A and NBS1 Leu150Phe, on tumorigenesis was evaluated by analyzing the occurrence of AI in the tumors of mutation carriers. Additionally, the effect of these mutations on genomic instability was assessed by cytogenetic analysis of peripheral blood T-lymphocytes. The AI analysis of eight RAD50 687delT, one RAD50 IVS3-1G>A and two NBS1 Leu150Phe carriers provided no evidence for the loss of the wild-type allele, whereas the cytogenetic analysis of four RAD50 687delT, two RAD50 IVS3-1G>A and two NBS1 Leu150Phe carriers showed a significant increase in the number of chromosomal aberrations (p=0.006) (Figure 6). In particular, both simple and complex chromosomal rearrangements were found more frequently in mutation carriers than in healthy controls (p=0.002 and p=0.010 respectively), whereas for chromosome/chromatid breaks and deletions the difference did not reach statistical significance (p=0.20). In contrast, telomeric associations were rare, as they were seen only once in one RAD50 687delT and in one NBS1 Leu150Phe carrier (p=0.20). All observed chromosomal aberrations were considered random, as no preference for a specific break site or evidence for clonality was observed.

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Fig. 6. Increase in spontaneous chromosomal aberrations in T-lymphocytes of heterozygous RAD50 687delT, RAD50 IVS3-1G>A and NBS1 Leu150Phe carriers. Comparison of (A) the frequency of cells with chromosomal aberrations and (B) the frequency of aberration type per 100 metaphases. Bars represent the mean ± SD of pooled data for mutation carriers (n=8) and healthy controls (n=6). A minimum of 50 Giemsa-banded metaphases was analyzed for each individual. Simple chromosomal rearrangements include inversions, ring chromosomes and translocations (≤ 3 break rearrangements), and complex chromosomal rearrangements include translocations (≥ 4 break rearrangements) and marker chromosomes.

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6 Discussion

6.1 ATM mutations and breast cancer susceptibility (I, II)

BRCA1 and BRCA2 mutations have been observed in only about 20% of the breast cancer families in Finland (Vehmanen et al. 1997a, Vehmanen et al. 1997b, Huusko et al. 1998), suggesting involvement of additional susceptibility genes. ATM has long been considered a good candidate gene, based on its integral role in the maintenance of genomic integrity. Moreover, epidemiological studies have consistently shown that female relatives of A-T patients are at increased risk of breast cancer (Swift et al. 1987; Swift et al. 1991; Easton 1994; Olsen et al. 2001). However, the evidence for ATM mutations’ involvement in breast cancer susceptibility in general population has been quite controversial (reviewed in Khanna & Chenevix-Trench 2004). In Finland, the contribution of ATM mutations to breast cancer predisposition has previously been studied by analyzing breast cancer cases from Northern Finnish families for the occurrence of ATM germline mutations originally identified in Finnish A-T patients (Laake et al. 2000; Allinen et al. 2002). The results suggested some contribution of these mutations to familial aggregation of breast cancer.

Of eight different mutations two, 6903insA and 7570G>C, were observed in the studied cohort, but the overall frequency of these mutations was considered low, as 6903insA was observed in one and 7570G>C in two families (Allinen et al. 2002). Therefore, the aim of the current study was first to investigate whether ATM mutations apart from those identified in Finnish A-T patients could explain an additional fraction of hereditary predisposition to breast cancer (study I). Second, we wanted to clarify the role of A-T-related mutations in breast cancer susceptibility in the Finnish population (study II). Since the previous study was conducted with a relatively small and geographically constrained cohort, the evaluation of the contribution and impact of the observed ATM mutations on breast cancer predisposition was limited. Moreover, the involvement of those ATM mutations not detected (IVS14+3-4delAT, IVS37+9A>G, 6779-6780delTA, 8710-8715delGAGACA and 9139C>T) could not be ruled out. The eighth ATM mutation studied in Allinen et al. (2002) leading to a 200-bp deletion in the transcript was later found to derive from an immigrant parent, and was therefore not studied further.

55

In study I, the entire coding region of ATM was screened for mutations in 121 Northern Finnish breast and breast-ovarian cancer families. No clearly pathogenic mutations were identified, the criteria for pathogenicity being based on both the mutations’ predicted effect on the protein as currently known today, and prevalence in cancer cases versus controls. However, the results suggest a cancer risk modifying effect for a common ATM 5557G>A (Asp1853Asn) variant when occurring in cis position, i.e. contained in the same haplotype, with IVS38-8T>C. In the current cohort the 5557G>A, IVS38-8T>C combination allele was associated with bilateral breast cancer (OR 10.2, 95% CI 3.1-33.8, p=0.001). Among altogether 176 breast cancer patients studied 16 had a bilateral disease, five of which (31.3%) were heterozygous for the 5557G>A, IVS38-8T>C compound allele. Of the remaining eleven bilateral cases, one was found to carry the CHK2 1100delC low-penetrance allele, which has been associated with bilateral breast cancer (Vahteristo et al. 2002; Broeks et al. 2004), and two cases were found positive for either the previously identified BRCA2 999del5 or TP53 Asn235Ser mutation (Huusko et al. 1998; Huusko et al. 1999). Previous studies have also suggested a functional role for the 5557G>A variant. It has been indicated to modulate the penetrance of HNPCC in carriers of MLH1 and MSH2 germline mutations (Maillet et al. 2000), and it was the most frequently occurring ATM polymorphism in individuals with idiopathic or radiation-induced ocular telangiectasia of either choroidal or retinal origin, without family history of A-T (Mauget-Faysse et al. 2003). In addition, 5557G>A homozygotes were over-represented in breast cancer patients who had an adverse response to radiotherapy (Angèle et al. 2003b). These studies did not, however, report what proportion of the 5557A alleles was contained in the same haplotype with IVS38-8T>C.

As the 5557G>A change has been reported to affect an exonic splicing enhancer (ESE) (Thorstenson et al. 2003), it was hypothesized that the observed compound allele could have some effect on the correct splicing of exon 39. Many ATM exons have poor agreement to the splicing consensus sequences and are also small in size, which predicts that efficient splicing is likely to require enhancers to promote splicing at unfavorable sites. Indeed, ESEs have been identified in every exon of the ATM gene (Thorstenson et al. 2003). Disruption of ESEs may alter the correct splicing, which might be the result of the 5557G>A transition, and the intronic change IVS38-8T>C in relatively close proximity of the intron/exon boundary might strengthen this effect. No aberrant transcripts were, however, detected, and the exact mechanism that could lead to association of 5557G>A, IVS38-8T>C with bilateral breast cancer remains unclear. However, the results from study I suggest that the 5557G>A, IVS38-8T>C compound allele might result in lowered ATM expression levels (p=0.09). The variation in ATM dosage may affect the DNA damage recognition and response pathways as well as other functions important for the maintenance of genome integrity. Possibly in combination with certain genetic background and environmental factors it could modify the cancer risk by increasing genetic instability or by altering the effect of the normal DNA damage response.

The preliminary observations of study I have subsequently been investigated by two other studies. In the WECARE Study the primary hypothesis is that women who carry a mutant ATM allele and who received radiation therapy as treatment for their first primary breast cancer have increased risk of developing a second primary breast cancer. This study, however, failed to support the association of the haplotype containing 5557G>A

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and IVS38-8T>C with bilateral breast cancer (Langholtz et al. 2006). The authors provided several potential explanations for this discrepancy. First, it is possible that instead of being causal, the 5557G>A and IVS38-8T>C variants are markers of a risk haplotype harboring other risk-modifying alleles. However, no evidence for significant linkage disequilibrium (LD) between either of these variants and alleles at any flanking site was observed. However, it is noted that the patterns of LD in the ATM gene within the Finnish population may differ from those observed in the WECARE population originating from the USA and Denmark. Second, the natures of the two study populations are different, as the WECARE Study is population-based and unselected for family history, while the cases in study I were ascertained based on their membership in high to moderate risk breast cancer families. Therefore, difference in background risk factors may contribute to differing abilities to observe a risk-modifying effect of the 5557G>A, IVS38-8T>C haplotype. The recent study by Tommiska et al. (2006a) did not, however, confirm the association of 5557G>A, IVS38-8T>C with bilateral breast cancer in a Southern Finnish breast cancer cohort comprising both familial and unselected cases, arguing against these hypotheses. However, it is always possible that there are regional differences between Southern and Northern Finland in the occurrence of different ATM haplotypes. In the breast cancer cohort from Southern Finland 5557G>A, IVS38-8T>C was found in three different haplotypes: haplotype 8 contained only the combined variant, while haplotype 13 also contained a Ser333Phe change, and haplotype 9 also contained Pro1526Pro (Tommiska et al. 2006a). In contrast, in study I Ser333Phe was not observed, whereas there were altogether eight carriers of Pro1526Pro. However, none of them carried the 5557G>A, IVS38-8T>C compound allele, arguing against that these three SNPs were contained in the same haplotype. Consequently, the haplotyping of the 5557G>A, IVS38-8T>C positive individuals identified in study I would be necessary in order to investigate whether they share a unique, founder haplotype associated with risk. Nevertheless, we also have to acknowledge the possibility that the failure to confirm the association of 5557G>A, IVS38-8T>C allele with bilateral breast cancer by two different studies (Langholtz et al. 2006; Tommiska et al. 2006a) might be due to the fact that the result of study I is a false positive.

Taken together, the results of study I and Allinen et al. (2002) indicate that breast cancer associated ATM alterations seem to be mainly restricted to those reported in A-T. Subsequently, in study II we investigated the prevalence of seven Finnish A-T-related mutations in a large cohort of 541 breast cancer families, 1124 breast cancer patients unselected for family history, together with 1107 matched control subjects originating from Northern, Southern and Central Finland. Consistent with previous results (Allinen et al. 2002), 6903insA and 7570G>C were the only A-T-related mutations observed, thus providing more convincing evidence that they are breast cancer susceptibility alleles outside the A-T families as well. The other five A-T mutations were not detected, which might be due to their low frequency, but it is also possible that these alleles do not predispose to breast cancer. Besides 6903insA and 7570G>C another ATM mutation, 8734A>G, was observed in two families. It has previously been associated with breast cancer susceptibility (Teraoka et al. 2001; Thorstenson et al. 2003) and has been suggested, although not yet confirmed, to be causative for A-T (Thorstenson et al. 2003). The 6903insA, 7570G>C and 8734A>G mutations were observed altogether in six breast cancer families (6/541) (OR 12.4, 95% CI 1.5-103.3, p=0.006). The study of additional

57

family members, however, showed incomplete segregation with the disease, as both unaffected mutation carriers and mutation-negative breast cancer patients were observed. In addition, 6903insA and 7570G>C were observed in breast cancer patients without known familial background of the disease (7/1124) (OR 6.9, 95% CI 0.85-56.4, p=0.070), and 7570G>C was also found in one healthy control, further suggesting that the penetrance of these mutations is incomplete. Thus the risk associated with the observed ATM mutations most likely depends on environmental factors and/or susceptibility alleles in other genes. This is consistent with the recent study that found an approximately twofold increase in risk of breast cancer associated with ATM mutations causing A-T. This risk appears to be similar to that of low-penetrance susceptibility allele CHK2 1100delC (Renwick et al. 2006).

When combining the results of Allinen et al. (2002) and study II, ATM mutations have been observed in altogether 1.4% (9/630, p=0.0003, OR 18.9, 95% CI 2.4-149.7) of familial and 0.6% (7/1209, p=0.03, OR 7.6, 95% CI 0.9-61.9) of unselected breast cancer cases compared to 0.1% (1/1307) in healthy controls. Consequently, the ATM mutations show an apparent, yet overall limited contribution to breast cancer susceptibility in Finland. The relatively low frequency of these alleles is not, however, surprising when considering the fact that at least two of them are also pathogenic for A-T, thereby limiting their population frequency. Nevertheless, as the observed alleles show geographical clustering, their contribution to familial breast cancer in certain regions might be significant. In particular, the 6903insA and 8734A>G mutations cluster to the area of Tampere, and together their frequency in the breast cancer families studied from this region is 3.0% (5/168). In contrast, 7570G>C mainly clusters to the Oulu region (Laake et al. 2000; Allinen et al. 2002; study II).

It has been suggested that mutations with dominant-negative effect, particularly missense changes, are mainly responsible for the increased cancer risk in ATM carriers (Meyn 1999; Gatti et al. 1999). Consistent with this idea, two out of three currently identified breast cancer associated ATM changes were missense mutations. Nevertheless, all three alterations had different effects at protein level. Only 7570G>C leading to Ala2524Pro substitution showed a dominant-negative effect on the kinase activity. Substitution of the evolutionarily conserved (e.g. Mus musculus, Xenopus laevis and Tel1p protein of Schizosaccharomyces pombe) Ala2524 residue with proline in the FAT domain leads to a stable protein with defective kinase activity. As FAT and FATC domains have been suggested to fold together to ensure the proper function of the interposed kinase domain (Bosotti et al. 2000), the pathogenic nature of 7570G>C could be explained by the failure in correct folding, thus leading to kinase inactivation. A dominant-negative effect has also been shown for another A-T-related and breast cancer associated mutation, 7271T>G (Val2424Gly), located in the FAT domain (Chenevix-Trench et al. 2002; Stankovic et al. 1998). The breast cancer risk associated with 7271T>G has been estimated to be 13-fold in both heterozygotes and homozygotes compared to healthy controls (Stankovic et al. 1998; Chenevix-Trench et al. 2002). In contrast, the current results suggest that despite the dominant-negative effect, 7570G>C is not a high-risk breast cancer susceptibility allele. Only one case with moderate familial risk for breast cancer was found positive for 7570G>C, and she also carried the CHK2 1100delC low-penetrance allele (Vahteristo et al. 2002). In addition, two breast cancer cases without family history of disease were found positive, and 7570G>C was the only

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ATM mutation observed in controls, although the age and the family history of cancer of the one identified healthy carrier are unknown. Nevertheless, the association of 7570G>C with breast cancer susceptibility is supported by the two A-T families exhibiting this mutation, as both also displayed signs of hereditary predisposition to breast cancer, and by the two previously identified breast cancer families (Allinen et al. 2002).

For ATM 6903insA carriers, haploinsufficiency might be a more plausible explanation for the cancer predisposition, as no truncated protein was present in the heterozygous LCLs. The total amount of endogenous ATM was reduced to half, which seems sufficient for normal function of the ATM checkpoint signaling pathway, but not to ensure normal level of cell survival after IR-induced damage. This differential impact on cellular radiosensitivity has been reported previously by over-expression of a fragment of ATM containing the leucine zipper domain that can act in a dominant-negative manner to influence cell survival, but not p53 stabilization and cell cycle checkpoints (Morgan et al. 1997). Thus, if a truncated protein product was expressed, although below the level of the detection method used, it would contain the leucine zipper and could potentially act in a dominant-negative way to influence cell survival. However, it is more likely that 6903insA mutation acts effectively as null. If the transcript is truly translated, it seems to result in an unstable protein that is only present in very small amounts. Consequently, the current results suggest that different biological endpoints and functions have different threshold requirements for ATM, which has also been reported previously (Delia et al. 2000; Fernet et al. 2004). Thus, whilst one cellular pathway that might promote tumorigenesis is altered, others may function apparently normally. The geographically confined high frequency among breast cancer patients and absence among healthy controls strongly suggests that ATM 6903insA is associated with increased risk of developing cancer. Consequently, this indicates that the ATM gene-dosage effect is sufficient to exert a cellular phenotype that promotes tumorigenesis.

The role of 8734A>G as a breast cancer susceptibility allele has been suggested by two previous studies (Teraoka et al. 2001; Thorstenson et al. 2003), and is further supported by the current results. 8734A>G leads to Arg2912Gly substitution in the catalytic kinase domain and alters a residue highly conserved between different species and also in most other members of the PIK-family kinases. However, the LCL heterozygous for this mutation showed no defects in the phosphorylation of ATM on Ser1981 or p53 on Ser15, whereas defective CHK1 Ser317 phosphorylation was observed. Thus, Arg2912Gly substitution does not impair the phosphorylation of all ATM substrates, and the cancer-predisposing effect of this mutation is not due to the dominant-negative mechanism. Nevertheless, Arg2912Gly may impair some other protein-protein interactions required for optimal ATM kinase activity. Interestingly, it has been shown that after irradiation the phosphorylation of CHK1 Ser317 by ATM is dependent on NBS1 (Gatei et al. 2003), and it has been suggested that NBS1 assists ATM in targeting some of its substrates. Consistent with this, it has been reported that phosphorylation of p53 by ATM occurs through NBS1-independent mechanism (Gatei et al. 2003; Lee & Paull 2004). Even though 8734A>G has been observed in several populations, it has not so far been reported in A-T patients. Based on the evidence from the current functional data, there might be a simple explanation for this. Although 8734A>G seems to predispose to breast cancer, it is not pathogenic enough to result in A-T clinical phenotype when paired with itself or other mutant ATM alleles. This suggests that

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functionally and possibly clinically relevant missense mutations exist beyond those identified within A-T families, which was also demonstrated in the study by Scott et al. (2002). The idea that 8734A>G might impair only NBS1-dependent ATM-mediated phosphorylation events but still lead to breast cancer predisposition, further emphasizes the essential role of the Mre11 complex in the maintenance of genomic integrity and prevention of cancer.

All in all, current functional evidence suggests that breast cancer susceptibility is not restricted to ATM mutations with dominant-negative effect on the kinase activity. This observation has been supported by two recent studies from A-T families. The study by Thompson et al. (2005) showed a moderate risk of breast cancer for A-T heterozygotes and gave some evidence for an excess risk of other cancers, but provided no support for large mutation-specific differences in risk. Additionally, another A-T study concluded that whereas the risk of breast cancer is higher in women heterozygous for pathogenic ATM alterations, it does not differ significantly according to the type of mutation. However, the risk appeared to associate with the position of some truncating mutations in certain binding domains of the protein (Cavaciuti et al. 2005).

Overall the results from study I and II indicate that ATM mutations penetrant enough to result in multiple-case breast cancer families seem to be mainly restricted to those pathogenic for A-T. However, the relative rarity of the individual A-T alleles limits their overall contribution to breast cancer susceptibility. It is likely that other functionally relevant missense mutations exist as well, but these seem to be rare variants, such as the 8734A>G, rather than common polymorphisms. For these rare variants, very large case-control studies are required to find significant association with breast cancer susceptibility. In addition, functional studies are needed in order to assess the importance of different ATM alterations as breast cancer susceptibility alleles, and to find out more about the mechanisms through which they operate. Finally, some common SNPs or the haplotypes that they represent, such as 5557G>A and IVS38-8T>C, might have some effect, at least as modifiers, on breast cancer susceptibility. It is possible that environmental factors, certain exposures such as IR, increase the risk of cancer in individuals with these modifier ATM genotypes.

6.2 ATR mutations and breast cancer susceptibility (III)

Based on its biological function, ATR is a plausible candidate gene to explain some of the remaining familial aggregation of breast cancer. The mutation analysis performed in study III was the first to investigate the possible association of germline mutations in this gene with predisposition to cancer. The results, however, suggest that ATR defects are not involved in hereditary susceptibility to breast and/or ovarian cancer. This conclusion has been supported by a recent study that screened 54 French Canadian familial breast cancer cases for ATR germline mutations and did not observe any deleterious mutations (Durocher et al. 2006). However, neither of the ATR screening studies performed can completely exclude the possibility that some of the identified rare variants might have low to moderate effect on cancer predisposition which has to be demonstrated by more extensive case-control studies. It is, however, also possible that breast and/or ovarian

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cancer is not the primary cancer phenotype associated with germline defects in ATR, as somatic mutations have so far only been reported in gastric and endometrial tumors displaying microsatellite instability (MSI). These mutations occur in the coding region of ATR in a stretch of (A)10 repeats in exon 10, leading to subsequent truncation of the protein. The MSI-associated frameshift mutations in ATR are functionally relevant as they are capable of abrogating ATR-dependent DNA damage response. Importantly, the wild-type sequence is still present in these tumors, and it has been proposed that ATR serves as a haploinsufficient tumor suppressor in the mismatch repair-deficient background (Menoyo et al. 2001; Vassileva et al. 2002; Fang et al. 2004; Lewis et al. 2005).

Altogether, the current data suggest that germline mutations in the essential ATR gene are very rare. This could reflect its fundamental role in cell viability (Abraham 2001; Shechter et al. 2004). Correspondingly, no germline mutations have ever been reported in CHK1, which is an important ATR substrate. In fact, despite the numerous players in DNA DSB repair pathway (chapter 2.4.2), thus far only a few have been associated with breast cancer susceptibility, and the identification of the common nominator for these genes would be of utmost importance.

6.3 Mutations in the Mre11 complex genes and breast cancer susceptibility (IV, V)

Given the importance of the Mre11 complex in the maintenance of genomic integrity and preventing cells from malignancy, we have evaluated the role of Mre11 complex alterations in hereditary susceptibility to breast cancer. The initial screening study IV revealed three novel, potentially pathogenic alterations: MRE11 913C>T (Arg305Trp), NBS1 448C>T (Leu150Phe) and RAD50 687delT (stop codon at 234). However, because of the types of cancer reported in the MRE11 Arg305Trp positive family, the alteration was only considered to be potentially pathogenic for gynecological cancers, which is why it was not studied further in the additional breast cancer cohort (study V). In contrast, the results of the follow-up study V indicated that NBS1 Leu150Phe and RAD50 687delT, in addition to the newly identified RAD50 IVS3-1G>A, associate with breast cancer susceptibility.

The association of NBS1 Leu150Phe with breast cancer susceptibility was indicated in study IV, where the allele was observed in a single breast cancer patient. This was supported by study V, since two more carriers were identified in a cohort of 317 consecutive, newly diagnosed breast cancer patients. The NBS1 Leu150Phe carrier frequencies were similar in both studies, approximately 0.6%, indicating that it is a rare allele. Its penetrance could not be evaluated, as only one additional DNA sample from the three Leu150Phe positive families was available for mutation testing. However, this individual, diagnosed with ovarian cancer at the age of 17, did carry the NBS1 Leu150Phe allele. Although NBS1 Leu150Phe has so far only been observed in affected individuals, its frequency is too low to reach statistical significance when compared to controls (0/1000, p=0.058). Nevertheless, its pathogenicity is supported by the increase in chromosomal instability in peripheral lymphocytes of mutation carriers studied.

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In NBS1 and MRE11 deleterious germline mutations have previously been reported in the context of NBS and ATLD, respectively (Varon et al. 1998; Stewart et al. 1999). In contrast, study IV was the first publication to suggest that germline mutations also exist in the third member of the Mre11 complex, RAD50, and moreover, possibly associate with disease susceptibility in humans. Despite the expected deleterious effect on the protein, the relatively high frequency of RAD50 687delT in healthy population controls suggested that it has incomplete penetrance. In addition, as there was no difference in the age at diagnosis between the RAD50 687delT carriers and non-carriers, it further emphasized that the consequences of RAD50 687delT differ from those of BRCA1 or BRCA2 mutations, which are more likely to cause early onset of cancer. Overall, RAD50 687delT was estimated to confer a four-fold increase in breast cancer risk (OR 4.3, 95% CI 1.5-12.5, p=0.008). Thus, the results indicate that RAD50 687delT is a new low-penetrance allele which multiplies cancer risk, possibly in cooperation with susceptibility alleles in other genes and/or environmental factors, as suggested by the polygenic model for breast cancer susceptibility (Pharoah et al. 2002). Consistent with this, in the mutation-positive families the clustering of the disease could not be explained by RAD50 687delT alone. In study IV, one of the affected RAD50 687delT carriers even showed parallel involvement of the previously identified BRCA1 3745delT truncation mutation (Huusko et al. 1998). In this family it is possible that both RAD50 and BRCA1 truncation mutations act independently on the disease phenotype. However, co-occurrence of RAD50 687delT could also strengthen the effect of the BRCA1 mutation, as double heterozygosity for defects in functionally related proteins has been suggested to have an additive effect on tumor development (Smilenov 2006 and refs. therein). Interestingly, a previous comparative genome hybridization-targeted linkage analysis has revealed a possible BRCA1 penetrance-modifier locus on 5q in a location close to RAD50 (Dolganov et al. 1996; Nathanson et al. 2002).

According to the polygenic model for breast cancer susceptibility, each of the low-penetrance alleles confers a modest independent risk (Pharoah et al. 2002). Consequently, these alleles might account for a substantial fraction of breast cancer incidence without necessarily causing evident familial clustering. This also suggests the possibility that a significant portion of seemingly sporadic cancer cases could in fact be due to genetic predisposition (Balmain et al. 2003). The results of study V indicated that the risk associated with RAD50 687delT is apparent in women unselected for family history. No difference in allele frequency between cases with (2/70) or without (6/247) family history of breast and/or ovarian cancer was observed. However, this might be at least partially due to the low number of cases considered as familial.

The screening of additional breast cancer cohorts from Nordic countries and the shared haplotype of the carriers suggest that RAD50 687delT is a Finnish founder mutation. Consistent with this, a recent study by Tommiska et al. (2006b) did not identify RAD50 687delT in 435 UK familial breast cancer patients, but did observe this allele in 3/590 Southern Finnish breast cancer families compared to 1/560 in controls. As the prevalence of RAD50 687delT in the Southern Finnish cohort was low (Tommiska et al. 2006b), it appears that this allele clusters mainly to Northern Finland. The difference in geographical distribution is also observed with other susceptibility alleles, including BRCA1 and BRCA2 mutations (Sarantaus et al. 2000).

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Next, the effect of the observed mutations on genomic instability was investigated. It seems that RAD50 687delT does not produce a stable protein product (J.Petrini, personal communication; Tommiska et al. 2006b) and the carrier LCLs show a decreased overall abundance of the full-length protein compared with normal control LCLs (Tommiska et al. 2006b). Also the RAD50 IVS3-1G>A transcript appears to be unstable at mRNA level, which indicates that the amount of RAD50 is reduced in the carriers of this allele. This dosage variation may have direct influence on DNA damage recognition and response pathway activities (Fodde & Smits 2002), which is supported by the increase in genetic instability in RAD50 687delT and RAD50 IVS3-1G>A carriers. In contrast, the exact functional consequences of NBS1 Leu150Phe are unknown, but it is possible that substitution of the evolutionarily conserved Leu150 residue is critical for BRCT domain function. This domain seems essential for the interaction between NBS1 and histone γ-H2AX after IR (Kobayashi et al. 2002), suggesting that it plays an important role in DNA damage recognition. Indeed, other changes in the conserved residues of the BRCT domain, GlyGly136-137GluGlu and Tyr176Ala, have been shown to disrupt the Mre11 complex nuclear focus formation and to block NBS1 phosphorylation after irradiation (Cerosaletti & Concannon 2003). Additionally, the NBS1 Ile171Val mutation that is pathogenic for ALL and aplastic anemia (Varon et al. 2001; Shimada et al. 2004) changes a conserved residue in the BRCT domain, and it has been associated with an increase in numerical and structural chromosomal aberrations (Shimada et al. 2004), suggesting a defect in the DNA damage response.

The studied heterozygous carriers of RAD50 687delT, RAD50 IVS3-1G>A and NBS1 Leu150Phe all showed an increase in genomic instability compared to healthy controls. Although based on a small number of individuals analyzed, the total number of chromosome aberrations observed in mutation carrier T-lymphocytes (mean 13.6, SD 8.4) was increased 7-fold compared with noncarrier healthy controls (mean 1.9, SD 1.9) (p=0.006). In particular, the number of chromosomal rearrangements was significantly increased (p=0.002). As the Mre11 complex has been implicated in multiple cellular responses to DNA damage (Stracker et al. 2004), the elevated frequency of spontaneous translocations in mutation carriers could reflect a deficiency in DNA repair. Indeed, experimental analyses have shown that DSBs are the principal lesions involved in the process of chromosomal aberration formation. Defects in the DSB repair pathways may lead to broken chromosomes and chromosome rearrangements, and this genomic instability could be related to increased mutation rates of cancer-related genes, ultimately contributing to tumorigenesis (Obe et al. 2002). Interestingly, epidemiological studies have shown that people with elevated frequency of chromosomal aberrations in their peripheral blood lymphocytes have a significantly elevated risk of developing cancer. Consequently, it has been suggested that the occurrence of chromosomal aberrations could be a relevant biomarker for cancer risk, reflecting either early biological effects of genotoxic carcinogens or individual cancer susceptibility (Bonassi et al. 2000).

The elevated spontaneous chromosomal instability in heterozygous mutation carriers suggests an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity. Indeed, it has previously been indicated that in the case of tumor suppressor genes participating in the maintenance of the genomic stability the gene-dosage effect rather than biallelic inactivation is sufficient to exert a cellular phenotype that leads to tumorigenesis. However, these genes may show differences in tissue specific protein-dosage thresholds

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below which they fail to operate normally (reviewed in Fodde & Smits 2002). For NBS1 haploinsufficiency and cancer predisposition there is supporting evidence from studies with mice. In addition to lymphomas, heterozygous NBS1 knockdown mice developed a wide array of tumors affecting the liver, mammary gland, prostate and lung. Additionally, it was demonstrated that haploinsufficiency, not LOH, was the mechanism underlying tumor development. Moreover, this study provided evidence that NBS1 heterozygosity predisposes cells to malignancy most likely because of chromosomal instability and defects in DNA repair (Dumon-Jones et al. 2003).

As the current results suggest that constitutional mutations in the Mre11 complex genes act in a haploinsufficient manner, it raises the question about the importance of inactivation of these genes in sporadic cancer cases as well. The first suggestion of reduced dosage of RAD50 at the somatic level already comes from acute myeloid leukemia (AML), since a strong correlation between AML and chromosomal aberrations involving the RAD50 containing 5q23-q31 region has been observed (Dolganov et al. 1996). However, no direct implication of RAD50 defects in AML has yet emerged. Nevertheless, it is rather interesting that the currently identified RAD50 IVS3-1G>A positive family also shows one case of leukemia, as does one family with RAD50 687delT (Tommiska et al. 2006b). In addition, the NBS1 Leu150Phe allele has been recently identified in one ovarian cancer patient, diagnosed at the age of 45, who had two first-degree relatives with leukemia (unpublished results). However, the carrier status of all these leukemia cases is currently unknown. The haploinsufficiency model might also change the interpretation of the role of NBS1 as a tumor suppressor in colorectal carcinoma. It has previously been observed that AI at NBS1 locus is frequent in proximal colorectal carcinoma (Uhrhammer et al. 2000), but as the sequencing of the coding region of NBS1 in the cases showing AI failed to show any sequence anomaly, the authors ruled out the possibility that NBS1 is the tumor suppressor contained in the region (Varon et al. 2002). However, the current results suggests that RAD50 and NBS1 do not act as classical tumor suppressor genes in which both alleles need to be inactivated for functional relevance, at least in breast tumorigenesis.

Altogether, the present results suggest that germline mutations in Mre11 complex genes contribute to some extent to hereditary breast cancer susceptibility in Northern Finland. In MRE11 the potentially pathogenic germline mutations seem very rare, and their overall contribution, if any, is low and possibly only associated with predisposition to gynecological cancers. So far pathogenic MRE11 germline mutations have only been reported in ATLD patients, whereas somatic MRE11 alterations have been suggested to have an occasional role in the development of breast cancer and in mismatch repair-deficient tumorigenesis of colorectal cancer and gastric carcinomas (Fukuda et al. 2001; Giannini et al. 2002; Giannini et al. 2004; Ottini et al. 2004). In NBS1 one potential breast cancer susceptibility allele, NBS1 Leu150Phe, with at least moderate disease penetrance was observed. However, the families displaying NBS1 Leu150Phe do not show exclusively breast cancer but also other malignancies, including gynecological and stomach cancer. This suggests that NBS1 Leu150Phe might predispose to several malignancies. Finally, based on the current results RAD50 seems to be a novel breast cancer susceptibility gene. The prevalence of RAD50 687delT, a new low-penetrance susceptibility allele, in the unselected breast cancer cohort studied was relatively high (2.5%). Consequently, an important question to resolve in the future studies is whether

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the breast tumors of RAD50 687delT carriers display any common characteristics, and whether there is an effect on the response to therapy. Analogous to NBS1 Leu150Phe, the families displaying the 687delT mutation do not exclusively show breast cancer. Thus, further studies are warranted to determine the role of RAD50 687delT as a susceptibility allele in other types of cancer as well. The identification of yet another RAD50 mutation, IVS3-1G>A, associated with genomic instability further suggests the existence of additional cancer susceptibility alleles in this evolutionarily conserved gene. These may, however, be very rare, affecting perhaps one or two families, but also population-specific. Consistent with this idea, Tommiska et al. (2006b) identified a novel RAD50 truncation mutation (Gln350Stop) in one breast cancer family originating from the UK. Consequently, the screening of the entire RAD50 gene for additional germline mutations in other populations might be beneficial.

In conclusion, the discovery of additional breast cancer susceptibility alleles, such as CHK2 1100delC and RAD50 687delT, is a challenging task, as there are probably many genes that are involved and whose contribution may vary considerably in different populations. Moreover, as the effect of different alleles is likely to vary substantially, it is important to determine the risks associated with these alterations. This might be straightforward for individual polymorphisms given sufficiently large case-control studies. Defining the risks associated with genotype combinations is, however, a more complex problem (Pharoah et al. 2004), as simple association studies fail to identify interactions unless the appropriate interacting loci have also been tested (Balmain et al. 2003). The identification of new susceptibility genes helps to understand the genetic background contributing to the etiology of breast cancer, and may ultimately lead to the identification of novel pharmaceutical targets and possibly to genetic screening in order to identify individuals at increased risk. Hopefully, this will lead to improved prevention efforts and treatment.

7 Concluding remarks

The major observations and conclusions of this study are as follows:

1. The mutation screening of the ATM gene indicated that pathogenic ATM mutations penetrant enough to result in multiple-case breast cancer families seem to be mainly restricted to those reported in A-T. However, an association of ATM 5557G>A and IVS38-8T>C combination allele, or the risk haplotype that these two SNPs represent, with bilateral breast cancer was observed (OR 10.2, 95% CI 3.1-33.8, p=0.001). Consequently, we propose a cancer modifying effect for this allele, although this still warrants further investigation.

2. Two of the seven known Finnish A-T-related ATM mutations seem to be breast cancer susceptibility alleles also outside the A-T families. ATM 6903insA was observed in 3/541 familial and 5/1124 unselected cases but not among healthy controls (0/1107), while 7570G>C (Ala2524Pro) occurred in 1/541 familial and 2/1124 unselected cases compared to 1/1107 in controls. Additionally, one ATM mutation, 8734A>G (Arg2912Gly), previously associated with breast cancer susceptibility was observed in two families. In combination with previous study (Allinen et al. 2002) ATM mutations have now been observed altogether in 1.4% (9/630) familial (p=0.0003, OR 18.9, 95% CI 2.4-149.7) and in 0.6% (7/1209) unselected Finnish breast cancer cases (p=0.03, OR 7.6, 95% CI 0.9-61.9) compared to 0.1% (1/1307) controls. This suggests an apparent, yet overall limited contribution of ATM mutations to breast cancer predisposition. However, as the observed alleles show geographical clustering, their contribution to hereditary breast cancer susceptibility in certain regions might be significant.

3. The breast cancer associated ATM mutations are not restricted to those with a dominant-negative effect on kinase activity, as ATM 6903insA, 7570G>C and 8734A>G mutations all displayed different functional consequences at protein level. Only 7570G>C acted in a dominant-negative manner, whereas 8734A>G showed a partial defect in the phosphorylation of ATM substrates and 6903insA seemed to be a null allele.

4. The analysis of ATR did not reveal any clearly pathogenic mutations, which indicates that it is not a breast cancer susceptibility gene.

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5. The mutation screening of the Mre11 complex genes revealed two novel potentially breast cancer associated mutations, NBS1 448C>T (Leu150Phe) and RAD50 687delT (stop codon at 234), in 2.0% (3/151) of the families analyzed. The study of a cohort of newly diagnosed unselected breast cancer cases indicated that RAD50 687delT is a relatively common low-penetrance susceptibility allele in Northern Finland (cases 8/317 vs. controls 6/1000, OR 4.3, 95% CI 1.5-12.5, p=0.008). NBS1 Leu150Phe was also observed in the unselected cohort (2/317), further supporting its role as breast cancer susceptibility allele. The identification of yet another RAD50 mutation, IVS3-1G>A, (1/317) suggests that RAD50 might harbor also other breast cancer related alterations. Thus the screening of the entire RAD50 gene for additional germline mutations in other populations might be beneficial.

6. The retention of the wild-type allele in the tumors of individuals carrying RAD50 687delT, NBS1 Leu150Phe and RAD50 IVS3-1G>A mutations suggests that RAD50 and NBS1 do not act as classical tumor suppressor genes in breast tumorigenesis. Instead, the increase in chromosomal aberrations in peripheral blood lymphocytes suggests an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity.

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Original papers

I Heikkinen K, Rapakko K, Karppinen S-M, Erkko H, Nieminen P & Winqvist R (2005) Association of common ATM polymorphism with bilateral breast cancer. Int J Cancer 116: 69-72.

II Pylkäs K, Tommiska J, Syrjäkoski K, Kere J, Gatei M, Waddell N, Allinen M, Karppinen S-M, Rapakko K, Kääriäinen H, Aittomäki K, Blomqvist C, Mustonen A, Holli K, Khanna KK, Kallioniemi O-P, Nevanlinna H & Winqvist R (2006) Evaluation of the role of Finnish ataxia-telangiectasia mutations in hereditary predisposition to breast cancer. Carcinogenesis, Dec 13 [epub ahead of print].

III Heikkinen K, Mansikka V, Karppinen S-M, Rapakko K & Winqvist R (2005) Mutation analysis of the ATR gene in breast and ovarian cancer families. Breast Cancer Res 7: R495-R501.

IV Heikkinen K, Karppinen S-M, Soini Y, Mäkinen M & Winqvist R (2003) Mutation screening of Mre11 complex genes: indication of RAD50 involvement in breast and ovarian cancer susceptibility. J Med Genet 40: e131.

V Heikkinen K, Rapakko K, Karppinen S-M, Erkko H, Knuutila S, Lundán T, Mannermaa A, Børresen-Dale A-L, Borg Å, Barkardóttir RB, Petrini J & Winqvist R (2006) RAD50 and NBS1 are breast cancer susceptibility genes associated with genomic instability. Carcinogenesis 27: 1593-1599.

The permission of Wiley InterScience (I), BioMed Central Ltd (III), BMJ Publishing Group Ltd (IV) and Oxford University Press (II, V) to reprint the original papers is gratefully acknowledged.

Original publications are not included in the electronic version of the dissertation.

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