Fu Zhenming. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 138-162). / Abstracts in English and Chinese. / List of abbreviations --- p.i / Abstract (in English) --- p.ii / 摘要（中文） --- p.iii / Acknowledgement --- p.iv / Chapter Chapter 1. --- Introduction and Objectives of Study --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma in general --- p.2 / Chapter 1.1.1 --- "Epidemiology, risk factors" --- p.2 / Chapter 1.1.2 --- Pathology and staging --- p.4 / Chapter 1.1.3 --- Treatment --- p.6 / Chapter 1.1.4 --- Improvement of early detection and treatment of HCC --- p.7 / Chapter 1.2 --- General aspects of mitochondria and mitochondrial DNA (mtDNA) --- p.10 / Chapter 1.2.1 --- Structure and dynamics of mitochondria --- p.10 / Chapter 1.2.1.1 --- General introduction of mitochondria --- p.10 / Chapter 1.2.1.2 --- Respiration chain of mitochondria --- p.11 / Chapter 1.2.2 --- The mitochondrial genome --- p.14 / Chapter 1.2.2.1 --- Strucure --- p.14 / Chapter 1.2.2.2 --- Genes for structure proteins --- p.16 / Chapter 1.2.2.3 --- Genes for translation --- p.17 / Chapter 1.2.2.4 --- Imported proteins and RNAs --- p.17 / Chapter 1.2.3 --- Mitochondrial DNA maintenance --- p.19 / Chapter 1.2.4 --- Mitochondrial DNA replication --- p.25 / Chapter 1.2.5 --- Mitochondrial DNA transcription --- p.30 / Chapter 1.2.6 --- Mitochondrial DNA translation --- p.32 / Chapter 1.3 --- MtDNA diseases --- p.35 / Chapter 1.4 --- MtDNA mutation and HCC --- p.35 / Chapter 1.5 --- Aims of the study --- p.39 / Chapter Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Chemicals --- p.42 / Chapter 2.1.2 --- Primers --- p.42 / Chapter 2.1.3 --- Enzymes --- p.45 / Chapter 2.1.4 --- Cell line --- p.45 / Chapter 2.1.5 --- Collection of specimens --- p.46 / Chapter 2.2 --- Methodology --- p.47 / Chapter 2.2.1 --- "DNA extraction from hcc tissues, cell line Hep3B and PBMCs" --- p.47 / Chapter 2.2.1.1 --- DNA extraction from HCC tissues --- p.47 / Chapter 2.2.1.2 --- DNA extraction from cell line Hep3B --- p.49 / Chapter 2.2.1.3 --- DNA extraction from and PBMCs --- p.50 / Chapter 2.2.1.3.1 --- Preparation of PBMCs --- p.50 / Chapter 2.2.1.3.2 --- DNA extraction from and PBMCs --- p.51 / Chapter 2.2.2 --- Detection of mt whole genome mutation by direct sequencing --- p.51 / Chapter 2.2.2.1 --- Design of mtDNA primers --- p.51 / Chapter 2.2.2.2 --- PCR amplification of the whole mt genome --- p.51 / Chapter 2.2.2.3 --- Direct sequencing of the whole mt genome --- p.52 / Chapter 2.2.2.3.1 --- Primer used in sequencing --- p.52 / Chapter 2.2.2.3.2 --- Purification of the PCR products of the whole mt genome --- p.53 / Chapter 2.2.2.3.3 --- Dye terminator cycle sequencing reaction --- p.53 / Chapter 2.2.2.3.4 --- Purification of extension products --- p.54 / Chapter 2.2.3 --- Detection of mtDNA control region mutation --- p.55 / Chapter 2.2.3.1 --- PCR amplification of D310 in the mtDNA control region --- p.55 / Chapter 2.2.3.2 --- Screening of D310 mutation by PFLDA --- p.55 / Chapter 2.2.3.2.1 --- Making 8% denatured gel mixture --- p.55 / Chapter 2.2.3.2.2 --- Setting up and Pouring the denatured gel --- p.56 / Chapter 2.2.3.2.4 --- Preparing and Loading the PCR products --- p.57 / Chapter 2.2.3.2.5 --- Electrophoresis --- p.57 / Chapter 2.2.3.2.6 --- "Gel fixing, silver staining and color development " --- p.58 / Chapter 2.2.3.3 --- Direct sequencing of D310 in the mtDNA control region --- p.59 / Chapter 2.2.4 --- Detection of mt DNA coding region mutation --- p.60 / Chapter 2.2.4.1 --- PCR amplification of the 5 respiratory chain subunit genes --- p.60 / Chapter 2.2.4.2 --- Restriction enzyme digestion of 5 genes in mtDNA coding region --- p.60 / Chapter 2.2.4.3 --- Screening of mtDNA coding region mutation by SSCP --- p.61 / Chapter 2.2.4.3.1 --- Making 6% 49:1 acrylamide/Bis SSCP gel mixture --- p.61 / Chapter 2.2.4.3.2 --- "Setting up the SSCP gel, loading sample, fixing, staining and developing of the gel " --- p.62 / Chapter 2.2.4.4 --- Sequencing conformation of the mtDNA coding region mutation --- p.62 / Chapter 2.2.5 --- Statistics --- p.63 / Chapter 2.2.5.1 --- The chi-square test --- p.63 / Chapter 2.2.5.2 --- The Friedman test --- p.63 / Chapter 2.2.5.3 --- Wilcoxon signed ranks test --- p.63 / Chapter Chapter 3. --- Results --- p.64 / Chapter 3.1 --- Detection mt DNA whole genome mutation --- p.65 / Chapter 3.1.1 --- Identification of mtDNA whole genome by direct sequencing --- p.65 / Chapter 3.2 --- Detection mt DNA D-loop mutation --- p.76 / Chapter 3.2.1 --- Screening of C-tract alteration in HCC tissus by PCR fragments length detection assay (PFLDA) --- p.76 / Chapter 3.2.2 --- Screening of coding region alteration in HCC tissues by SSCP --- p.77 / Chapter 3.2.2.1 --- Identification of C-tract alterations in HCC and non-tumorous tissues by direct sequencing --- p.77 / Chapter 3.2.3 --- Identification of C-tract alterations by direct sequencing --- p.82 / Chapter 3.2.3.1 --- Identification of C-tract alterations in HCC tissues by direct sequencing --- p.82 / Chapter 3.2.3.2 --- Identification of C-tract alteration in PBMC of normal subjects by direct sequencing --- p.82 / Chapter 3.2.3.3 --- Identification of C-tract alteration in PBMC of HCC patients by direct sequencing --- p.82 / Chapter 3.2.4 --- Statistics of the analysis of C-tract alterations --- p.82 / Chapter 3.3 --- Detection mt DNA mutation in the coding region --- p.87 / Chapter Chapter 4. --- Discussion --- p.98 / Chapter 4.1 --- Detection mtDNA whole genome mutation --- p.99 / Chapter 4.2 --- Detection mtDNA D-loop mutation --- p.107 / Chapter 4.3 --- Detection mtDNA mutation in the coding region --- p.119 / Chapter 4.4 --- Possible mechanisms of mtDNA mutation in HCC carcinogenesis --- p.125 / Chapter 4.5 --- Proposals for prospective studies --- p.126 / Chapter 4.5.1 --- Function of C7 in D310 --- p.128 / Chapter 4.5.2 --- Function changes of mtDNA coding region mutation --- p.130 / Chapter 4.5.3 --- Detection of D310 C-tract mutation in patients' plasma --- p.131 / Chapter 4.5.4 --- Relationship between nMSl and mtMSI --- p.132 / Chapter 4.6 --- Summary --- p.134 / References --- p.137

CCCTC-binding factor (CTCF) binds DNA, thereby helping to partition the mammalian genome into discrete structural and regulatory domains. In doing so, it insulates chromatin and fine-tunes gene activation, repression, and silencing. Complete removal of CTCF from mammalian cells causes catastrophic genomic dysregulation, most likely due to widespread collapse of 3D chromatin looping within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction in CTCF expression are viable but have an increased incidence of spontaneous multi-lineage malignancies. In addition, CTCF is mutated in many human cancers and is thus implicated as a tumour suppressor gene. This study aimed to interrogate the genome-wide consequences of a reduced genomic concentration of Ctcf and its implications for carcinogenesis. In a genetically engineered mouse model, Ctcf hemizygous cells showed modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these were enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, several hundred genes concentrated in cancer-related pathways were dysregulated due to changes in transcriptional regulation. Global chromatin structure was preserved but some loop interactions were destabilised, often around differentially expressed genes and their enhancers. Importantly, these transcriptional alterations were also seen in human cancers. These findings were then examined in a hepatocyte-specific mouse model of Ctcf hemizygosity with diethylnitrosamine-induced liver tumours. Ctcf hemizygous mice had a subtle liver-specific phenotype, although the overall tumour burden in Ctcf hemizygous and wild-type mice was the same. Using whole genome sequencing, the highly reproducible mutational signature caused by DEN exposure was characterised, revealing that Braf(V637E), orthologous to BRAF(V600E) in humans, was the predominant oncogenic driver in these liver tumours. Taken together, while Ctcf loss is partially physiologically compensated, chronic CTCF depletion dysregulates gene expression by subtly altering transcriptional regulation. This study also represents the first comprehensive genome-wide and histopathological characterisation of this commonly used liver cancer model.

Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 4 uppsatser.

Cancer development is driven by somatic genome alterations, ranging from single point mutations to larger structural variants (SV) affecting kilobases to megabases of one or more chromosomes. Studies of somatic rearrangement have previously been limited by a paucity of whole genome sequencing data, and a lack of methods for comprehensive structural classification and downstream analysis. The ICGC project on the Pan-Cancer Analysis of Whole Genomes provides an unprecedented opportunity to analyse somatic SVs at base-pair resolution in more than 2500 samples from 30 common cancer types. In this thesis, I build on a recently developed SV classification pipeline to present a census of rearrangement across the pan-cancer cohort, including chromoplexy, replicative two-jumps, and templated insertions connecting as many as eight distant loci. By identifying the precise structure of individual breakpoint junctions and separating out complex clusters, the classification scheme empowers detailed exploration of all simple SV properties and signatures. After illustrating the various SV classes and their frequency across cancer types and samples, Chapter 2 focuses on structural properties including event size and breakpoint homology. Then, in Chapter 3, I consider the SV distribution across the genome, and show patterns of association with various genome properties. Upon examination of rearrangement hotspot loci, I describe tissue-specific fragile site deletion patterns, and a variety of SV profiles around known cancer genes, including recurrent templated insertion cycles affecting TERT and RB1. Turning to co-occurring alteration patterns, Chapter 4 introduces the Hierarchical Dirichlet Process as a non-parametric Bayesian model of mutational signatures. After developing methods for consensus signature extraction, I detour to the domain of single nucleotide variants to test the HDP method on real and simulated data, and to illustrate its utility for simultaneous signature discovery and matching. Finally, I return to the PCAWG SV dataset, and extract SV signatures delineated by structural class, size, and replication timing. In Chapter 5, I move on to the complex SV clusters (largely set aside throughout Chapters 2—4) , and develop an improved breakpoint clustering method to subdivide the complex rearrangement landscape. I propose a raft of summary metrics for groups of five or more breakpoint junctions, and explore their utility for preliminary classification of chromothripsis and other complex phenomena. This comprehensive study of somatic genome rearrangement provides detailed insight into SV patterns and properties across event classes, genome regions, samples, and cancer types. To extrapolate from the progress made in this thesis, Chapter 6 suggests future strategies for addressing unanswered questions about complex SV mechanisms, annotation of functional consequences, and selection analysis to discover novel drivers of the cancer phenotype.

As neoplasias mieloproliferativas (NMPs) BCR-ABL1 negativas compreendem a mielofibrose primária (PMF), trombocitemia essencial (TE) e a policitemia vera (PV). A patogênese e progressão dessas NMPs não estão completamente elucidadas. As metaloproteinases de matriz (MMPs) degradam a matriz extracelular, ativando citocinas e fatores de crescimento que, por sua vez, participam da tumorigênese e angiogênese. O objetivo deste estudo foi avaliar a relação da expressão gênica das MMPs, TIMPs, HIF1-&#945; e SPARC com os marcadores angiogênicos bFGF e VEGFA em pacientes com MF e TE, considerando o status mutacional; bem como avaliar a regulação desses genes em camundongos submetidos à hipóxia, e em modelos HIF1-&#945;(-/-) e VHL(-/-). Foram incluídos 21 pacientes com MF, 21 com MF pós-TE, 6 com MF pós-PV, 23 com TE e 78 indivíduos controle. As análises realizadas foram: dosagem sérica e expressão de RNAm de MMP2, MMP9, TIMP1, TIMP2 e SPARC, hemograma, determinação da proteína C reativa ultrassensível, determinação das concentrações de VEGFA e bFGF e avaliação das mutações nos genes JAK2, cMPL e CALR. A avaliação da densidade microvascular da medula óssea foi feita em 30 dos pacientes incluídos. Os pacientes com MFP, MFPTE e TE apresentaram maior expressão de MMP2, SPARC, TIMP1, TIMP2 e bFGF quando comparados aos seus controles (P<0,05), enquanto MMP9 foi mais expressa nos pacientes com MFPTE e TE (P= 0,011 e P=0,047, respectivamente). Os pacientes com TE apresentaram maior expressão de HIF1-&#945; e VEGFA em relação ao grupo controle (P<0,05). Pacientes com MF JAK2V617F positivos apresentaram maiores concentrações de MMP9, TIMP2, bFGF e VEGFA quando comparados aos pacientes portadores de mutações na CALR (P<0,05). Os pacientes com TE JAK2V617F positivos apresentaram maiores concentrações de MMP2 e TIMP2 (P=0,049 e P=0,020, respectivamente). As concentrações das proteínas estudadas não apresentaram correlação com a carga alélica de JAK2V617F e nem com a densidade microvascular da medula óssea. Células de medula óssea de camundongos submetidos à hipóxia apresentaram maior expressão de MMP2 e TIMP1 comparados aos camundongos em normóxia. Camundongos VHL(-/-) apresentaram aumento na expressão dos genes MMP2, MMP9, TIMP1, TIMP2 e VEGFA. Diferentemente, embriões HIF1-&#945;(-/-) não foram considerados um bom modelo para este estudo devido ao envolvimento das MMPs na embriogênese/organogênese. Frente aos resultados encontrados, pode-se sugerir que a maior expressão de MMP2, SPARC e de bFGF estão associadas às NMPs. A mutação JAK2V617F foi associada a maiores concentrações de MMPs, TIMP2 VEGFA e bFGF. HIF1-&#945; foi mais expresso na PV e na TE, sugerindo uma possível regulação da expressão das MMPs e TIMPs nessas doenças. / Myeloproliferative neoplasms (MPNs) BCR-ABL1-negative include primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV). The mechanisms underlying the pathology and disease progression in MPN are not completely elucidated. The matrix metalloproteinases (MMPs) cleave extracellular matrix, activating cytokines and growth factors that, in turn, regulate tumorigenesis and angiogenesis. The aim of this study was to evaluate the relationship of MMPs, TIMPs, HIF1-&#945 and SPARC gene expression with angiogenic markers bFGF and VEGFA in patients with MPN considering their mutational status; as well as to assess the regulation of these genes in animal models HIF1-&#945 and VHL knockouts. Twenty-one MF, 21 MF post-ET, 6 MF post-PV, 23 ET patients and 78 controls were enrolled. The analysis performed in peripheral blood were: serum and mRNA expression of MMP2, MMP9, TIMP1, TIMP2 and SPARC, blood count, high-sensitivity C-reactive protein determination and VEGFA and bFGF measurements in plasma. We also evaluate mutations in JAK2, MPL and CALR. The assessment of microvascular density (MVD) in bone marrow was performed in 30 patients. Patients with MFP, MFPET and ET presented higher expression of MMP2, SPARC, TIMP1, TIMP2 and bFGF compared to their controls (P <0.05), while MMP9 expression was higher in patients with MFPET and ET (P=0.011 and P=0.047, respectively). Higher expression of HIF1-&#945; and VEGFA was found in ET patients compared to the controls (P <0.05). PMF JAK2V617F patients had higher concentrations of MMP9, TIMP2, bFGF and VEGFA compared to CALR mutated ones (P <0.05). ET patients JAK2V617F positive had higher levels of MMP2 and TIMP2 (P=0.049 and P=0.020, respectively). The JAK2V617F allele burden was not associated with MVD in the bone marrow. Bone marrow cells from mice in hypoxia condition showed higher MMP2 and TIMP1 expression compared to the control. VHL(-/-) mice exhibited increased expression of MMP2, MMP9, TIMP1, TIMP2 and VEGFA. In contrast, the HIF1-&#945;(-/-) embryos were not considered an applicable model for this study due to MMPs role in embryogenesis/organogenesis. In view of these findings, we can conclude that increased expression of MMP2, SPARC and bFGF are associated with MPN. The JAK2V617F mutation was associated with higher concentrations of MMPs, TIMP2 VEGFA and bFGF. HIF1-&#945; is upregulated in PV and ET and perhaps regulate the MMPs and TIMPs expression in these diseases.

Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

Abstract Approximately 5–10% of all breast cancer cases are estimated to result from a hereditary predisposition to the disease. Currently no more than 25–30% of these familial cases can be explained by mutations in the known susceptibility genes, BRCA1 and BRCA2 being the major ones. Additional predisposing genes are therefore likely to be discovered. This study evaluates whether germline alterations in three BRCA1-associated genes, BACH1 (i.e. BRIP1/FANCJ), BARD1 and TOPBP1, contribute to familial breast cancer. Altogether 214 Finnish patients having breast and/or ovarian cancer were analysed for germline mutations in the BACH1 gene. Nine alterations were observed, four of which located in the protein-encoding region. The previously unidentified Pro1034Leu was considered a possible cancer-associated alteration as it appeared with two-fold higher frequency among cancer cases compared to controls. All the other observed alterations were classified as harmless polymorphisms. Mutation analysis of the BARD1 gene among 126 Finnish patients having family history of breast and/or ovarian cancer revealed seven alterations in the protein-encoding region. The Cys557Ser alteration was seen at an elevated frequency among familial cancer cases compared to controls (p = 0.005, odds ratio [OR] 4.2, 95% confidence interval [CI] 1.7–10.7). The other alterations appeared to be harmless polymorphisms. To evaluate further the possible effect of Cys557Ser on cancer risk, a large case-control study was performed, consisting of 3,956 cancer patients from the Nordic countries. The highest prevalence of Cys557Ser was found among breast and ovarian cancer patients from BRCA1/BRCA2 mutation-negative families (p &lt; 0.001, OR 2.6, 95% CI 1.7–4.0). In contrast, no significant association with male breast cancer, ovarian, colorectal or prostate cancer was observed. The current study is the first evaluating the role of TOPBP1 mutations in familial cancer predisposition. The analysis of 125 Finnish patients having breast and/or ovarian cancer revealed one putative pathogenic alteration. The commonly occurring Arg309Cys allele was observed at a significantly higher frequency among familial cancer cases compared to controls (p = 0.002, OR 2.4, 95% CI 1.3–4.2). The other 18 alterations observed were classified as harmless polymorphisms.

Abstract In Western countries, breast and ovarian cancer are among the most frequent malignancies affecting women. Approximately 5–10% of the cases in the general population have been suggested to be attributed to inherited disease susceptibility. BRCA1 and BRCA2 are the main genes associated with predisposition to breast and ovarian cancer. Mutations in these two genes explain a major part of the families displaying a large number of early-onset breast and/or ovarian cancers, but at least one third of the cases appear to be influenced by other, as yet unidentified genes. Therefore, it is likely that defects in other cancer predisposing genes, perhaps associated with lower disease penetrance and action in a polygenic context, will also be discovered. In the present study, the contribution of germline mutations in putative breast and/or ovarian cancer susceptibility genes, based on their biological function, has been investigated in Finnish breast cancer families. The role of large genomic deletions or other rearrangements in the BRCA1 and BRCA2 genes was evaluated by Southern blot analysis, and mutation analysis of TP53, RAD51, the BRC repeats of BRCA2, and 53BP1 was performed by conformation sensitive gel electrophoresis and DNA sequencing. Germline TP53 mutations were searched for in 108 Finnish breast cancer families without BRCA1 or BRCA2 alterations. In this study, the pathogenic TP53 germline mutation, Arg248Gln, was identified in only one family. This family showed a strong family history of breast cancer and other cancers also fulfilling the criteria for Li-Fraumeni-like syndrome. Germline TP53 mutations are expected to be found in cancer families with clinical features seen in Li-Fraumeni or Li-Fraumeni-like syndromes. In this study, large deletions in BRCA1 and BRCA2 were not observed in 82 breast and/or ovarian cancer families. Likewise, no disease-related aberrations were detected in RAD51, the BRC repeats of BRCA2 or 53BP1 in the 126 breast and/or ovarian cancer families studied. The obtained results were validated by comparing to the occurrence in 288–300 female cancer-free control individuals. These results do not support the hypothesis that alterations in these particular genomic regions play a significant role in breast cancer predisposition in Finland. Thus, there are still genes to be discovered to explain the molecular background of breast cancer.