京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22341号 / 医博第4582号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 羽賀 博典, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Aproximadamente 4% das pacientes com câncer de mama têm o diagnóstico abaixo dos 36 anos. Mutação germinativa nos genes BRCA1 ou BRCA2 pode ser um dos fatores de predisposição e a identificação de pacientes carreadoras pode permitir o direcionamento do tratamento e o aconselhamento familiar para medidas de prevenção e detecção precoce. Entretanto, a maioria das pacientes apresenta câncer esporádico e fatores predisponentes são menos evidentes. Para estas pacientes, a identificação de mutações somáticas pode permitir a melhor compreensão da biologia da doença e o desenvolvimento de tratamentos dirigidos a alvos moleculares. Assim, nossos objetivos foram avaliar em pacientes jovens com câncer de mama: história familiar, hábitos de vida, mutação germinativa nos genes BRCA1 e BRCA2 e mutações somáticas no tumor. Oitenta e três pacientes diagnosticadas com câncer de mama entre 18-35 anos foram entrevistadas usando um questionário. O DNA foi extraído do sangue periférico e toda região codificadora dos genes BRCA1/2 foi sequenciada pelo método de sequenciamento de Sanger e a pesquisa de grandes deleções e inserções foi realizada por Amplificação Dependente da Ligação de Múltiplas Sondas (MLPA). Os resultados foram analisados utilizando-se os programas Mutation Surveyor v.3.20 e Chromas version 2.13 e as mutações foram caracterizadas utilizando-se os bancos de dados BIC, LOVD, LOVD-IARC, UMD e ClinVar. O sequenciamento completo do exoma foi realizado em amostras de sangue e tumor fresco congelado de oito pacientes (receptor hormonal positivo, HER2 negativo e BRCA1/2 tipo selvagem) usando Nextera Rapid Capture Enrichment e sequenciadas no Illumina HiSeq 1000. Para detecção de alterações somáticas provenientes do sequenciamento completo do exoma, foi utilizado o programa SomaticSniper (v1.0.2). Foi também analisada a presença de mutação somática no gene PIK3CA em amostras tumorais em blocos de parafina de pacientes jovens e pacientes mais idosas com câncer de mama. Além disso, foi realizada uma meta análise para avaliar se mutação somática nos cinco genes mais frequentemente mutados em câncer de mama estão associados à idade precoce. A idade mediana das 83 pacientes ao diagnóstico foi 32 anos (22-35 anos); idade mediana da menarca foi 13 anos (8-19 anos); 71,1% já passaram por pelo menos uma gestação a termo com idade mediana de 22 anos (14-34 anos); 80,7% nunca fumaram; 74,7% não ingerem bebida alcoólica regularmente; a média para índice de massa corpórea foi 25,26 (10,78-41,57). A maioria das pacientes teve diagnóstico de carcinoma ductal invasivo (89,3%), grau histológico III (49,4%), subtipo luminal (65,9%) e com expressão de Ki67 >14% (89,2%). Aproximadamente 52% das pacientes relataram história familiar para câncer de mama, ovário ou próstata. Mutações deletérias nos genes BRCA1/2 foram detectadas em 13 pacientes (BRCA1, n= 4 e BRCA2, n= 9). Uma mutação nova, que gera um códon de terminação (c.483T>A; p.Cys161Ter), foi detectada no exon 6 do gene BRCA2. O sequenciamento do exoma foi realizado em amostras de oito pacientes carreadoras de BRCA1/2 tipo selvagem e tumor subtipo luminal e revelou uma média de 39 alterações somáticas por tumor (variando entre 19-74). Foram observadas alterações com potencial driver em 53 genes, como PIK3CA, PRKD1, PRKAR1A e PIK3AP1, que codificam proteínas tirosina quinase ou proteínas envolvidas na regulação de proteínas quinases; e no gene POLD1, que codifica a subunidade catalítica da polimerase delta, com papel na replicação e reparo do DNA. Em uma meta análise observamos que a mutação somática no gene PIK3CA é relativamente frequente em pacientes jovens e idosas. Concluindo, mais de 15% das pacientes jovens são carreadoras de mutação deletéria nos genes BRCA1 ou BRCA2. Em pacientes com câncer esporádico foram encontradas mutações somáticas em genes que codificam proteínas tirosina quinase ou proteínas envolvidas em reparo de DNA. Em consonância com o observado em tumores de pacientes mais idosas, mutação somática no gene PIK3CA é relativamente frequente em tumores de pacientes jovens / Approximately 4% of the breast cancer patients have their diagnosis under the age of 36 years and germline mutations in BRCA1 or BRCA2 genes is one of the predisposing factors. Identification of patients who are mutation carriers may contribute for the adoption of targeted therapies and for genetic counseling of their family members for early detection or preventive measures. However, most patients present with sporadic cancer where predisposing factors are less understood. In the latter group of patients, the identification of somatic mutations may contribute to a better understanding of the biology of the disease and to the development of molecular targeted therapies. Therefore, our goals were to characterize early onset breast cancer patients for family history, lifestyle, germline mutations in BRCA1 and BRCA2 genes and somatic mutations. Eighty-three breast cancer patients aged 18-35 years were interviewed using a questionnaire. DNA was extracted from peripheral blood and all coding regions of BRCA1/2 genes were screened by Sanger sequencing and large deletions and insertions were examined by Multiplex Ligation-Dependent Probe Amplification (MLPA). Results were analyzed through Mutation Surveyor v.3.20 and Chromas version 2.13 and searched in BIC Database, LOVD, LOVD-IARC, UMD and ClinVar. Whole exome sequencing was performed in blood and fresh-frozen tumor samples from eight patients (hormone receptor positive, HER2 negative and BRCA1/2 wild type) using Nextera Rapid Capture Enrichment in an Illumina HiSeq 1000. To detect somatic SNVs from whole exome sequencing data, SomaticSniper (v1.0.2) was used. Somatic mutation in PIK3CA gene was then searched in Formaldehyde Fixed-Paraffin Embedded samples from another group of young and older breast cancer patients. In addition, a metaanalysis was performed to evaluate whether somatic mutations in the five genes most frequently mutated in breast cancer patients were associated with an early age onset. Median age of 83 patients at diagnosis was 32 years (22-35y), median age of menarche was 13 years (8-19y); 71.1% patients had at least one born child, median age of first pregnancy was 22 years (14-34y); 80.7% were never smokers; 74.7% reported no regular alcoholic drinking; median body mass index was 25.26 (10.78-41.57). Most patients presented with invasive ductal carcinoma (89.3%), histological grade III (49.4%), luminal subtype (65.9%) and Ki67 expression > 14% (89.2). Approximately 52% of the patients reported a positive family history for breast, ovarian or prostate cancer. Deleterious mutations in BRCA1/2 genes ware detected in 13 patients (BRCA1, n= 4 and BRCA2, n= 9). One novel mutation was detected in BRCA1 gene: a stop codon in exon 6 (c.483T > A; p.Cys161Ter). Exome sequencing was evaluated in eight luminal tumors fromBRCA1/2 wild type patients and a median of 39 somatic alterations/tumor was detected, varying from 17 to 74. Potential driver alterations were detected in 53 genes, such as PIK3CA, PRKD1, PRKAR1A and PIK3AP1, that encode tyrosine kinase proteins or proteins involved in tyrosine kinases regulation; and POLD1 gene, that encodes the catalytic subunit of DNA polymerase delta which plays a critical role in DNA replication and repair. A Meta-analysis showed that somatic mutation in PIK3CA gene was frequently detected in both young and older patients. In conclusion, more than 15% of the young breast cancer patients are BRCA1 or BRCA2 mutation carriers. In patients with sporadic cancer somatic mutations were found in genes that encode tyrosine kinase proteins or are involved in the DNA repair. In agreement with what was observed in tumors from older patients, somatic mutation in PIK3CA gene is relatively frequent in tumors from young patients

Aproximadamente 4% das pacientes com câncer de mama têm o diagnóstico abaixo dos 36 anos. Mutação germinativa nos genes BRCA1 ou BRCA2 pode ser um dos fatores de predisposição e a identificação de pacientes carreadoras pode permitir o direcionamento do tratamento e o aconselhamento familiar para medidas de prevenção e detecção precoce. Entretanto, a maioria das pacientes apresenta câncer esporádico e fatores predisponentes são menos evidentes. Para estas pacientes, a identificação de mutações somáticas pode permitir a melhor compreensão da biologia da doença e o desenvolvimento de tratamentos dirigidos a alvos moleculares. Assim, nossos objetivos foram avaliar em pacientes jovens com câncer de mama: história familiar, hábitos de vida, mutação germinativa nos genes BRCA1 e BRCA2 e mutações somáticas no tumor. Oitenta e três pacientes diagnosticadas com câncer de mama entre 18-35 anos foram entrevistadas usando um questionário. O DNA foi extraído do sangue periférico e toda região codificadora dos genes BRCA1/2 foi sequenciada pelo método de sequenciamento de Sanger e a pesquisa de grandes deleções e inserções foi realizada por Amplificação Dependente da Ligação de Múltiplas Sondas (MLPA). Os resultados foram analisados utilizando-se os programas Mutation Surveyor v.3.20 e Chromas version 2.13 e as mutações foram caracterizadas utilizando-se os bancos de dados BIC, LOVD, LOVD-IARC, UMD e ClinVar. O sequenciamento completo do exoma foi realizado em amostras de sangue e tumor fresco congelado de oito pacientes (receptor hormonal positivo, HER2 negativo e BRCA1/2 tipo selvagem) usando Nextera Rapid Capture Enrichment e sequenciadas no Illumina HiSeq 1000. Para detecção de alterações somáticas provenientes do sequenciamento completo do exoma, foi utilizado o programa SomaticSniper (v1.0.2). Foi também analisada a presença de mutação somática no gene PIK3CA em amostras tumorais em blocos de parafina de pacientes jovens e pacientes mais idosas com câncer de mama. Além disso, foi realizada uma meta análise para avaliar se mutação somática nos cinco genes mais frequentemente mutados em câncer de mama estão associados à idade precoce. A idade mediana das 83 pacientes ao diagnóstico foi 32 anos (22-35 anos); idade mediana da menarca foi 13 anos (8-19 anos); 71,1% já passaram por pelo menos uma gestação a termo com idade mediana de 22 anos (14-34 anos); 80,7% nunca fumaram; 74,7% não ingerem bebida alcoólica regularmente; a média para índice de massa corpórea foi 25,26 (10,78-41,57). A maioria das pacientes teve diagnóstico de carcinoma ductal invasivo (89,3%), grau histológico III (49,4%), subtipo luminal (65,9%) e com expressão de Ki67 >14% (89,2%). Aproximadamente 52% das pacientes relataram história familiar para câncer de mama, ovário ou próstata. Mutações deletérias nos genes BRCA1/2 foram detectadas em 13 pacientes (BRCA1, n= 4 e BRCA2, n= 9). Uma mutação nova, que gera um códon de terminação (c.483T>A; p.Cys161Ter), foi detectada no exon 6 do gene BRCA2. O sequenciamento do exoma foi realizado em amostras de oito pacientes carreadoras de BRCA1/2 tipo selvagem e tumor subtipo luminal e revelou uma média de 39 alterações somáticas por tumor (variando entre 19-74). Foram observadas alterações com potencial driver em 53 genes, como PIK3CA, PRKD1, PRKAR1A e PIK3AP1, que codificam proteínas tirosina quinase ou proteínas envolvidas na regulação de proteínas quinases; e no gene POLD1, que codifica a subunidade catalítica da polimerase delta, com papel na replicação e reparo do DNA. Em uma meta análise observamos que a mutação somática no gene PIK3CA é relativamente frequente em pacientes jovens e idosas. Concluindo, mais de 15% das pacientes jovens são carreadoras de mutação deletéria nos genes BRCA1 ou BRCA2. Em pacientes com câncer esporádico foram encontradas mutações somáticas em genes que codificam proteínas tirosina quinase ou proteínas envolvidas em reparo de DNA. Em consonância com o observado em tumores de pacientes mais idosas, mutação somática no gene PIK3CA é relativamente frequente em tumores de pacientes jovens / Approximately 4% of the breast cancer patients have their diagnosis under the age of 36 years and germline mutations in BRCA1 or BRCA2 genes is one of the predisposing factors. Identification of patients who are mutation carriers may contribute for the adoption of targeted therapies and for genetic counseling of their family members for early detection or preventive measures. However, most patients present with sporadic cancer where predisposing factors are less understood. In the latter group of patients, the identification of somatic mutations may contribute to a better understanding of the biology of the disease and to the development of molecular targeted therapies. Therefore, our goals were to characterize early onset breast cancer patients for family history, lifestyle, germline mutations in BRCA1 and BRCA2 genes and somatic mutations. Eighty-three breast cancer patients aged 18-35 years were interviewed using a questionnaire. DNA was extracted from peripheral blood and all coding regions of BRCA1/2 genes were screened by Sanger sequencing and large deletions and insertions were examined by Multiplex Ligation-Dependent Probe Amplification (MLPA). Results were analyzed through Mutation Surveyor v.3.20 and Chromas version 2.13 and searched in BIC Database, LOVD, LOVD-IARC, UMD and ClinVar. Whole exome sequencing was performed in blood and fresh-frozen tumor samples from eight patients (hormone receptor positive, HER2 negative and BRCA1/2 wild type) using Nextera Rapid Capture Enrichment in an Illumina HiSeq 1000. To detect somatic SNVs from whole exome sequencing data, SomaticSniper (v1.0.2) was used. Somatic mutation in PIK3CA gene was then searched in Formaldehyde Fixed-Paraffin Embedded samples from another group of young and older breast cancer patients. In addition, a metaanalysis was performed to evaluate whether somatic mutations in the five genes most frequently mutated in breast cancer patients were associated with an early age onset. Median age of 83 patients at diagnosis was 32 years (22-35y), median age of menarche was 13 years (8-19y); 71.1% patients had at least one born child, median age of first pregnancy was 22 years (14-34y); 80.7% were never smokers; 74.7% reported no regular alcoholic drinking; median body mass index was 25.26 (10.78-41.57). Most patients presented with invasive ductal carcinoma (89.3%), histological grade III (49.4%), luminal subtype (65.9%) and Ki67 expression > 14% (89.2). Approximately 52% of the patients reported a positive family history for breast, ovarian or prostate cancer. Deleterious mutations in BRCA1/2 genes ware detected in 13 patients (BRCA1, n= 4 and BRCA2, n= 9). One novel mutation was detected in BRCA1 gene: a stop codon in exon 6 (c.483T > A; p.Cys161Ter). Exome sequencing was evaluated in eight luminal tumors fromBRCA1/2 wild type patients and a median of 39 somatic alterations/tumor was detected, varying from 17 to 74. Potential driver alterations were detected in 53 genes, such as PIK3CA, PRKD1, PRKAR1A and PIK3AP1, that encode tyrosine kinase proteins or proteins involved in tyrosine kinases regulation; and POLD1 gene, that encodes the catalytic subunit of DNA polymerase delta which plays a critical role in DNA replication and repair. A Meta-analysis showed that somatic mutation in PIK3CA gene was frequently detected in both young and older patients. In conclusion, more than 15% of the young breast cancer patients are BRCA1 or BRCA2 mutation carriers. In patients with sporadic cancer somatic mutations were found in genes that encode tyrosine kinase proteins or are involved in the DNA repair. In agreement with what was observed in tumors from older patients, somatic mutation in PIK3CA gene is relatively frequent in tumors from young patients

Abstract Two major genes are involved in hereditary predisposition to breast and ovarian cancer – BRCA1 and BRCA2. However, germline mutations in these tumor suppressors account for a maximum 20% of the familial breast cancer cases. A significant portion of the genes predisposing to this disease is unknown and therefore needs to be discovered. The aim of this study was to identify novel breast cancer susceptibility genes from the interweaving BRCA/Fanconi anemia (FA) pathway. Five candidate genes – MERIT40, ABRAXAS, BRIP1, CHK1, and FANCA – were screened for mutations by utilizing conformation-sensitive gel electrophoresis and sequencing, or with multiplex ligation-dependent probe amplification in blood DNA samples of Finnish familial breast cancer patients. Investigation of the MERIT40 gene revealed novel nucleotide changes, being the first report on mutation screening of this gene. None of the observed alterations, however, appeared to be disease related, suggesting that germline mutations in MERIT40 are rare or absent in breast cancer patients. A missense alteration (c.1082G>A, leading to Arg361Gln) was identified in ABRAXAS in 3 out of 125 Northern Finnish breast cancer families (2.4%), but not in any of the 867 healthy controls. The prevalence of the mutation between familial and control cases was statistically significantly different (p=0.002). ABRAXAS c.1082G>A appears to have pathological significance based on its exclusive occurrence in cancer cases, evolutionary conservation, disruption of a putative nuclear localization signal, reduced nuclear localization of the protein, and defective accumulation at DNA damage sites. The BRIP1 (FANCJ) and CHK1 genes were screened for large genomic rearrangements, but no abnormalities were detected, ruling out a significant contribution to breast cancer susceptibility in the Northern Finnish population. A novel large heterozygous deletion was identified in the FANCA gene in one out of 100 breast cancer families, removing the promoter and the first 12 exons. The deletion allele was not present in the tested controls, suggesting that it might contribute to breast cancer susceptibility. This is the first report on the association of a large-size germline deletion in a gene acting in the upstream part of the FA signaling pathway with familial breast cancer. / Tiivistelmä BRCA1 ja BRCA2 ovat kaksi tärkeintä perinnöllisen rinta- ja munasarjasyövän alttiusgeeniä. Niissä esiintyvät ituradan muutokset selittävät kuitenkin vain noin 20 % familiaalisista rintasyöpätapauksista. Suurin osa alttiusgeeneistä on edelleen tunnistamatta ja näitä tekijöitä etsitään aktiivisesti. Tämän tutkimuksen tarkoituksena on ollut tunnistaa uusia alttiustekijöitä toisiinsa läheisesti liittyviltä BRCA/Fanconin anemia (FA) signaalinsiirtoreiteiltä. Viisi kandidaattigeeniä - MERIT40, ABRAXAS, BRIP1, CHK1 ja FANCA – kartoitettiin mutaatioiden suhteen suomalaisissa rintasyöpäperheissä käyttämällä konformaatiosensitiivistä geelielektroforeesia ja sekvensointia, tai multiplex ligation-dependent probe amplification- menetelmää. MERIT40-geenissä havaittiin useita aikaisemmin raportoimattomia nukleotidimuutoksia, mutta yhdenkään niistä ei havaittu liittyvän rintasyöpäalttiuteen. MERIT40-geenimuutosten mahdollista yhteyttä rintasyöpäalttiuteen ei ole tutkittu aikaisemmin. ABRAXAS-geenissä havaittiin missense-mutaatio (c.1082G>A, joka johtaa Arg361Gln aminohappokorvautumiseen) kolmessa pohjoissuomalaisessa rintasyöpäperheessä (3/125, 2.4 %). Muutosta ei havaittu terveissä kontrolleissa (N=867), ja ero mutaation esiintyvyydessä familiaalisten rintasyöpätapausten ja terveiden kontrollien välillä oli tilastollisesti merkitsevä (p=0.002). ABRAXAS c.1082G>A-muutos on todennäköisesti patogeeninen, sillä kyseinen aminohappopaikka on evolutiivisesti konservoitunut ja sijaitsee todennäköisellä tumaanohjaussignaalialueella. Funktionaaliset kokeet osoittivat, että mutatoitunut proteiinituote lokalisoitui villityypin proteiinia heikommin tumaan ja sen ohjautuminen DNA-vaurioalueille oli puutteellista. BRIP1- (FANCJ) ja CHK1-geeneistä etsittiin laajoja genomisia uudelleenjärjestelyjä, mutta niitä ei havaittu. Näin ollen kyseisillä muutoksilla ei ole merkittävää roolia perinnöllisessä rintasyöpäalttiudessa suomalaisessa väestössä. FANCA-geenissä havaittiin laaja heterotsygoottinen deleetio yhdessä tutkitusta 100 rintasyöpäperheestä. Deleetio poistaa geenin promoottorialueen lisäksi sen 12 ensimmäistä eksonia. Deleetioalleelia ei havaittu terveissä kontrolleissa, joten se mahdollisesti liittyy perinnölliseen rintasyöpäalttiuteen. Tutkimus on ensimmäinen, jossa raportoidaan laaja genominen deleetio FA-signaalinsiirtoreitin ylävirran geenissä familiaalisessa rintasyövässä.

No description available.

Abstract Around 5−10% of all breast cancer cases are estimated to result from a strong hereditary predisposition to the disease. However, mutations in the currently known breast cancer susceptibility genes account for only 20−30% of all familial cases. Additional factors contributing to the pathogenesis of breast cancer, therefore, await discovery. Aims of this study were to evaluate variations of the AATF and MRG15 genes as novel potential candidates for breast cancer susceptibility, to further examine the prevalence of the cancer-related PALB2 c.1592delT mutation among BRCA-negative high-risk breast cancer families counselled at the Department of Clinical Genetics, Oulu University Hospital, to identify Finnish Fanconi anaemia patients complementation groups as well as causative mutations, and to evaluate the potential role of these mutations in breast cancer susceptibility. The analysis of 121 familial breast cancer cases revealed altogether seven different sequence changes in the AATF gene. However, none of them were considered pathogenic, suggesting that germline mutations in AATF are rare or absent in breast cancer patients. Investigation of the MRG15 gene among familial breast cancer cases revealed seven previously unreported variants, but in silico analyses revealed that none of these variants appeared to modify the function of MRG15. The results suggest that MRG15 alterations are unlikely to be significant breast cancer susceptibility alleles. A previously identified pathogenic PALB2 mutation, c.1592delT, was identified in three patients from a cohort of 62 high-risk BRCA1/2-negative breast cancer patients from the Department of Clinical Genetics. PALB2 c.1592delT mutation testing should thus be a routine part of the genetic counselling protocol, particularly for BRCA1/2-negative high-risk breast cancer patients. Investigation of the complementation groups of Finnish Fanconi anaemia patients revealed a total of six different causative mutations. These mutations were examined further by analysing their prevalence in large cohorts of breast (n=1840) and prostate (n=565) cancers. However, no significant association emerged between cancer predisposition and these FA mutations. / Tiivistelmä Arviolta 5−10 prosenttia kaikista rintasyöpätapauksista aiheutuu merkittävästä perinnöllisestä alttiudesta sairauteen. Tällä hetkellä tiedossa olevien rintasyövälle altistavien geenivirheiden ajatellaan kuitenkin selittävän vain noin 20−30 prosenttia kaikista perinnöllisistä tapauksista. On todennäköistä, että uusia tekijöitä, jotka osallistuvat rintasyövän patomekanismiin, on vielä löytymättä. Tämän tutkimuksen tarkoituksena oli arvioida AATF- ja MRG15-geeneissä esiintyvien muutosten mahdollista vaikutusta rintasyöpäalttiuteen, tutkia tarkemmin PALB2 c.1592delT -mutaation esiintymistä BRCA-mutaationegatiivisten korkean rintasyöpäriskin potilaiden joukossa (perinnöllisyyspoliklinikka, Oulun yliopistollinen sairaala) ja määrittää suomalaisten Fanconi-anemiapotilaiden komplementaatioryhmät, sairauden taustalla olevat mutaatiot sekä tutkia näihin mutaatioihin mahdollisesti liittyvää rintasyöpäriskiä. 121 familiaalisen rintasyöpätapauksen analyysissä löytyi yhteensä seitsemän erilaista sekvenssimuutosta AATF-geenissä. Näistä yksikään ei kuitenkaan ollut selvästi patogeeninen. Tuloksen perusteella perinnölliset rintasyövälle altistavat muutokset AATF-geenissä ovat joko erittäin harvinaisia tai niitä ei esiinny lainkaan. MRG15-geenin mutaatioanalyysissä havaittiin seitsemän aikaisemmin raportoimatonta muutosta, mutta in silico -analyysien perusteella millään muutoksista ei ole vaikutusta MRG15-proteiinin toimintaan. Tulosten perusteella on epätodennäköistä, että MRG15-geenin muutokset olisivat merkittäviä rintasyövälle altistavia muutoksia. Jo aiemmin patogeeniseksi todettu PALB2 c.1592delT -mutaatio löydettiin kolmelta niistä perinnöllisyyspoliklinikan korkean syöpäriskin 62 potilaasta, jotka olivat BRCA1/2-geenitestauksessa saaneet normaalin tuloksen. Tulostemme perusteella PALB2 c.1592delT -mutaatiotestaus tulisi Suomessa ottaa osaksi perinnöllisyyspoliklinikoiden tarjomaa tutkimusprotokollaa. Suomalaisten Fanconi-anemiapotilaiden komplementaatioryhmiä selvittävässä tutkimuksessa identifioitiin yhteensä kuusi erilaista tautia aiheuttavaa mutaatiota. Näiden muutosten esiintymistä tutkittiin myös laajoissa rinta- (n=1840) ja eturauhassyöpäaineistoissa (n=565). Tilastollisesti merkittävää assosiaatiota ei kuitenkaan todettu suomalaisten FA-mutaatioiden ja syöpäalttiuden välillä.

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles. / Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.

Abstract Hereditary mutations in DNA damage response (DDR) genes often lead to genomic instability and ultimately tumor development. However, the molecular mechanism of how these DDR deficiencies promote genomic instability and malignancy is not well understood. Thus, the specific aim of this thesis is to identify the functional and molecular framework behind the elevated breast cancer risk observed in heterozygous PALB2 and ABRAXAS mutation carriers. The heterozygous germline alteration in PALB2 (c.1592delT) causes a haploinsufficiency phenotype in the mutation carrier cells. Due to PALB2 haploinsufficiency, elevated Cdk activity and consequently aberrant DNA replication/damage response was observed in the PALB2 mutation carrier cells. Excessive origin firing that is indicative of replication stress was also seen in the PALB2 mutation carrier cells. In addition to replication stress, PALB2 mutation carrier cells also experience G2/M checkpoint maintenance defects. The increased malignancy risk in females associated with heterozygosity for the Finnish PALB2 founder mutation is likely to be due to aberrant DNA replication, elevated genomic instability and multiple different cell cycle checkpoint defects. The heterozygous germline alteration in ABRAXAS (c.1082G>A) causes a dominant-negative phenotype in the mutation carrier cells. Decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP was observed in the ABRAXAS mutation carrier cells. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2/M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. Thus, the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilization in the heterozygous carrier cells. / Tiivistelmä Perinnölliset muutokset DNA-vauriovasteen geeneissä johtavat usein genomin epävakauteen ja lopulta syövän kehittymiseen. Molekyylitason mekanismeja, joilla vauriovasteen vajaatoiminta ajaa genomin epävakautta ja syöpää, ei kuitenkaan ymmärretä kunnolla. Tämän väitöskirjan tavoitteena on tunnistaa solutoiminnan ja molekyylitason vaikuttajat heterotsygoottisten PALB2- ja ABRAXAS-geenimuutosten kantajien kohonneen rintasyöpäriskin taustalla. Heterotsygoottinen ituradan suomalainen perustajamuutos PALB2-geenissä (c.1592delT) aiheuttaa haploinsuffisienssin kantajahenkilöiden soluissa. PALB2:n haploinsuffisienssin seurauksena kantajasoluissa havaittiin kohonnutta Cdk-proteiinin aktiivisuutta ja siitä johtuvaa kiihtynyttä DNA:n kahdentumista. PALB2-mutaatiota kantavissa soluissa nähtiin myös liiallista replikaation aloituskohtien käyttöä, mikä viittaa replikaatiostressiin. Replikaatiostressin lisäksi PALB2-mutaation kantajasoluilla havaittiin vaikeuksia ylläpitää solusyklin G2/M-tarkastuspisteen toimintaa. Näiden solutoiminnan poikkeavuuksien takia heterotsygoottisen PALB2 c.1592delT -mutaation kantajilla todettiin genomin epävakautta ja kohonnut syöpäriski. Heterotsygoottinen ituradan mutaatio ABRAXAS-geenissä (c.1082G>A) aiheuttaa dominantti-negatiivisen fenotyypin mutaation kantajasoluissa. ABRAXAS-mutaatiota kantavissa soluissa havaittiin BRCA1-proteiinitasojen laskua sekä BRCA1- ja CtIP-proteiinien vähentynyttä lokalisaatiota tumaan ja DNA-vauriopaikoille. Tämä aiheuttaa häiriöitä BRCA1-A-kompleksin paikallistumisessa, mikä johtaa häiriöihin virhealttiiden DNA-kaksoisjuoste¬katkoksien korjausmekanismien käytössä, DNA-vauriovasteessa, G2/M-tarkastus-pisteen säätelyssä ja ohjelmoidussa solukuolemassa. Tärkeimpänä löydöksenä havaittiin mutaation kantajasoluissa muuttunut transkriptioprofiili, joka johtunee BRCA1-proteiinitasojen laskusta tumassa. Näin ollen suomalainen ABRAXAS-perustajamutaatio toimii dominantti-negatiivisena BRCA1:n suhteen, aiheuttaen genomin epävakautta heterotsygoottisissa kantajasoluissa.

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles. / Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.