Identification of new alleles in genes that predispose to breast and ovarian cancer

Breast cancer is the most common form of cancer affecting women in the developed world. Although approximately 5-10% of breast cancer cases are attributed in mutations in BRCA1 and BRCA2 genes, a significant fraction of these cases is explained by mutations in other high and medium penetrant genes. Consequently, genetic analysis of hereditary breast/ovarian cancer plays an important role in understanding the molecular mechanisms leading to carcinogenesis and it significantly contributes to the clinical management of patients with breast/ovarian cancer. The aim of the present thesis is the identification and characterization of novel mutations in breast cancer susceptibility genes in patients with severe breast/ovarian cancer family history, the detection of new large genomic rearrangements as well as the evaluation of mutations that appear frequently in Greek population. The total number of breast cancer patients tested for point mutations in the BRCA1 and BRCA2 genes was 252 and 196, respectively. Pathogenic mutations in the BRCA1 gene were detected in 7.53% of patients while 8 women (4.1%) carried a BRCA2 pathogenic mutation. In addition, 4 unclassified variants were identified in the BRCA1 gene (1.58%) and 2 in the BRCA2 gene, respectively. Moreover, all the patients that were negative for mutations, were tested for the presence of large genomic rearrangements by another method and consequently two patients carried a deletion encompassing both exons 23 and 24 of the BRCA1 gene. Furthermore, in a series of 2,092 high-risk families fully screened for BRCA1 and BRCA2 germline mutations, the deletion of exons 23 and 24 of the BRCA1 gene was found in 35 families (1.68%) and haplotype analysis showed that it was a founder mutation for the Greek population. All the BRCA1 carriers were originated from the Aegean islands and the Asia Minor coast. Moreover, 226 patients with a strong family history of breast/ovarian cancer were analyzed by massively parallel sequencing using BROCA panel, which captured the entire loci of 50 genes. Of the 226 patients, 28 (12.4%) carried loss-of-function mutations in one of the 50 genes while 20 carried variants of unknown clinical significance. Additionally, 40 women diagnosed with early onset breast and/or ovarian cancer or with a severe family history were analyzed using “TruSight Cancer panel” that captured the entire loci of 94 genes. The experimental approach was carried out at Molecular diagnostics laboratory, NCSR Demokritos. In total, two pathogenic mutations were identified in 2 patients while 4 patients carried unclassified variants. Moreover, a novel BRCA2 LGR encompassing exons 12 and 13 was identified in the Greek population. Firstly, the genomic breakpoints for the deletion were determined by Long range PCR and subsequently 2,386 women diagnosed with breast cancer were screened by a customized PCR assay. The deletion was detected in 0.25% (6/2,386) of cases while haplotype analysis showed that it was a Greek founder mutation. All mutation carriers were originated from Crete Island.CHEK2 is a well-studied breast cancer susceptibility gene, beyond BRCA1 and BRCA2 genes. In the present study, the medium penetrance gene CHEK2 was analyzed in order to characterize the mutation spectrum in Greek population. Initially, 2,408 women diagnosed with breast cancer were screened for CHEK2 c.1100delC mutation and it was detected in 0.16% (4 of 2,408) of females, indicating that the specific allele did not contribute substantially to hereditary breast cancer in patients of Greek descent. Moreover, a novel CHEK2 LGR of a ~7.5kb deletion encompassing exon 6 was identified by BROCA panel. Subsequently, the genomic breakpoints of the latter were determined and 2,355 breast cancer patients and 1,580 controls were genotyped by a custom-designed PCR. The deletion was detected in 0.22% (5/2,355) of patients but in none of the 1,580 controls while haplotype analysis showed that all carriers shared a common disease-associated haplotype for a stretch of 1,850kb, suggesting a single common ancestor and a founder mutation for the Greek population. All carriers were originated from Western Greece. In silico analysis, results of CHEK2-yeast functional assay and structure–function relationship prediction were consistent with impairment of the mutant protein. An important aspect of the pathogenicity of this mutation was the effect that had been confirmed on the RNA level, where an aberrant isoform was being produced. It is quite possible that this mutation has a dominant-negative effect, rather than being destroyed by nonsense mRNA decay.In conclusion, in the present thesis three rare genomic events were characterized in BRCA1, BRCA2 and CHEK2 genes that were identified in ~2% of Greek women diagnosed with early onset breast cancer or/and with severe breast/ovarian cancer family history. The aforementioned genomic rearrangements were present in specific regions and showed geographical distribution, thus this information is useful in diagnostic level. Moreover, massively parallel sequencing of many genes was developed resulting in the identification of many genetic alterations in well-studied as well as in new breast cancer susceptibility genes, using next generation sequencing technology. Consequently, through these technologies, a significant progress was made in broadening the spectrum of breast cancer-related genes (e.g. CHEK2 and PALB2), beyond BRCA1 and BRCA2, resulting in better understanding of the underlying mechanisms of familial breast cancer in Greece.Ο καρκίνος του μαστού αποτελεί τον πιο συνηθισμένο τύπο καρκίνου που διαγιγνώσκεται στις γυναίκες στις ανεπτυγμένες χώρες. Περίπου 5-10% των περιστατικών καρκίνου του μαστού οφείλεται σε μεταλλάξεις των γονιδίων BRCA1 και BRCA2, ενώ ένα σημαντικό μέρος αυτών εξηγείται από μεταλλάξεις άλλων γονιδίων, υψηλής και μεσαίας διεισδυτικότητας. Έτσι, η γονιδιακή ανάλυση στον κληρονομικό καρκίνο του μαστού συμβάλει στην κατανόηση των μοριακών μηχανισμών της καρκινογένεσης αλλά και τη βελτιστοποίηση της διαχείρισης των ασθενών.Σκοπός της παρούσας διδακτορικής διατριβής αποτελεί η ταυτοποίηση και ο χαρακτηρισμός νέων μεταλλάξεων σε γονίδια προδιάθεσης καρκίνου του μαστού σε ασθενείς με βεβαρυμμένο οικογενειακό ή ατομικό ιστορικό, η ανίχνευση σπάνιων γενετικών φαινομένων, καθώς και ο χαρακτηρισμός μεταλλάξεων που εμφανίζονται με μεγάλη συχνότητα στον Ελληνικό πληθυσμό. Αρχικά αναλύθηκε το γενετικό υλικό 252 και 196 ασθενών με την αλληλούχιση κατά Sanger για μεταλλάξεις του γονιδίου BRCA1 και του γονιδίου BRCA2, αντίστοιχα όπου και ανιχνεύθηκαν 19 (7,53%) και 8 (4,1%) παθογόνοι μεταλλάξεις, αντίστοιχα. Οι ασθενείς εκείνες που ήταν αρνητικές για μεταλλάξεις, υποβλήθηκαν σε έλεγχο με ανεξάρτητη μέθοδο για την παρουσία γονιδιακών αναδιατάξεων, όπου και ανιχνεύθηκε απαλοιφή των εξονίων 23 και 24 του γονιδίου BRCA1 σε δύο ασθενείς. Στη συνέχεια, προσδιορίσθηκε η συχνότητα της απαλοιφής αυτής σε 2.092 ασθενείς με σοβαρό ιστορικό καρκίνου μαστού/ωοθηκών, η οποία υπολογίστηκε στο 1,68%. Η ανάλυση απλοτύπων έδειξε πως αποτελεί ιδρυτική μετάλλαξη για τον Ελληνικό πληθυσμό και πιο συγκεκριμένα, με καταβολή από τα νησιά του Αιγαίου και την περιοχή της Μικράς Ασίας. Προκειμένου να αποσαφηνιστεί το γονιδιακό υπόβαθρο 226 ασθενών με ηλικία διάγνωσης καρκίνου <35 ετών ή/και βεβαρυμμένο οικογενειακό ιστορικό καρκίνου μαστού/ωοθηκών χρησιμοποιήθηκε η μεθοδολογία της νέας γενιάς αλληλούχισης (Next Generation Sequencing) και πιο συγκεκριμένα το BROCA πάνελ, το οποίο αναλύει ταυτόχρονα 50 γονίδια προδιάθεσης καρκίνου. Συνολικά, ανιχνεύθηκαν 29 παθογόνοι μεταλλάξεις σε 28 ασθενείς (12,4%) στα υπό μελέτη γονίδια προδιάθεσης, ενώ 20 ασθενείς έφεραν γενετικές αλλαγές αγνώστου παθογένειας. Εν συνεχεία, αναλύθηκαν 40 δείγματα γυναικών με σοβαρό οικογενειακό ιστορικό ή νεαρή ηλικία διάγνωσης (