ADAMO B

AGNESE, Valentina

AUGELLO, Claudia

BARBERA, Floriana

BAZAN, Viviana

BRUNO, Loredana

CALO', Valentina

CASCIO, Sandra

COLUCCI G

CORSALE S

FRICANO S

GARGANO, G

RUSSO, Antonio

SUMARCZ E

TERRASI, Marianna

VALERIO, Maria Rosaria

Archivio istituzionale della ricerca - Università di Palermo

Annals of Oncology 17 (Supplement 7): vii30–vii33, 2006doi:10.1093/annonc/mdl946symposium articleY179C, F486L and N550H are BRCA1 variants that maybe associated with breast cancer in a Sicilian family:results of a 5-year GOIM (Gruppo Oncologico dell’ItaliaMeridionale) prospective studyC. Augello1#, L. Bruno1#, V. Bazan1, V. Calo`1, V. Agnese1, S. Corsale1, S. Cascio1,5,G. Gargano1, M. Terrasi1, F. Barbera1, S. Fricano2, B. Adamo3, M. R. Valerio1,G. Colucci4, E. Sumarcz5 & A. Russo1*1Section of Medical Oncology and 2Section of Surgical Oncology, Department of Surgical and Oncology, Universita` di Palermo, Italy; 3Unit of Medical Oncology,A.O. Universitaria Policlinico ‘G.Martino’, Universita` di Messina, Italy; 4Division of Medical Oncology, National Institute of Oncology, Bari, Italy; 5Sbarro Institutefor Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USABackground: Over 600 different pathogenic mutations have been identified in the BRCA1 gene. Nevertheless,numerous missense mutations of unknown biological function still exist. Understanding of biological significance ofthese mutations should help in genetic counselling to carriers and their families.Patients and methods: A total of 104 patients with breast and/or ovarian cancer whose genetic counsellinganswered the criteria of the American Society of Clinical Oncology (ASCO 2003), were prospectively screened formutations in all coding exons of the BRCA1 gene by automatic direct sequencing.Results: During these mutational screening procedures one case presented three mutations classified in the BreastCancer Information Core Database as unknown variants. These were 655A/G found in exon 8 of BRCA1, 1575T/Cand 1767A/C found in exon 11 of the same gene. The identification of the three unknown variants in the proband(16SIRIO) and in her mother and sister indicates that such alterations exist in cis.Conclusions: Our results suggest that the charge and stechiometry variations determined by the changes in theamino acids Y179C, F486L and N550H might produce an effect on the conformation of the protein and, consequently,on its function.Key words: breast cancer, BRCA1, germinal mutations, unknown variantintroductionThe 5%–10% of cases of breast/ovarian carcinoma areassociated with hereditary genetic mutations and a highpenetrance of the oncosuppressor BRCA1 [1]. Geneticalterations in this gene lead to a higher predisposition to breastand ovarian cancer and confer a significantly higher risk ofendometrial, pancreas, cervix and prostate gland cancers [2].The BRCA1 gene consists of 22 exons encoding a nuclear 1863amino acid phosphoprotein. BRCA1 protein contains severalfunctional domains, which interact directly or indirectly witha variety of molecules, including tumour suppressors,oncogenes, DNA damage repair proteins, cell cycle regulators,transcriptional activators and repressors [3, 4].Over 600 different pathogenic mutations identified in thegene BRCA1 have been reported in the Breast CancerInformation Core Database (BIC) [5]; most of these areframeshift or nonsense mutations that give rise to theformation of truncated proteins; others instead are missensesubstitutions or intronic variants, including those involved insplicing, which have been linked with pathogenesis.Recently, many studies have focused on the interpretation ofthe unknown biological significance of missense mutations inthe gene BRCA1, defined as unknown variant (UV), withthe aim of determining whether they had a pathogenic or aneutral role [6, 7]. Moreover, these functional and biochemicalalterations within the different protein domains must beacquired in order to understand their role in cancerpredisposition and aid in genetic counselling.During genetic screening of BRCA1 in Sicilian patientswith breast/ovarian cancer (BC/OC), selected according tohereditary breast/ovarian cancer syndromes (HBOC) criteria,the patient 16SIRIO presented three UVs in the gene. Wetherefore examined other members of her family to assesswhether the UVs were present in cis and if their segregationwas associated with other neoplasias diagnosed in thesesubjects.symposiumarticle*Correspondence to: Dr A. Russo, Section of Medical Oncology, Department ofOncology, Universita` di Palermo, Via del Vespro 127, 90127 Palermo, Italy.Tel: +39-091-6552500; Fax: +39-091-6554529; E-mail: lab-oncobiologia@usa.net#C. Augello and L. Bruno have equally contributed to this work.ª 2006 European Society for Medical Oncologypatients and methodsA consecutive series of 650 patients resident in Sicily with breast/ovariancancer diagnosed between 1999 and 2004 were prospectively recruited at the‘Regional Reference Centre for the Characterisation and Genetic Screeningof Hereditary Tumours’ at the University of Palermo. Written informedconsent to genetic counselling was obtained from all patients recruited. Thisprotocol was conducted by an oncologist, a geneticist and a psychologist andincluded information regarding personal and family history in order toevaluate risk factors and genealogical tree. The latter was updated every yearand investigated retrospectively for at least three generations in patients withbreast/ovarian cancer or other types of tumours related toBRCA1 mutations.After considering different family relationships, a total of 104 patients,selected during genetic counselling according to the criteria of the AmericanSociety of Clinical Oncology (ASCO) [8], were screened for mutations inall coding exons of the BRCA1 gene by automatic direct sequencing. In casesof positive testing, the patients were invited to follow preventativeprogrammes for the early diagnosis of BRCA1-associated tumours and tocommunicate the results to their first-degree relatives so that they might alsoundergo genetic counselling. Even when genetic testing proved negative,the relatives of such patients were encouraged to take a regular part in theearly diagnosis programmes because of their familial cancer risk. If thegenetic testing was unclear, the patient was informed in order to decidewhether or not to proceed to other types of biomolecular investigation.The genomic DNA was extracted from peripheral blood according to theinstructions contained in the QIAamp DNA mini Kit (Qiagen, Hilden,Germany). A 9700 thermal cycler (Applied Biosystem, Foster City, CA) wasused to perform the polymerase chain reaction (PCR) of all the exons and,after dividing exon 11, of the 12 fragments of the gene BRCA1. Thefragments of amplicons were analysed by agarose gel electrophoresis ata concentration ranging from 1.5% to 2%. Direct sequencing of the PCRproducts was performed with a 3100 Avant Analyzer automatic sequencer(Applied Biosystems, Foster City, CA).The DNA samples from the 16SIRIO family members were screenedfor BRCA1 mutations in exons 8 and 11. Screening of the 50 DNA controlswith any familial cancer history, sequenced for the positions 655A/C,1575T/C and 1767A/C, were performed in order to verify that the threevariants were present together only in the 16SIRIO family.resultsDuring mutational screening of the gene BRCA1 in a groupof 104 Sicilian patients with hereditary and familial ovarianand/or breast cancer selected in the Regional Reference Centrefor the Characterisation and Genetic Screening of HereditaryTumours (University of Palermo, Italy), one case presentedthree missense mutations classified in the BIC database asunknown variants. These were 655A/G found in exon 8 ofBRCA1 and 1575T/C and 1767A/C found in exon 11 of the samegene (Table 1). Furthermore, the same patient showed theintronic variation IV7-34 C>T, which was reported severaltimes in the BIC both as a polymorphism and as an UV; in ournegative controls, this intronic variant was detected in 70% ofthe cases (35/50), both in homozygotes and in heterozygotes.The patient, indicated by an arrow in the geneological tree,was a nulliparous, non-Askenazi woman aged 35, with aductal infiltrating carcinoma of the left breast (T1NoMx),intermediate grade (G2), positive to the test foroestroprogestinic receptors (40%) and for MIB-1 (10%) andnegative for Erb-B2 (Table 2).During genetic counselling, the patient’s geneological treewas reconstructed together with her individual and familyclinical history, retrospectively for three generations. Thepatient was included in our study because she was diagnosedwith breast cancer at an early age (<40 years old) and alsobecause of the tumour history in the first generation. Thepatient’s mother had previously been diagnosed witha tumour of the uterus (spinocellular, cheratoblastic andendocervical infiltrating carcinoma) at the age of 42. Allmembers of the family, including the proband’s father,mother and sister, underwent analysis of exons 8 and 11 ofthe gene BRCA1. Direct automatic sequencing of DNAextracted from the lymphocytes showed exactly the same UVs:Y179C, F486L and N550H in the mother and the sister(Figure 1). Furthermore, the patient’s father presented thesame polymorphic intronic variant IV7-34 C>T as the proband,while the sister and the father showed S694S polymorphism(Table 1). The identification of the three UVs both in themother and in the sister of the proband indicates that suchalterations exist in cis. None of the control cases presentedany of the three UVs.discussionThe BRCA1 germinal mutations are responsible for the majorityof hereditary breast and/or ovarian cancers and they have alsorecently been associated with a high risk for several other typesof tumours, such as those involving the prostate gland, thepancreas and the endometrium [9, 10].Since carriers of these mutations have a high risk ofdeveloping these tumours at an early age, the American Societyof Clinical Oncology recommended genetic testing to beperformed to patients before the age of 40 years [11].The patient 16SIRIO developed breast carcinoma at an earlyage and analysis of her genealogical tree indicated that hermother had been affected by endometrial carcinoma at a similarage. This suggested that the patient was probably a carrier ofTable 1. Details of the five sequence variants of BRCA1 gene detected in the 16SIRIO familySequence variants Exon/intron Nucleotide no. Base change No. times in BIC Amino acid change EffectMissense mutation Exon 8 655 A>G 35 Y179C UVs probably related to BCwhen present simultaneouslyMissense mutation Exon 11 1575 T>C 36 F486LMissense mutation Exon 11 1767 A>C 34 N550HIntronic variant Intron 7 IVS7-34 C>T 7 – NeutralSilent mutation Exon 11 2201 C>T 12 S694S NeutralAnnals of Oncology symposium articleVolume 17 | Supplement 7 | June 2006 doi:10.1093/annonc/mdl946 | vii31a BRCA1 mutation. Genetic tests on the patient 16SIRIOshowed the presence of three UVs: 655A/G, 1575T/C and1767A/C, which were also found in the patient’s mother andyounger sister, whereas none of these UVs were found in any ofthe control cases. Furthermore, both the proband and her fatherpresented the intronic variation IVS7-34C/T, frequentlyreported in the BIC both as a polymorphism and also as an UV.This variant was found in 70% (35/50) of our control cases, bothin homozygotes and in heterozygotes, indicating therefore thatit was a benign nucleotide alteration. Moreover, both thepatient’s sister and father showed the nucleotide variation2201C/T, which does not involve any amino acid variation(S694S).The analysis of the control cases with the same ethnicbackground was important for determining whether the changesdetected in the BRCA1 genomic sequence are variants associatedwith disease or benign variants that are typically found in theSicilian population.It is known that the conformation and therefore the finalstructure of a protein is the result of its amino acid compositionand consequently of the physico-chemical properties andstechiometry of the amino acids. The three UV identified in thisreport are mentioned several times in the BIC and determineimportant amino acid changes: in fact, 655A/G gives rise to theTable 2. Proband’s clinical featuresAge at initial diagnosis 35Family history of breast cancer NegativeFamily history of ovarian cancer NegativeLaterality of breast cancer LeftHistology of breast cancer Ductal infiltratingTumour stage T1Number of nodes involved 0Histological grade 2Oestrogen receptor status PositiveProgesterone receptor status PositiveErb-B2 NegativeMIB-1 Positive (10%)Figure 1. (A) Geneological tree with 16SIRIO proband indicated by arrow. Black circles indicate women affected by cancer. White square indicatesman without any mutation. Br, breast cancer; Ut, uterine cancer. Numbers following abbreviations indicate age at diagnosis. (B) Electropherogramof exon 11 sequence (1767A/C) of BRCA1 gene in the proband.Figure 2. Schematic diagram of BRCA1 polypeptide showing main functional domains (in grey). In the circles are indicated some of the proteinsinteracting with BRCA1, whose interaction domains include the amino acids Y179, F486 and H550.symposium article Annals of Oncologyvii32 | Augello et al. Volume 17 | Supplement 7 | June 2006substitution of thyrosine 179 characterised by the presence of anaromatic ring, by cisteine, which possesses oxydrilic lateralchains (Y179C, n. 35 times in the BIC); 1575T/C brings aboutthe substitution of phenylalanine 486, which also possesses anaromatic ring, by leucine, which has aliphatic lateral chains(F486L, n. 36 times in the BIC); and 1767A/C leads to thesubstitution of the amino acid not charge asparagin 550 by theamino acid basic histidine (N550H, n. 34 times in the BIC).Several studies have been conducted to understand thepathogenicity of BRCA1 unknown variants. The studies werebased on the alignment of the orthologous sequences and onin vitro embryonal engineering (Xenophus and Mouse), whereUVs have been inserted in trans compared with deleteriousmutations of the same gene. These analyses demonstrated that ifY179C, F486L and N550H are considered separately, they can beclassified either as neutral alterations or as changes with onlylow clinical significance [12–14].Combining these results and considerations with the studyof the 16SIRIO family, we suggest that three amino acidchanges identified might alter the charge and stechiometry ofprotein, and in consequence its function. The latter might berelated to the change in BRCA1 interactions with proteinswhose binding domains include the amino acids Y179, F486 andN550, for instance, TP53, SW1/SNF, ZBRK1/GADD45,importin alpha, STAT1, ER and cMYC. These factors areinvolved in DNA damage repair, cell cycle regulation andtranscriptional regulation [3, 4]. In particular, cMyconcogene interacts with two BRCA1 domains that includeY179 and F486 (Figure 2).An accurate follow-up of this family, and particularly of theproband’s sister, might confirm the hypothesis that the threeamino acid alterations could play a role in determining breastand/or ovarian cancer susceptibility. It might also be interestingto study the segregation of the three variants in the carriersaffected by disease compared with those detected in the healthymembers of the family.references1. Hadjisavvas A, Adamou A, Phanis CO et al. Q356R and S1512I are BRCA1variants that may be associated with breast cancer in a Cypriot family. OncologyReports 2002; 9: 383–386.2. Rosen EM, Fan S, Isaacs C. BRCA1 in hormonal carcinogenesis: basic andclinical research. Endocrine-Related Cancer 2005; 12: 533–548.3. Wang Q, Zhang H, Kajino K, Greene MI. BRCA1 binds c-Myc and inhibits itstranscriptional and transforming activity in cells. Oncogene 1998; 17: 1939–1948.4. Hedenfalk IA, Ringner M, Trent JM, Borg A. Gene expression in inherited breastcancer. Adv Cancer Res 2002; 84: 1–34.5. Breast Cancer Information. Core:http//www.nchgr.nih.gov//dir/Intramural_research_Lab_Transfer/Bic/index.html6. Arnold N, Peper H, Bandick K et al. Establishing a control population to screen forthe occurrence of nineteen unclassified variants in the BRCA1 gene bydenaturing high-performance liquid chromatography. J Chromatog 2002; B 782:99–104.7. Calo` V, Agnese V, Gargano G et al. A new germline mutation in BRCA1 genein a Sicilian family with ovarian cancer. Breast Cancer Res Treat 2006; 96:97–100.8. Trepanier A, Ahrens M, McKinnon W et al. Genetic cancer risk assessment andcounseling: recommendations of the National Society of Genetic Counselors. JGenet Couns 2004; 13: 83–114.9. Weinreb I, Colban TJ. BRCA mutations and pathology in female genital tract. CurrDiagn Pathol 2004; 10: 326–335.10. Thompson D, Easton DF. The Breast Cancer Linkage Consortium: Cancerincidence in BRCA1 mutation carriers. J Natnl Cancer Inst 2002; 94.11. Yang Q, Yoshimura G, Nakamura M et al. BRCA1 in non-inherited breastcarcinoma. Oncol Rep 2002; 9: 1329–1333.12. Abkevich V, Zarkikh A, Deffenbaugh AM et al. Analysis of missense variation inhuman BRCA1 in the context of interspecific sequence variation. J Med Genet2004; 41: 492–507.13. Figge MA, Blankenship L. Missense mutations in the BRCT domain of BRCA-1from high-risk women frequently perturb strongly hydrophobic amino acidsconserved among mammals. Cancer Epidemiol Biomarkers Prev 2004; 13:1037–41.14. Judkins T, Hendrickson BC, Deffenbaugh AM, Scholl T. Single nucleotidepolymorphisms in clinical genetic testing: the characterization of the clinicalsignificance of genetic variants and their application in clinical research forBRCA1. Mutat Res 2005; 573: 168–179.Annals of Oncology symposium articleVolume 17 | Supplement 7 | June 2006 doi:10.1093/annonc/mdl946 | vii33

Y179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study

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Y179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study

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