Sonja Levant

Levant, Sonja

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25Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancerMOLECULAR DIAGNOSTICS OF HEREDITARY              BREAST CANCERSonja Levanat Laboratory for Hereditary Cancer, Division of Molecular Medicine,Rudjer Boskovic Institute, Zagreb, CroatiaSummaryMutations in BRCA1 and BRCA2 genes are associated with the family predisposition to breast and ovarian cancer. The main purpose of genetic testing is an early detection of the predisposition,  because even early detection increases the effectiveness of treatment. Keywords: breast cancer;  hereditary breast cancer; BRCA1 and BRCA2 genes; mu-tations.INTRODUCTIONBreast cancer is a leading cancer in female population worlwide. Epide-miological data indicate 5-10 % of all breast and /or ovarian cancer cases are hereditary, and germline mutations in BRCA genes account for the majority of hereditary breast nad ovarian cancers [1]. By today, those two genes Breast Cancer Gene 1 (BRCA1) and Breast Cancer Gene 2 (BRCA2) are only known breast cancer predisposing genes, whose mutations can be first sign of fami-lial setting, before cancer occurs. Those genes are tumor suppressors, invol-ved in DNA repair processes, and are the major breast and ovarian cancer susceptibility genes.The penetrance of deleterious BRCA mutations has been variably estima-ted; a recent combined analysis of different reports [2] estimates the average cumulative risk (by the age of 70) in BRCA1 mutation cariers to be about  70 % for breast cancer and 40 % for ovarian cancer, whereas the corresponding risk for BRCA2 are 45 % and 11 %. So, person with inherited mutation in UDK 618.19-006-07Original scientific paper  Received: 4 June 2014Accepted: 24 September 201426Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancerBRCA1 and BRCA2 genes have 45-85% probability of developing breast can-cer, and 11-62% probability of developing ovarian cancer by age 70, compa-ring to general population risk of 10%.In familial setting, breast cancer occurs at young age and risk of bilateral breast and ovarian cancer is increased. Age of onset in subsequent genera-tions of BRCA mutations carrying families is lower by 7,9 years [3].  Also, carriers of BRCA1 and BRCA2 mutations are at increased risk for other can-cers: uterine, cervical, prostate, pancreatic, male breast, bile duct, stomac can-cer and melanoma [4]. Breast cancer can occur in men as well, but the risk is 100 times lower than in women.In general population the overall prevalence of BRCA1 and BRCA2 mu-tation carriers is estimated to be from 1 in 400 to1 in 800, the quite variable ratio among etnic groups and by geographic region.Figure 1. Shematic presentation of the location of BRCA 1 gene mapped on chromosome 17 and BRCA 2 gene on chromosome 13. There are listed also few data about size of the gene, exon number and protein size they code. • 1863 ak• 5589 pb• protein ~380kDa17q21-q24• 24 exonsBRCA1chromosome 17• 2329 ak13q12-q13• 10254 pb• protein ~220kDa• 27 exonsBRCA2chrromosome 13chromosome 17 chromosome 1327Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancerMost reported disease-associated alleles of BRCA1 and BRCA2 genes (Fi-gure 1) have been attributed to frameshifts, nonsense or missense mutations, large rearrangements and splice alterations. They usually lead to truncated BRCA1 or BRCA2 protein or affect critical aminoacids  for its structure or function. However, a large number of sequence variants, particularly mi-ssense variants, routinely encountered in clinical and research laboratories, cannot be readily distinguished  as either disease-causing (deleterious) mu-tations or  benign polymorphisms (clinically not significant) and thus are classified as variants of unknown clinical significance.To date, more than 3600 BRCA1 and BRCA2 variants have been reported in locus-specific databases like Breast Cancer Infromation Core (BIC) - http://research.nhgri.nih.gov/bic/, than  Universal Mutation Database (UMD) - http://www.umd.be/BRCA1/ and  http://www.umd.be/BRCA2/, Leiden Open Variation Database - http://chromium.liacs.nl/LOVD2/cancer/home.php and kConFab - http://www.kconfab.org/Progress/Mutations.aspx.Considering these numbers, genetic testing represents invaluable step forward in defynig these malignant disease. The testing is especially impor-tant in predicting the risk of developing ovarian cancer, as this kind of can-cer is difficult to detect until late, when the chances of cure are less efficient.Unfortunately, every year in Croatia 2500 women develop breast cancer and 450 develop ovarian cancer, and more than 900 die of breast cancer and almost 400 deaths of ovarian cancer. According to statistical estimates based on 4,3 million Croatian population, at least two hundred breast cancers have familial predisposition, and those estimates follow general worldwide stati-stics (data from Central Bureau of Statistics of Republic of Croatia and Croa-tian National Institute of Public Health, 2006). No data on BRCA variants in affected population of Croatia have been gathered so far.EXPERIENCE WITH GENETIC TESTING IN CROATIAIn Croatia few years ago conditions were appropriate for providing gene-tic testing of inherited predisposition for breast and ovarian cancer . It was organized in Laboratory for Hereditary Cancer at Rudjer Boskovic Institute thanking to funds of Terry Fox donation and support by Croatian League against Cancer and Europa Donna Croatia. The screening was performed by high-resolution melting approach, di-rect sequencing and semi-quantitative multiplex PCR method and provides 28Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancera comprehensive analysis on the type and distribution of BRCA1 and BRCA2 mutations and allelic variants [5].The test entails very complex molecular-genetic methods that have been used in most developed countries for decades. The Laboratory is a member of EMQN (European Molecular Genetics Quality Network), international organization focused on standardizing protocols for molecular genetics di-agnostics and on quality assurance of its members. Therefore, the methods used are at the European level of quality. The main purpose of this testing was early detection of predisposition, because it lowers the cost of treatment as well as declines the need for long hospitalizations, chemotherapy, radiation and multiple surgeries. Even early detection increases the effectiveness of treatment. Targeted and preventive therapy, as well as the lifestyle changes can significantly decrease the mor-tality.CANDIDATES FOR TESTINGEach candidate has to undergo genetic counseling before and after te-sting. The test is mainly for breast and ovarian cancer prone family mem-bers and is not informative for sporadic cases of breast and ovarian cancer. BRCA1 and BRCA2 mutations are inheritable with the same probability from either parent.Potential candidates for BRCA1 and BRCA2 mutation testing are:- persons with two or more relatives with breast cancer - persons with early (before age of 50) breast cancer in the family- persons with breast cancer in the family in more than one generation- persons with bilateral breast cancer in the family- persons with multiple ovarian cancer in the family- persons with early (before age of 40) bilateral ovarian cancer- persons with one or more relatives with BRCA1 and/or BRCA2 gene mutation in the family.FIRST STUDY IN CROATIAThe results of BRCA1 and BRCA2 analysis in a group of 168 candidates from Croatia were selected for increased risk of breast and ovarian cancer on the basis of their personal or family history of the diseases. Candidates were 29Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancervolunteers from several national associations and support groups for breast cancer patients, selected according to the set criteria.In this study, 168 women from 147 families with positive family or per-sonal history of breast and ovarian cancer were screened for mutations in BRCA1 and BRCA2 genes. All candidates gave their informed consent to per-form DNA analysis on their blood samples before the samples were taken. The study was conducted according to the Declaration of Helsinki Principles.The candidates were recruited from mostly all locations in Croatia (Ka-štel Novi, Rijeka, Ploče, Šibenik, Osijek, Zagreb, Dubrovnik, Zadar, Čakovec, Karlobag, Gospić) and were classified into nine categories according to fa-mily or personal history of disease. Table 1. shows the specific characteristics of the candidates according to the inclusion criteria.Table 1. Inclusion criteria for the candidates included in this study (from 6).Group Inclusion criteria Cases (%)A affected individual with breast or ovarian cancer before 35 years of age 9 (5.3)B affected individual with breast cancer before the age of 35 with at least one case of breast or ovarian cancer in the family4 (2.4)C affected individual with breast or ovarian cancer between the age of 35 and 55 with at least one case of breast cancer in the family14 (8.3)D affected individual with breast or ovarian cancer between the age of 35 and 55 with at least one case of breast and at least one case of ovarian cancer in the family7 (4.2)E unaffected individual with at least two cases of breast cancer in the family 65 (38.7)F unaffected individual with at least one case of breast cancer in the family before 50 years of age39 (23.2)G unaffected individual with at least one case of breast and at least one case of ovarian cancer in the family18 (10.7)H unaffected individual with at least one ovarian cancer  in the family 8 (4.8)I unaffected individual with at least one case of breast cancer and male breast cancer in the family4 (2.4)Total 168 (100)Candidates in group A are considered early onset breast cancer patients, as the usual cut off for early onset is 35 years of age. From the 168 candidates which participated in this study, 32 (19%, age range 25-58, median 41) were affected with breast cancer, 2 (1.2%, ages 31 and 53) with ovarian cancer, and one with both breast and ovarian cancer (0.6%, ovarian cancer in 51 and breast cancer in 54). The remaining candidates were unaffected (133) but with positive family history (groups E-I). 30Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancerMutations in BRCA1 and BRCA2 genes detectedFigure 2.  BRCA1 gene shematic presentation with marked type and position of detected mutations.Figure 3.  BRCA2 gene shematic presentation with marked type and position of detected mutations.Al together (in 10 %) genetic testing identified 14 pathogenic mutations in17 candidates, 9 in BRCA1 and 5 in BRCA2 (Figures 1 and 2).  Of those, 11 have been previously described and 3 were novel [6]. Nine pathogenic mutations in BRCA1 and five in BRCA2 were detected [6]. Croatia shares most of the mutations with Slovenia and Germany . No founder mutations were detected in Croatia, although one is a candidate. Further analyses on a larger number of samples is necessary. The benefit of this testing is clear, since mutations were detected in young unaffected women who will be closely monitored. 31Rad 520. Medical Sciences, 40 (2014) : 25-31S. Levant: Molecular diagnostics of hereditary breast cancerReferences[1]  Ford D et al (1998) Genetic heterogeneity  and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. Am J Hum Genet. 62:676-689.[2]  Antoniou A et al (2003) Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet. 72(5):1117-1130.[3]  Litton JK et al (2011) Earlier age of onset of BRCA mutation-related cancers in subse-quent generations. Cancer. 118(2):321-325.[4] Arnold MA, Goggins M (2001)BRCA2 and predisposition to pancreatic and other cancers. Exp Rev Mol Med. 3:1-10.[5]  Levacic-Cvok M, Cretnik M, Musani V, Ozretic P, Levanat S(2008) New sequence vari-ants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis in an elderly healthy female population in Coatia. Clin Chem Lab Med. 46(10):1376-1383. [6]  Levanat S, Musani V, Cvok ML, Susac I, Sabol M, Ozretic P, Car D, Eljuga D, Eljuga L, Eljuga D. (2012) Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia. Gene. 498(2):169-176SažetakMolekularna dijagnostika nasljednog raka dojkeMutacije u genima BRCA1 i BRCA2 povezuju se sa nasljeđenom sklonošću za na-stanak raka dojke i jajnika. Svrha genetičkog testiranja je utvrditi dovoljno rano postoji li predispozicija, čime bi se dovoljno rano mogle poduzeti učinkovite mjere prevencije.Ključne riječi: rak dojke; nasljedni rak dojke; geni BRCA1 i BRCA2; mutacije gena.Corresponding author: Sonja Levanat E-mail: levanat@irb.hr

Molekularna dijagnostika nasljednog raka dojke

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