brca1 and brca2 germline mutations are associated with high penetrance for both breast and ovarian cancer ( miki et al . the overall prevalence of brca1/2 alterations in general population varies considerably among different ethnic groups , with respect to specific founder mutations . in 2000 , three founder alleles of brca1 ( c.5266dup , c.181t > g , c.4034dela ) were reported in polish families with a strong aggregation of breast or ovarian cancers and subsequently incorporated into the standard genetic screening panel ( gorski et al . further investigations were expanded in a large population of unselected female breast cancer patients ( brozek et al . 2011 ; gaj et al . 2012 ; lubinski et al . 2006 ; szwiec et al . 2014 ) as well as in consecutive ovarian cancer series ( brozek et al . 2008 ; majdak et al . 2005 ; menkiszak et al . 2003 ) . a relatively high germline mutation frequency at the level of 13.5  % for brca1 and 13.9  % for brca1/2 among cases with unselective primary ovarian carcinoma has been reported in studies by menkiszak et al . albeit both studies confirmed the strong founder effect for brca1 5266dup and c.181t > g alterations , the latter reported other recurrent brca1/2 mutations in the group of polish patients with ovarian cancer . in line with the recommendations of the american society of clinical oncology testing for recurrent brca1 mutations is required in each case of ovarian cancer in the polish population ( 1996 ) . according to the reports of the latest clinical trials patients with brca mutations and recurrent serous ovarian carcinoma may benefit from specific therapies targeting brca signaling pathways ( audeh et al . for the purpose of the most effective treatment as well as genetic counseling and prophylactic strategies for patients and their families , mutational analysis of brca1 and brca2 needs to be highly sensitive and cost - efficient . the aim of this study was to establish the frequency of germline brca1/2 mutations in consecutive ovarian cancer series from northern poland . additionally , it was of our interest to investigate whether an application of next generation sequencing can significantly improve brca1/2 mutation detection rate and subsequently effectiveness of further prophylactic and treatment strategies . the study comprises 134 unselected ovarian cancer patients who were referred to the university hospital in gdansk and the red cross hospital in gdynia between 2012 and 2013 . within the studied group 77.6 % ( n  =  104/134 ) of patients were diagnosed with serous ovarian cancer , average age at diagnosis was 60.8 ( 2487 ) years . informed consent was obtained from all of the patients and the study was approved by the medical review board of medical university of gdansk . genomic dna was extracted from the whole blood using red - blood - cells lysis buffer followed by the standard phenol - chloroform procedure as described elsewhere . brca1 and brca2 mutation screening was performed using the brca mastr assay v1.2 ( multiplicom , niel , belgium ) followed by miseq targeted re - sequencing at minimum of 99x coverage ( illumina inc . ) . the analysis was performed with illumina variant studio software ( illumina inc . ) and geneious software ( biomatters ltd ) . presence of the brca1/2 mutations detected by ngs analysis was confirmed by pcr followed by bi - directional sanger sequencing ( abi prism 3130 , life technologies , inc . ) . the study comprises 134 unselected ovarian cancer patients who were referred to the university hospital in gdansk and the red cross hospital in gdynia between 2012 and 2013 . within the studied group 77.6 % ( n  =  104/134 ) of patients were diagnosed with serous ovarian cancer , average age at diagnosis was 60.8 ( 2487 ) years . informed consent was obtained from all of the patients and the study was approved by the medical review board of medical university of gdansk . genomic dna was extracted from the whole blood using red - blood - cells lysis buffer followed by the standard phenol - chloroform procedure as described elsewhere . brca1 and brca2 mutation screening was performed using the brca mastr assay v1.2 ( multiplicom , niel , belgium ) followed by miseq targeted re - sequencing at minimum of 99x coverage ( illumina inc . ) . the analysis was performed with illumina variant studio software ( illumina inc . ) and geneious software ( biomatters ltd ) . presence of the brca1/2 mutations detected by ngs analysis was confirmed by pcr followed by bi - directional sanger sequencing ( abi prism 3130 , life technologies , inc . ) . in the group of 134 patients with unselected primary ovarian cancer , pathogenic brca1 or brca2 mutations were found in 20 individuals ( 14.9  % ) . in addition , two variants of unknown significance were detected ( brca1 : c.301 + 7g > a ; brca2 : c.9486_9488del ) . among the brca1/2 positive cases , 16 carried brca1 and four brca2 mutation , which accounts for 80 and 20  % , respectively . thirteen alterations ( n  =  13/20 ; 65  % ) were recurrent brca1 mutations included in the standard genetic screening panel used in northern poland , as previously reported ( ratajska et al . the remaining seven mutations ( n  =  7/20 ; 35  % ) , three located in brca1 and four in brca2 are not included in the standard targeted mutation analysis , which overall gives seven out of the 20 mutation positive cases ( 35  % ) . detailed clinical , histopathological and molecular data of brca1/2 positive patients are presented in table  1.table 1clinical , histopathological , and molecular data of the ovarian cancer patients with brca1/2 mutationno.case noexon / intronmutation in corresponding cdna 
 predicted amino acid sequencemutation type 
 rs number 
 age ( years)figo stagehistologyfamily history 
 brca1 gene  11155c.181t > g ( 300t > g)p.cys61glym2889767236iiicserousnegative  229654iiicserouspositive  32211c.3700_3704del ( 3819del5)p.val1234glnfs*8f8035760961iiibserousunknown  43843iiicserousnegative  56652unknownserousunknown  65020c.5266dup ( 5382insc)p.gln1756profs*74f39750724652iiicserouspositive  710863unknownserousunknown  813865unknownserouspositive  931446iiicendometrioidpositive  1032337iiicserouspositive  1136860unknownserousunknown  1237466unknownserousunknown  1337836unknownserousunknown  14789c.594 - 2a > cr.[=;594_670del]p.val179cysfs*3f8035803324unknownserousunknown  158510c.1793t > ap.leu598*n8035711843unknownendometrioidnegative  169513c.4357 + 2t > g;r.[=;4186_4357del]p.arg1377tyrfs*2f8035815245iiicserousnegativebrca2 gene  17311c.3975_3978dupp.ala1327cysfs*4f39751563645ivserouspositive  1893c.2808_2811delp.ala938profs*21f8035935182unknownserouspositive  19103c.5042_5043delp.val168glufs*7f8035947862iiicserousnegative  208714c.7180a > tp.arg2394*n8035894667iiicendometrioidunknown 
 mutation type according to the hgvs nomenclature ; hgvs , human genome variation society . nomenclature commonly used is shown in brackets 
 f , frameshift ; m , missense ; n , nonsense 
 a reference snp number 
 a positive family history was determined when at least one family member with breast / ovarian cancer was present besides the patient clinical , histopathological , and molecular data of the ovarian cancer patients with brca1/2 mutation 
 mutation type according to the hgvs nomenclature ; hgvs , human genome variation society . nomenclature commonly used is shown in brackets 
 f , frameshift ; m , missense ; n , nonsense 
 a reference snp number 
 a positive family history was determined when at least one family member with breast / ovarian cancer was present besides the patient out of the 20 brca1/2 positive patients , 17 were diagnosed with ovarian serous adenocarcinoma ( 85  % ) , whereas the other three with adenocarcinoma endometroides ( 15  % ) . overall , the frequency of brca1/2 mutations was 40  % ( n  =  9/22 ) in the group diagnosed before or at age of 50 and approximately 10  % among patients older than 50  years ( n  =  11/112 ) . the mean age of cancer diagnosis was 52 ( 2482 ) years in the group of mutation carriers and 62.3 ( 2787 ) years in patients with brca1/2 wild - type . finally , detailed family history was available only in 60  % ( n  =  12/20 ) of brca1/2 positive patients , of whom in five individuals it was negative for breast / ovarian cancer in first and second degree relatives ( 41  % ) . the percentage of brca1/2 mutations in unselected ovarian cancer patients identified in the current study is comparable to that previously reported ( 14.9 vs 13.5 and 13.9  % ) ( brozek et al . the worldwide prevalence of brca1/2 mutations in consecutive ovarian cancer series is estimated at 5 to 15  % and varies markedly depending on the population s ethnic background ( berchuck et al . 2001 ) . effective and cost - efficient targeted mutation analysis is widely performed in patients with familial history of breast and ovarian cancer , including population - specific founder mutations . since the implementation of genetic screening panel covering the three founder mutations in 2000 , there have been subsequent reports on the prevalence of other recurrent brca1/2 mutations in poland ( brozek et al . 2012 ; gorski et al . 2004 ; perkowska et al . 2003 ; ratajska et al . 2008 ; szwiec et al . the standard testing panel has been expanded to five brca1 alterations in northern poland ( ratajska et al . 2008 ) . 2014 ) proposed to test six brca1 mutations in a group of women diagnosed with breast cancer at the age of 50 or below . it is widely accepted that the differences in frequency and spectrum of brca1/2 mutations may result from an application of different molecular techniques or ethnic diversity even within the same country ( brozek et al . , we performed a next generation sequencing in the group of 134 patients with unselected ovarian cancer . based on the results we conclude that the sensitivity of genetic screening covering the three founder brca1 mutations ( c.5266dup , c.181t > g , c.4034dela ) is established at 50  % ( only ten out of 20 mutations ) . expanded panel , comprising two additional recurrent mutations in brca1 ( c.3700_3704del and c.68_69delag ) increases detection rate to 65  % . in the present study fairly high frequency of previously selected mutations c.5266dup was the most common brca1 alteration detected in the studied group ( n  =  8/13 ; 62  % ) . the second most common brca1 mutation was a frame shift deletion c.3700_3704del ( n  =  3/13 ; 23  % ) . the c.3700_3704del mutation is frequently detected in the caucasian populations and has been reported as one of the three most prevalent alterations in north - eastern and south - central poland , along with the c.5266dup and c.181t > g ( brozek et al . furthermore , a relatively low frequency of the other two founder mutations , c.4034dela and c.68_69delag in our region denotes heterogenity of brca1 alterations in polish patients with ovarian cancer . the remaining seven mutations detected in brca1 and brca2 genes were identified only in unique patients . however , all detected genetic variants have been previously reported in the breast cancer information core ( bic ) database ( szabo et al . 2000 ) . interestingly , within this study a relatively high proportion of deleterious brca2 mutations ( 20  % ) was observed . for instance , a c.2808_2811del deletion identified in an 82-year old patient is a recurrent mutation , frequently detected in non - ashkenazi breast / ovarian cancer patients and described to have multiple origins ( infante et al . the other brca2 genetic variants detected in the current study , although less frequent , were also previously reported ( szabo et al . 2000 ) . a substantial percentage of brca1/2 positive patients diagnosed after the age of 60 ( n  =  8/20 ) is consistent with the previous reports , indicating that the incidence of ovarian cancer in older age does not exclude brca germline mutation , especially as far as the brca2 gene is concerned ( brozek et al . limited significance of age at diagnosis as well as patient s family history justifies genetic screening for the founder brca1/2 mutations in all ovarian cancer patients . however , according to the current study , as many as 35  % of mutation positive individuals can be missed in the routine screening . this proportion can be even higher after taking into account possible large rearrangements in brca genes that were not examined in the present study . 2013 ) these brca1/2 mutations are relatively rare ( 3.7 and 4.8  % of all brca1 alterations , respectively ) in the polish population , we can not entirely exclude their occurrence . moreover , a deletion comprising exon 17 has been detected in two unrelated polish high - risk breast and ovarian cancer families ( rudnicka et al . the importance of mutation detection , apart from genetic counselling and prophylactic management arises in the considerance of recent clinical trials , with the implementation of poly ( adp - ribose ) polymerase 1 ( parp1 ) inhibitors . parp1 is a member of chromatin - associated polymerases involved in posttranslational adp - ribosylation and dna strand breaks repair ( el - khamisy et al . it has been reported that tumor cells with brca1/2 mutation , since their deficiency in homologous recombination , are markedly sensitized to the parp inhibition , resulting in chromosomal instability and consequent apoptosis ( bryant et al . 2005 ; farmer et al . 2005 ) . according to the results of phases 1 and 2 trials with parp1 inhibitor olaparib , brca mutation positive patients with platinum - sensitive relapsed serous ovarian cancer respond preferentially to parp inhibition , with a significant reduction in risk of disease progression in comparison to patients with wild - type brca ( audeh et al . these observations raise promising perspectives for personalized therapy in brca carriers and further emphasizes the necessity for proper mutational analysis of brca1/2 in a group of ovarian cancer patients . in conclusion , we propose routine genetic screening for at least five recurrent brca1 mutations ( c.5266dup , c.181t > g , c.3700_3704del , c.68_69delag , c.4034dela ) in all ovarian cancer patients in the polish population . in addition , in the negative cases for above - mentioned alterations analysis of the entire brca1/2 genes with an application of new generation sequencing should be performed . unfortunately , given the potential costs of such expanded diagnostics it will probably need to be limited to the selected group of patients with specific clinical and histopathological characterization . to establish recommendations for the entire brca1/2 genes mutational analysis further studies in larger ovarian cancer series with detailed family history are required . moreover , based on the results we confirm that next generation sequencing is a sensitive , reliable tool which can be applied in molecular diagnostics of brca1/2 genes in a group of selected patients .
the importance of proper mutational analysis of brca1/2 in individuals at risk for hereditary breast and ovarian cancer syndrome is widely accepted . 
 standard genetic screening includes targeted analysis of recurrent , population - specific mutations . 
 the purpose of the study was to establish the frequency of germline brca1/2 mutations in a group of 134 unrelated patients with primary ovarian cancer . 
 next generation sequencing analysis revealed a presence of 20 ( 14.9  % ) mutations , where 65  % ( n  =  13 ) were recurrent brca1 alterations included in the standard diagnostic panel in northern poland . 
 however , the remaining seven brca1/2 mutations ( 35  % ) would be missed by the standard approach and were detected in unique patients . a substantial proportion 
 ( n  =  5/12 ; 41  % ) of mutation - positive individuals with complete family history reported no incidence of breast or ovarian cancer in their relatives . 
 this observation , together with the raising perspectives for personalized therapy targeting brca1/2 signaling pathways indicates the necessity of comprehensive genetic screening in all ovarian cancer patients . 
 however , due to the limited sensitivity of the standard genetic screening presented in this study ( 65  % ) an application of next generation sequencing in molecular diagnostics of brca1/2 genes should be considered .