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BRCA germline mutations in an unselected nationwide cohort of Chinese patients with ovarian cancer and healthy controls

Open AccessPublished:August 02, 2018DOI:https://doi.org/10.1016/j.ygyno.2018.07.024

      Highlights

      • 22.4% (17.1% BRCA1, 5.3% BRCA2) OC patients and 0.4% (0.3 BRCA1, 0.1% BRCA2) controls carried deleterious variants in Chinese.
      • The dense cluster of mutations in BRCA1 exon10 indicated that hypermutation was a characteristic of Chinese OC patients.
      • The estimated odds ratio (OR) of OC associated with BRCA1 positive variants was 34.6 (95% CI, 12.5–95.7) in age < 40 group.
      • The OR was 42.4 (95% CI, 5.9–305.2) in age ≥ 50 group.

      Abstract

      Objective

      To investigate the BRCA status in Chinese patients with ovarian cancer (OC). Though there were two large prevalence studies in Chinese OC patients, this was the first time to observe it in healthy controls.

      Methods

      We performed BRCA mutation screening using next-generation sequencing to determine the prevalence of BRCA germline deleterious mutations in an unselected cohort of Chinese OC patients (n = 1331) versus healthy controls (n = 1763) and describe the types and spectrum of BRCA deleterious variants.

      Results

      Among the 1331 patients with OC, 227 (17.1%) carried deleterious variants in BRCA1 and 70 (5.3%) carried deleterious variants in BRCA2. Of 1763 control subjects, 6 (0.3%) and 2 (0.1%) had deleterious variants in BRCA1 and BRCA2. No patient carried mutations in both BRCA1 and BRCA2 simultaneously. Sixty-three novel mutations were identified, and three Chinese specific hot-spot mutations were notified as BRCA1 c.5470_5477delATTGGGCA, BRCA1 c.981_982delAT, and BRCA1 c.3770_3771delAG. Interestingly, all these high-frequency recurrent mutations were distributed on exon 10, which may also be the Chinese OC BRCA mutations' distinct characteristics. In addition, in our study, the estimated odds ratio (OR) of OC associated with BRCA1 positive variants were approximately 34.6 (95% CI, 12.5–95.7) in age group under 40 and 42.4 (95% CI, 5.9–305.2) in group older than 50 in the Chinese population, respectively.

      Conclusions

      We recommend BRCA testing to all Chinese OC patients and those general Chinese who have family members with hereditary breast and ovarian related cancer (HBOC)-related cancers. Variants carriers would not only benefit from early prevention of OC but also for the medical management.

      Keywords

      1. Introduction

      Ovarian cancer (OC) was ranked the fifth cancer mortality in US women (14,070 estimated deaths in 2018) due to the late diagnosis stage and the quick resistance to existing medical treatment [
      • Siegel R.L.
      • Miller K.D.
      • Jemal A.
      Cancer statistics, 2018.
      ]. In China, there were 52,100 women diagnosed with OC, and about 22,500 OC-related deaths reported in 2015 [
      • Chen W.
      • Zheng R.
      • Baade P.D.
      • Zhang S.
      • Zeng H.
      • Bray F.
      • Jemal A.
      • Yu X.Q.
      • He J.
      Cancer statistics in China, 2015.
      ]. Furthermore, no early screening strategy has been proven effective [
      • Buys S.S.
      • Partridge E.
      • Black A.
      • Johnson C.C.
      • Lamerato L.
      • Isaacs C.
      • Reding D.J.
      • Greenlee R.T.
      • Yokochi L.A.
      • Kessel B.
      • Crawford E.D.
      • Church T.R.
      • Andriole G.L.
      • Weissfeld J.L.
      • Fouad M.N.
      • Chia D.
      • O'Brien B.
      • Ragard L.R.
      • Clapp J.D.
      • Rathmell J.M.
      • Riley T.L.
      • Hartge P.
      • Pinsky P.F.
      • Zhu C.S.
      • Izmirlian G.
      • Kramer B.S.
      • Miller A.B.
      • Xu J.-L.
      • Prorok P.C.
      • Gohagan J.K.
      • Berg C.D.
      • PLCO Project Team
      Effect of screening on ovarian cancer mortality.
      ]. The unreliable evaluated screening interventions such as transvaginal ultrasound or/and CA-125 level made it difficult to diagnose OC at early stage [
      • Henderson J.T.
      • Webber E.M.
      • Sawaya G.F.
      Screening for ovarian cancer.
      ].
      Cumulative incidence of OC is 1.4% in general population [
      • Ries L.A.G.
      • Young J.L.
      • Keel G.E.
      • Eisner M.P.
      • Lin Y.D.
      • Horner M.J.
      Cancer survival among adults: US SEER Program, 1988–2001: patient and tumor characteristics.
      ]. Deleterious germline mutations in BRCA1 and BRCA2 have become the most important predisposing factors in OC [
      • Antoniou A.
      • Pharoah P.D.P.
      • Narod S.
      • Risch H.A.
      • Eyfjord J.E.
      • Hopper J.L.
      • Loman N.
      • Olsson H.
      • Johannsson O.
      • Borg A.
      • Pasini B.
      • Radice P.
      • Manoukian S.
      • Eccles D.M.
      • Tang N.
      • Olah E.
      • Anton-Culver H.
      • Warner E.
      • Lubinski J.
      • Gronwald J.
      • Gorski B.
      • Tulinius H.
      • Thorlacius S.
      • Eerola H.
      • Nevanlinna H.
      • Syrjäkoski K.
      • Kallioniemi O.-P.
      • Thompson D.
      • Evans C.
      • Peto J.
      • Lalloo F.
      • Evans D.G.
      • Easton D.F.
      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.
      ]. The risk of contracting OC by age 70 was estimated to be 39% for women with BRCA1 mutations and 11% for women with BRCA2 mutations [
      • Antoniou A.
      • Pharoah P.D.P.
      • Narod S.
      • Risch H.A.
      • Eyfjord J.E.
      • Hopper J.L.
      • Loman N.
      • Olsson H.
      • Johannsson O.
      • Borg A.
      • Pasini B.
      • Radice P.
      • Manoukian S.
      • Eccles D.M.
      • Tang N.
      • Olah E.
      • Anton-Culver H.
      • Warner E.
      • Lubinski J.
      • Gronwald J.
      • Gorski B.
      • Tulinius H.
      • Thorlacius S.
      • Eerola H.
      • Nevanlinna H.
      • Syrjäkoski K.
      • Kallioniemi O.-P.
      • Thompson D.
      • Evans C.
      • Peto J.
      • Lalloo F.
      • Evans D.G.
      • Easton D.F.
      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.
      ]. And it has been reported that the prevalence of BRCA mutations in OC patients varies among different ethnic groups [
      • Zhong Q.
      • Peng H.L.
      • Zhao X.
      • Zhang L.
      • Hwang W.T.
      Effects of BRCA1- and BRCA2-related mutations on ovarian and breast cancer survival: a meta-analysis.
      ]. There were hitherto only a few studies investigating its prevalence in Asian population [
      • Béroud C.
      • Collod-Béroud G.
      • Boileau C.
      • Soussi T.
      • Junien C.
      Mutational analysis of BRCA1 and BRCA2 genes in Chinese ovarian cancer identifies 6 novel germline mutations.
      ,
      • Khoo U.S.
      • Chan K.Y.K.
      • Cheung A.N.Y.
      • Xue W.C.
      • Shen D.H.
      • Fung K.Y.
      • Ngan H.Y.S.
      • Choy K.W.
      • Pang C.P.
      • Poon C.S.P.
      • Poon A.Y.A.
      • Ozcelik H.
      Recurrent BRCA1 and BRCA2 germline mutations in ovarian cancer: a founder mutation of BRCA1 identified in the Chinese population.
      ,
      • Than N.G.
      • Papp Z.
      Chap 11. Ethical issues in genetic counseling.
      ,
      • Kim Y.
      • Nam E.
      • Yoon B.
      • Kim S.
      • Kim S.
      • Kim J.
      • Kim H.
      • Koo J.
      • Kim J.
      Germline mutations of BRCA1 and BRCA2 in Korean sporadic ovarian carcinoma.
      ,
      • Shi T.
      • Wang P.
      • Xie C.
      • Yin S.
      • Shi D.
      • Wei C.
      • Tang W.
      • Jiang R.
      • Cheng X.
      • Wei Q.
      • Wang Q.
      • Zang R.
      BRCA1 and BRCA2 mutations in ovarian cancer patients from China: ethnic-related mutations in BRCA1 associated with an increased risk of ovarian cancer.
      ,

      X. Wu, L. Wu, B. Kong, J. Liu, R. Yin, H. Wen, N. Li, H. Bu, Y. Feng, Q. Li, X. Lu, J. Wei, X. Zhu, J. Mills, G. Ellison, T. Gutjahr, Y. Liu, The First Nationwide Multicenter Prevalence Study of Germline BRCA1 and BRCA2 Mutations in Chinese Ovarian Cancer Patients, (n.d.). doi:https://doi.org/10.1097/IGC.0000000000001065.

      ]. Moreover, the prevalence of BRCA1 mutations of 0.1%, and BRCA2 mutations of 0.2%, is low in other/western population [
      • Antoniou A.C.
      • Cunningham A.P.
      • Peto J.
      • Evans D.G.
      • Lalloo F.
      • Narod S.A.
      • Risch H.A.
      • Eyfjord J.E.
      • Hopper J.L.
      • Southey M.C.
      • Olsson H.
      • Johannsson O.
      • Borg A.
      • Passini B.
      • Radice P.
      • Manoukian S.
      • Eccles D.M.
      • Tang N.
      • Olah E.
      • Anton-Culver H.
      • Warner E.
      • Lubinski J.
      • Gronwald J.
      • Gorski B.
      • Tryggvadottir L.
      • Syrjakoski K.
      • Kallioniemi O.-P.
      • Eerola H.
      • Nevanlinna H.
      • Pharoah P.D.P.
      • Easton D.F.
      • Easton D.F.
      The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions.
      ], it is necessary to investigate their presentations in Chinese population prevalence. Additionally, the risk of OC is still rising in women with a family history of ovarian or breast cancer but without known mutations in susceptibility genes [
      • Permuth-Wey J.
      • Sellers T.A.
      Epidemiology of ovarian cancer.
      ].
      According to National Comprehensive Cancer Network (NCCN) guidelines [
      • Pilarski R.
      • Buys S.S.
      • Farmer M.
      • Friedman S.
      • Garber J.E.
      • Dana-Farber M.
      • Hutton M.L.
      • Kauff N.D.
      • Khan S.
      • Klein C.
      • Kohlmann W.
      • Kurian A.
      • Laronga C.
      • Litton J.K.
      • Madlensky L.
      • Mak J.S.
      • Merajver S.D.
      • Offit K.
      • Reiser G.
      • Shannon K.M.
      • Swisher E.
      • Thaker P.
      • Vinayak S.
      • Weitzel J.N.
      • Wick M.J.
      • Wiesner G.L.
      • Wisinski K.B.
      • Susan Darlow N.
      • Dwyer M.
      NCCN Guidelines Index Table of Contents Discussion NCCN Guidelines Version 1.2018 Panel Members Genetic/Familial High-Risk Assessment: Breast and Ovarian.
      ], all OC patients are recommended to take BRCA testing for genetic counseling before medical managements, given OC patients with BRCA mutations have higher sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors [
      • Audeh M.W.
      • Carmichael J.
      • Penson R.T.
      • Friedlander M.
      • Powell B.
      • Bell-McGuinn K.M.
      • Scott C.
      • Weitzel J.N.
      • Oaknin A.
      • Loman N.
      • Lu K.
      • Schmutzler R.K.
      • Matulonis U.
      • Wickens M.
      • Tutt A.
      Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial.
      ] and platinum-containing agents [
      • Ledermann J.
      • Harter P.
      • Gourley C.
      • Friedlander M.
      • Vergote I.
      • Rustin G.
      • Scott C.
      • Meier W.
      • Shapira-Frommer R.
      • Safra T.
      • Matei D.
      • Macpherson E.
      • Watkins C.
      • Carmichael J.
      • Matulonis U.
      Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer.
      ]. However, the cost is not included in national medical insurance in China. It is essential to accurately find the OC patients who are more likely to benefit from BRCA testing. Wide variations of BRCA gene and full spectrum of BRCA mutations, especially Chinese population-based, have far yet been fully investigated, which also makes genetic counseling even more difficult. Thus, to address these clinical questions and public health concerns, we performed analyses of BRCA germline mutation prevalence in unselected patients with OC and healthy controls to assess the risk of developing OC in BRCA1/2 mutated Chinese women using Next Generation Sequencing (NGS).

      2. Material and methods

      2.1 Study population and sample collection

      Patients with ovarian cancer and control group (all samples were collected in 2017) was included in our study. We recruited incident specimens and controls from Chinese national wide hospitals [
      • Wen W.X.
      • Allen J.
      • Lai K.N.
      • Mariapun S.
      • Hasan S.N.
      • Ng P.S.
      • Lee D.S.-C.
      • Lee S.Y.
      • Yoon S.-Y.
      • Lim J.
      • Lau S.Y.
      • Decker B.
      • Pooley K.
      • Dorling L.
      • Luccarini C.
      • Baynes C.
      • Conroy D.M.
      • Harrington P.
      • Simard J.
      • Yip C.H.
      • Mohd Taib N.A.
      • Ho W.K.
      • Antoniou A.C.
      • Dunning A.M.
      • Easton D.F.
      • Teo S.H.
      Inherited mutations in BRCA1 and BRCA2 in an unselected multiethnic cohort of Asian patients with breast cancer and healthy controls from Malaysia.
      ]. Of the 1360 patients with OC and 1783 control subjects recruited, 1331 and 1763 cases and controls, respectively, were contained in this study (see Fig. 1 for exclusion criteria).
      Fig. 1
      Fig. 1Flowchart illustrating the whole progress of the cohort study.

      2.2 DNA extraction

      Extraction of genomic DNA was from each case by the Mag-bind blood and tissue DNA HDQ 96 kit (Omega Bioservices, Norcross, GA, USA) according to the manufacturer's instructions. DNA purity was detected by a UV spectrophotometer (Nano Drop Technologies, Wilmington, DE) and quantification was performed using the Qubit 3.0.

      2.3 Library preparation and mutation screening using NGS approach

      The BRCA1/2 panel (Morgen, China) was used which covers the entire coding sequences of BRCA1 and BRCA2 including 10–50 bases of adjacent intronic sequence of each exon. According to the manufacturer's instructions, 120 amplicons were divided into two separate multiplex PCR amplification reactions (A/B Pools). 50 ng of sample DNA were added to each of the two reactions. PCR and the purification of PCR products were performed under the conditions according to the manufacturer's instruction. After adding barcode and adaptors for NextSeq (Illumina) using PCR reagents, the quality of PCR products was checked with LabChip GX Touch24 (PerkinElmer). The sequencing process was carried out according to Illumina's protocols (Illumina, San Diego, CA) using NextSeqCN500 (BerryGenomics, China) 300 cycle reagent cartridge corresponding to 2 × 150 bp paired end configuration. The average depth of each run was over 200×. After the sequencing, the FASTQ was used for alignment and variant calling. A variant was called when the minimum depth of coverage is 20×, with allele directional balance of 15%, and mutation threshold of 10%.

      2.4 Bioinformatics analysis

      Raw (sequence) reads were treated using FASTP (https://github.com/OpenGene/fastp). Hg19, the human genome reference sequence, aligned against the sequence reads with the transformation of Burrows-Wheeler Aligner [http://bio-bwa.sourceforge.net]. With the Genome Analysis Toolkit (GATK; https://www.broadinstitute.org/gatk), we accomplished subsequent local insertion/deletion (indel) realignment and data pre-processing, base quality score recalibration (BQSR). Genetic mutations were called with GATK UnifiedGenotyper using the default parameters except minIndelFrac (set to 0.05). Mutations were annotated by ANNOVAR (http://www.openbioinformatics.org/annovar) and VEP (http://asia.ensembl.org/Tools/VEP).

      2.5 BRCA1 and BRCA2 germline mutation classification

      Sequence variations were described according to the nomenclature (www.hgvs.org) with the use of reference sequence of NM_007294.3 (hg19) for BRCA1 and NM_000059 (hg19) for BRCA2. For the interpretations of missense mutations, mutation databases such as Breast Cancer Information Core (BIC, https://research.nhgri.nih.gov/bic/), Leiden Open Variation Database (LOVD, http://www.lovd.nl/), and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) were referred. Germline mutations fulfilled the referral condition described above were further classified as benign, likely benign, variants of undetermined significance (VUS), likely pathogenic and pathogenic (Classes 1 to 5, separately) following the classification protocol of American College of Medical Genetics (ACMG) professional practice and guidelines [
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • Grody W.W.
      • Hegde M.
      • Lyon E.
      • Spector E.
      • Voelkerding K.
      • Rehm H.L.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ]. All annotation processes were performed by at least two genetic experts independently.

      2.6 Sanger sequencing

      Sanger DNA sequencing was used to confirm identified variants (Classes 3–5) starting from the original samples using the specific gene primers.

      2.7 Statistical analysis

      Continuous and categorical variables were compared by performing Student's t-test and Pearson's χ2-test or the Fisher exact test, respectively. Each p value was based on two-sided hypothesis and p < 0.05 was inferred as statistically significant. The results are in case-control condition. Considering that breast cancer could be a cause of risk for the incidence of OC, we excluded the cases with personal history of breast cancer (n = 45) when calculating the odds ratios (ORs) to avoid the biased odds estimates. And the risk of OC incidence could elevate with increasing age, so the estimation of odds was stratified by age. Calculation in this study was based on the following assumption: 1) the hospital-based Chinese OC cases represent the general cases in Chinese OC; and 2) control group represents the general Chinese population. All analyses were performed using the SPSS Statistics software v23.0 and PRISM 7.0.

      3. Results

      3.1 Prevalence of BRCA1 and BRCA2 mutations and variants of undetermined significance (VUS)

      In this study, BRCA mutation carriers were notified as people who have likely pathogenic mutations or/and pathogenic mutations, also called as deleterious variants or BRCA positive. Of the 1331 patients with OC, 227 (17.1%) had pathogenic mutations in BRCA1 and 70 (5.3%) had deleterious mutations in BRCA2. Of 1763 control subjects, 6 (0.3%) carried deleterious variants in BRCA1 and 2 (0.1%) had deleterious variants in BRCA2 (Table 1). VUS in BRCA were presented in 85 cases (6.4%) versus 84 controls (4.8%) (Fig. 2a & b ). Cases were excluded if they carried both a BRCA1 and BRCA2 VUS mutation (n = 3) to avoid repeating count.
      Table 1Demographic characteristics and BRCA clinical status summary in Chinese ovarian cancer patients (left) and healthy controls (right).
      CategoryCases (n = 1331)p valueControls (n = 1763)p value
      Demographic factorsAll OC patients (n = 1331)BRCA1+

      (n = 227, 17.1%)
      BRCA2+ (n = 70, 5.3%)BRCA+

      (n = 297, 22.3%)
      BRCA

      (n = 1034, 77.7%)
      All healthy people (n = 1763)BRCA1+ (n = 6, 0.3%)BRCA2+ (n = 2, 0.1%)BRCA+

      (n = 8, 0.4%)
      BRCA-

      (n = 1755, 99.6%)
      Age (years, mean ± SD)51.5 ± 10.450.9 ± 8.554.1 ± 8.651.6 ± 8.652.2 ± 10.4BRCA+ vs BRCAp = 0.53 > 0.05

      BRCA1+ vs BRCA2+ p = 0.005 < 0.05
      37.5 ± 9.032.7 ± 9.130.0 ± 2.832.0 ± 7.937.5 ± 9.0BRCA+ vs BRCAp = 0.013 < 0.05

      BRCA1+ vs BRCA2+ p = 0.07 > 0.05
       ≤40143 (10.7%)22 (15.4%)0 (0)22 (15.4%)121 (84.6%)1160 (65.8%)5 (0.4%)2 (0.2%)7 (0.6%)1153 (99.4%)
       41–50424 (31.9%)85 (20%)24 (5.7%)109 (25.7%)315 (74.3%)425 (24.1%)1 (0.2%)0 (0)1 (0.2%)424 (99.8%)
       51–60465 (34.9%)82 (17.6%)29 (6.2%)111 (23.9%)354 (76.1%)158 (9.0%)0 (0)0 (0)0 (0)158 (100%)
       61–70195 (14.7%)24 (12.3%)11 (5.6%)35 (17.9%)160 (82.1%)20 (1.1%)0 (0)0 (0)0 (0)20 (100%)
       >7036 (2.7%)3 (8.3%)4 (11.1%)7 (19.4%)29 (80.6%)0 (0)0 (0)0 (0)0 (0)0 (0)
      Family history*
       No1148 (86.3%)170 (14.8%)55 (4.8%)224 (19.5%)924 (80.5%)BRCA+ vs BRCAp < 0.05

      BRCA1+ vs BRCA2+ p = 0.71 > 0.05
      1662 (94.3%)4 (0.2%)2 (0.1%)6 (0.4%)1656 (99.6%)BRCA+ vs BRCAp = 0.06 > 0.05

      BRCA1+ vs BRCA2+ p = 1.00 > 0.05
       Yes106 (7.9%)47 (44.3%)13 (12.3%)60 (56.6%)46 (43.4%)99 (5.6%)2 (2.0%)0 (0)2 (2.0%)97 (98.0%)
      Age: age of diagnosis (case group); age of detection (control group).
      Family history*: family members with HBOC-related tumours, female-relatives with breast, ovarian, and pancreatic cancer, male-relatives with breast, prostate and pancreatic cancers.
      +, positive, −, negative.
      Fig. 2
      Fig. 2BRCA1 and BRCA2 mutation status in Chinese ovarian cancer patients (a) and healthy controls (b).

      3.2 Chinese specific types and spectrum of deleterious mutations

      In this study, we found 131 pathogenic (class 5, 65.8%) and 68 likely-pathogenic (class 4, 34.2%) in patients (Fig. 2a), characterized by frameshifts, nonsense or splicing sites changes, or missense mutations according to existing database. Only 8 pathogenic (0.45%) and no likely pathogenic mutations in controls (Fig. 2b). 199 distinguishable deleterious mutations (including 138 BRCA1 and 61 BRCA2, separately) and 8 deleterious mutations (including 6 BRCA1 and 2 BRCA2, separately) occurred in OC cases and control subjects, respectively (Supplementary Table S1). Interestingly, three most notable recurrent deleterious mutations, BRCA1 c.5470_5477delATTGGGCA (18, 0.14%) [
      • Kim Y.C.
      • Zhao L.
      • Zhang H.
      • Huang Y.
      • Cui J.
      • Xiao F.
      • Downs B.
      • Wang S.M.
      Prevalence and spectrum of BRCA germline variants in mainland Chinese familial breast and ovarian cancer patients.
      ], BRCA1 c.981_982delAT (9, 0.68%) [
      • Kim Y.C.
      • Zhao L.
      • Zhang H.
      • Huang Y.
      • Cui J.
      • Xiao F.
      • Downs B.
      • Wang S.M.
      Prevalence and spectrum of BRCA germline variants in mainland Chinese familial breast and ovarian cancer patients.
      ] and BRCA1 c.3770_3771delAG (7, 0.53%) [
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • Grody W.W.
      • Hegde M.
      • Lyon E.
      • Spector E.
      • Voelkerding K.
      • Rehm H.L.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ] presented more predominate features as founder mutations. They were found in unrelated patients. In addition, they were observed with the highest frequencies in hereditary breast and ovarian related cancer (HBOC)-related families. Several other recurrent mutations in BRCA1 and BRCA2 were observed, each of which occurred in >3 patients (Supplementary Table S1). There were 22 mutations shared between OC patients and healthy controls (Supplementary Table S2). 62 novel mutations absent from publicly available databases (BIC, Clinvar, LOVD and ENIGMA) were defined in OC patients (Supplementary Table S3). The distribution of variants has a clear tendency of concentration. Most deleterious mutations in BRCA1 gene of Chinese population occurred within exon 10 (75/1331, 5.6%), whereas mutations in BRCA2 has the highest frequencies on exon 11 (36/1331, 2.7%).

      3.3 Clinicopathological characteristics

      The diagnostic age of OC patients ranged from 9 to 84 years (Table 1), the percentage of BRCA1 mutation carriers in all age groups, except the ones older than 70, is higher than 12% (p < 0.05). Whereas the age > 70 group has the highest percentage of BRCA2 mutation carriers, 11.1%. And the mean age at diagnosis of BRCA1 carriers was younger than BRCA2 carriers' (50.9 vs 54.1 years, p = 0.005), thus BRCA1 carriers may have a higher risk of developing OC than BRCA2 carriers. Moreover, of OC patients, 8.5% have HBOC-related cancers family history up to their third-degree relatives (p < 0.05). The proportion of BRCA1/2 mutation carriers is 56.6% (p < 0.05) in positive family history group. Regarding the histopathology of OC, 843 (63.3%) of the patients were serous carcinoma, among which, 27.2% (p < 0.05) were BRCA carriers. Out of these serous type cases, BRCA1 deleterious carriers were presented more than BRCA2 deleterious carriers (20.9% vs. 6.3%) (Table 2). 65.2%, 26.7%, 27.4% and 21.4% BRCA carriers were detected in the International Federation of Gynecology and Obstetrics (FIGO) stages I to IV (p < 0.01). And there is no significant difference of tumour stages in BRCA1 or BRCA2 carriers. When it comes to the control subjects, the age of healthy people participating in the study were younger than the OC group (mean ages at 51.49 and 37.47 years, separately). Although of 2.0% positive family history healthy people detected with BRCA mutation (p = 0.06), there is no significant difference in different healthy control groups with family history.
      Table 2Association between BRCA1 and BRCA2 mutation status with ovarian cancer clinical characteristics.
      CategoryCases (n = 1331)p value
      All OC patients (n = 1331)BRCA1+BRCA2+BRCA+BRCA
      (n = 228)(n = 70)(n = 297)(n = 1034)
      HistopathologyNo. (%)BRCA+ vs BRCA−, p < 0.01
       Serous843 (63.3)176 (20.9)53 (6.3)229 (27.2)614 (72.8)BRCA1+ vs BRCA2+,

      p = 0.39 > 0.05
       Adenocarcinoma_nonspecific_type120 (9.0)21 (17.5)6 (5.0)27 (22.5)93 (77.5)
       Clear_cell79 (5.9)6 (7.6)1 (1.3)6 (7.6)73 (92.4)
       Endometrioid65 (4.9)6 (9.2)1 (1.5)7 (10.8)58 (89.2)
       Mucinous57 (4.3)2 (3.5)2 (3.5)4 (7.0)53 (93.0)
       Other*40 (3.0)4 (10.0)0 (0)4 (10.0)36 (90.0)
       Mixed10 (0.8)0 (0)1 (10.0)1 (10.0)9 (90.0)
       NA117 (8.8)13 (0.1)6 (5.1)19 (16.2)98 (83.8)
      Mixed: (serous/endometrioid: 5, serous/endometrioid/mucinous: 1, serous/MMMT: 3, serous/mucinous: 1)
      FIGO stageBRCA+ vs BRCA−, p < 0.01
       I66 (5.0)34 (51.5)9 (13.6)43 (65.2)23 (34.8)BRCA1+ vs BRCA2+

      p = 0.55 > 0.05
       II217 (16.2)47 (21.7)11 (5.1)58 (26.7)159 (73.3)
       III518 (38.9)107 (20.7)36 (6.9)142 (27.4)376 (72.6)
       IV70 (5.3)13 (18.6)2 (2.9)15 (21.4)55 (78.6)
       NA460 (34.6)27 (5.9)12 (2.6)39 (8.5)421 (91.5)
      Other*: including six different rare pathology groups (malignant mixed müllerian tumours, transitional cell, and small cell carcinoma of the ovary of hypercalcemic type etc.).

      3.4 Estimated probability of Chinese BRCA carriers having ovarian cancer

      Deleterious variants in BRCA1 and BRCA2 were significantly more predominant in OC cases compared with control subjects, with estimated odd ratios (ORs) for OC BRCA1 carriers being 34.6 (95% CI, 12.5–95.7) and 42.4 (95% CI, 5.9–305.2) for age < 40 and age ≥50, respectively (Table 3). There is no enough number of BRCA2 carriers for OR calculation when they were grouped by age.
      Table 3Odds ratios.
      AgeClassCases (1286)Controls (1763)Odds ratio
      Compared with no mutation.
      (95% CI)
      <40 (case: 125; control: 1107)BRCA1 mutation carriers17534.6 (12.5–95.7)
      BRCA2 mutation carriers02
      Non-carriers1081100
      40–49 (case: 401; control: 448)BRCA1 mutation carriers800
      BRCA2 mutation carriers210
      Non-carriers300448
      ≥50 (case: 760; control: 208)BRCA1 mutation carriers121142.4 (5.9–305.2)
      BRCA2 mutation carriers480
      Non-carriers591207
      low asterisk Compared with no mutation.

      4. Discussion

      This is the first and largest study including full exon screening by NGS of BRCA1 and BRCA2 in an unselected series of Chinese OC patients and healthy controls. BRCA1 (227/1331, 17.1%) has a higher prevalence than BRCA2 (70/1331, 5.3%) in Chinese OC population, consistent with two previous large nationwide prevalence studies of germline BRCA in Chinese OC patients [
      • Shi T.
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      X. Wu, L. Wu, B. Kong, J. Liu, R. Yin, H. Wen, N. Li, H. Bu, Y. Feng, Q. Li, X. Lu, J. Wei, X. Zhu, J. Mills, G. Ellison, T. Gutjahr, Y. Liu, The First Nationwide Multicenter Prevalence Study of Germline BRCA1 and BRCA2 Mutations in Chinese Ovarian Cancer Patients, (n.d.). doi:https://doi.org/10.1097/IGC.0000000000001065.

      ]. Though three highest frequency mutations, BRCA1 c.5470_5477delATTGGGCA, BRCA1 c.981_982delAT and BRCA1 c.3770_3771delAG are recognized as Chinese hot-spot mutations [
      • Risch H.A.
      • McLaughlin J.R.
      • Cole D.E.C.
      • Rosen B.
      • Bradley L.
      • Kwan E.
      • Jack E.
      • Vesprini D.J.
      • Kuperstein G.
      • Abrahamson J.L.A.
      • Fan I.
      • Wong B.
      • Narod S.A.
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      ], haplotype assessment is under our further investigation for them being founder mutation of Chinese population. The 22 shared mutations between the case and control would be helpful for the notification of potential deleterious mutations in the further research.
      From the perspective of diagnosis age, family history of breast and/or ovarian cancer and pathological features of the cancers, our study showed consistent results with two previous OC studies although there were some minor differences in pathological classification between these studies [
      • Shi T.
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      • Shi D.
      • Wei C.
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      • Jiang R.
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      • Wei Q.
      • Wang Q.
      • Zang R.
      BRCA1 and BRCA2 mutations in ovarian cancer patients from China: ethnic-related mutations in BRCA1 associated with an increased risk of ovarian cancer.
      ,

      X. Wu, L. Wu, B. Kong, J. Liu, R. Yin, H. Wen, N. Li, H. Bu, Y. Feng, Q. Li, X. Lu, J. Wei, X. Zhu, J. Mills, G. Ellison, T. Gutjahr, Y. Liu, The First Nationwide Multicenter Prevalence Study of Germline BRCA1 and BRCA2 Mutations in Chinese Ovarian Cancer Patients, (n.d.). doi:https://doi.org/10.1097/IGC.0000000000001065.

      ]. We found that deleterious mutations in BRCA1 genes were more likely to occur in the cases with a younger diagnosis age (50.90 vs. 54.05, p < 0.05), family history of HBOC-related cancer, serous carcinoma and later tumour stage. Furthermore, BRCA mutation carriers were also more likely to have a family history of HBOC-related cancers.
      Based on our research and PARPi therapy candidates' identification, all Chinese OC patients should be offered genetic counseling. The percentages of BRCA carriers are all above 10% (Table 1, Table 2) in all subgroups of diagnosis age, family history, histopathology (excluding mucinous 7.0%, clear cell 7.6%) and FIGO stages, which are also consistent with previous BRCA mutation research in Chinese OC patients [
      • Shi T.
      • Wang P.
      • Xie C.
      • Yin S.
      • Shi D.
      • Wei C.
      • Tang W.
      • Jiang R.
      • Cheng X.
      • Wei Q.
      • Wang Q.
      • Zang R.
      BRCA1 and BRCA2 mutations in ovarian cancer patients from China: ethnic-related mutations in BRCA1 associated with an increased risk of ovarian cancer.
      ,

      X. Wu, L. Wu, B. Kong, J. Liu, R. Yin, H. Wen, N. Li, H. Bu, Y. Feng, Q. Li, X. Lu, J. Wei, X. Zhu, J. Mills, G. Ellison, T. Gutjahr, Y. Liu, The First Nationwide Multicenter Prevalence Study of Germline BRCA1 and BRCA2 Mutations in Chinese Ovarian Cancer Patients, (n.d.). doi:https://doi.org/10.1097/IGC.0000000000001065.

      ]. Generally, the probability of 10% for finding a germline BRCA mutation is believed to be cost-effective in gene testing, which also meets the standards in epithelial OC [
      • Moyer V.A.
      Risk assessment, genetic counseling, and genetic testing for BRCA-related cancer in women: U.S. Preventive Services Task Force recommendation statement.
      ,
      • Arts-De Jong M.
      • de Bock G.H.
      • van Asperen C.J.
      • Mourits M.J.E.
      • de Hullu J.A.
      • Kets C.M.
      Germline BRCA1/2 mutation testing is indicated in every patient with epithelial ovarian cancer: a systematic review.
      ].
      General Chinese people with family history of HBOC should also strongly be suggested to take BRCA testing. While regarding the healthy population, approximately 0.4% (8/1763) of deleterious variant carriers would be missed. No significant difference was found in controls with family history; however, positive family history healthy groups were significantly different (p < 0.05), when they were classified with relative degrees. Based on other ethnic population follow-up research, BRCA1 or BRCA2 mutation carriers are at high risk of developing OC, 40% and 18% separately [
      • Chen S.
      • Parmigiani G.
      Meta-analysis of BRCA1 and BRCA2 penetrance.
      ]. Overall there are 0.3% (6/1763) BRCA1 carriers and 0.1% (2/1763) BRCA2 carriers in healthy group, which is different from the prevalence of other ethnic population (0.12% and 0.2% for BRCA1 and BRCA2, respectively) [
      • Antoniou A.C.
      • Cunningham A.P.
      • Peto J.
      • Evans D.G.
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      • Pharoah P.D.P.
      • Easton D.F.
      • Easton D.F.
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      ]. The outcomes of this study were case-control status, therefore estimation of the probability that a BRCA carrier would contract OC was based on the ORs. And the odds of BRCA1 in age group under 40 and group older than 50 was 34.6 (95% CI, 12.5–95.7) and 42.4 (95% CI, 5.9–305.2). We should notice that if the controls were enriched with mutation carriers, the ORs in our analysis could be underestimated [
      • Satagopan J.M.
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      ]. Firstly, controls in this study are healthcare workers in hospitals (without retired workers, age ≥65) which would cause ascertainment bias. Secondly, individuals with family history were more likely to participate in this research. The real risk of OC in Chinese mutation carriers may be higher than our results. Full exon sequencing on unselected patients with OC enabled us to assess the frequency of possible missed carriers of BRCA1 and BRCA2 in clinical practice in China, a resource-constrained developing country with a population of around 1.4 billion. On the other hand, BRCA genetic testing in China is still not included in the national health care system, making this risk-stratified management approach costly and potentially unaffordable for most of the general Chinese people. Therefore, our research raised the urgent need for the identification of Chinese possible founder mutations or the integrated spectrum of Chinese BRCA mutations to help tailoring the specific genetic counseling for high risk OC Chinese families.
      The exon distribution frequencies of BRCA variations between Chinese and other populations were significantly different, which were reported to be a trend in certain regions conferring higher OC risk, ovarian cancer cluster region (OCCR) [
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      ]. According to our study, the mutations in BRCA1 exon 10 occurred in 75 of the 234 (32.1%) BRCA1 variation carriers (Supplementary Table S1). In BRCA2-variation cases, exon 11 frequencies were the highest in our research with 36 out of 72 (50.0%). The separation of the mutation exon distribution in our research is more distinct than previous Chinese OC BRCA germline mutation study [
      • Shi T.
      • Wang P.
      • Xie C.
      • Yin S.
      • Shi D.
      • Wei C.
      • Tang W.
      • Jiang R.
      • Cheng X.
      • Wei Q.
      • Wang Q.
      • Zang R.
      BRCA1 and BRCA2 mutations in ovarian cancer patients from China: ethnic-related mutations in BRCA1 associated with an increased risk of ovarian cancer.
      ]. Though a comprehensive BRCA variants literature analysis has reported that BRCA1 exon 11, exon 24, and BRCA2 exon 10 are the variation hot spots of the mainland Chinese familial breast and ovarian cancer patients [
      • Kim Y.C.
      • Zhao L.
      • Zhang H.
      • Huang Y.
      • Cui J.
      • Xiao F.
      • Downs B.
      • Wang S.M.
      Prevalence and spectrum of BRCA germline variants in mainland Chinese familial breast and ovarian cancer patients.
      ], their results were almost based on breast cancer studies. Interestingly, all the high frequencies recurrent mutations in our study almost completely lied within exon 10, an exon known to be involved in alternative splicing [
      • Grover A.
      • Houlden H.
      • Baker M.
      • Adamson J.
      • Lewis J.
      • Prihar G.
      • Pickering-Brown S.
      • Duff K.
      • Hutton M.
      5′ splice site mutations in tau associated with the inherited dementia FTDP-17 affect a stem-loop structure that regulates alternative splicing of exon 10.
      ,
      • Chen M.
      • Manley J.L.
      Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches.
      ]. These may offer indication of underlying mechanisms of this Chinese OC risk related location.
      There is a limitation in our study that large genomic rearrangement was not included. We did not perform multiplex ligation-dependent probe amplification analysis. What is more, some patients with deleterious mutations may have been overleaped owing to differences between the amplicon-based and capture-based sequencing approaches. Unfortunately, the high population mobility leading to the incomplete pathological information and the lack of public awareness of cancer risk related to hereditary may lead to the discrepancy with previous research and provide challenges for further assessment.
      The availability of Chinese-specific estimates of BRCA carriers in unselected OC patients and unaffected population may lead to the modifications of the existing risk models e.g. BRCAPRO [
      • Daniels M.S.
      • Babb S.A.
      • King R.H.
      • Urbauer D.L.
      • Batte B.A.L.
      • Brandt A.C.
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      Underestimation of risk of a BRCA1 or BRCA2 mutation in women with high-grade serous ovarian cancer by BRCAPRO: a multi-institution study.
      ] or the development of novel ones to predict BRCA carriers more accurately for Chinese population. On the other hand, accurate models to estimate the probabilities of BRCA mutations may help more Chinese OC patients benefit from cost-effective testing [
      • Tuffaha H.W.
      • Mitchell A.
      • Ward R.L.
      • Connelly L.
      • Butler J.R.G.
      • Norris S.
      • Scuffham P.A.
      Cost-effectiveness analysis of germ-line BRCA testing in women with breast cancer and cascade testing in family members of mutation carriers.
      ] and precision medical managements such as PARP-targeted therapy [
      • Vergote I.
      • Banerjee S.
      • Gerdes A.-M.
      • van Asperen C.
      • Marth C.
      • Vaz F.
      • Ray-Coquard I.
      • Stoppa-Lyonnet D.
      • Gonzalez-Martin A.
      • Sehouli J.
      • Colombo N.
      Current perspectives on recommendations for BRCA genetic testing in ovarian cancer patients.
      ]. Despite the prevalence of BRCA germline mutations in Chinese population is lower than other ethnic group, the high occurrence of OC among mutation carriers still point to the necessity of understanding causes of their likelihood of contracting the disease. ORs in our study indicate that BRCA screening is beneficial to clinical management such as early prevention, risk-reducing salpingo-oophorectomy [
      • Kauff N.D.
      • Satagopan J.M.
      • Robson M.E.
      • Scheuer L.
      • Hensley M.
      • Hudis C.A.
      • Ellis N.A.
      • Boyd J.
      • Borgen P.I.
      • Barakat R.R.
      • Norton L.
      • Castiel M.
      • Nafa K.
      • Offit K.
      Risk-reducing salpingo-oophorectomy in women with a BRCA1 or BRCA2 mutation.
      ] for BRCA germline mutation carriers.
      In conclusion, 22.3% of unselected Chinese OC patients and 0.4% of general Chinese population are BRCA1/2 carriers. With the healthy control, our study offers possibility of implementing OC risk evaluation for the Chinese population and emphasizes the need for understanding the contribution of exon 10 on BRCA's role in cancer development in Chinese OC patients.
      The following are the supplementary data related to this article.

      Funding

      This work was supported by the National Clinical Key Specialty Construction Program, Fujian Natural Science Foundation (Grant Number 2015J01381 ), and Training Project of Young Talents in Fujian Health System (Grant Number 2015-ZQN-JC-6 ).

      Compliance with ethical standards

      Ethical approval: All procedures performed in this study involving human participants were done in accordance with the Chinese ethical standards and with the 2008 Helsinki declaration. After the written informed consent signed, blood samples were collected from each of the cases.

      Conflict of interest statement

      None declared.

      Acknowledgement

      We are grateful to Dr. Lu Fan, Dr. Cigdem Selli and Dr. Yanni Zeng for their useful comments and suggestions. We thank Mindy Swift, Haiyan Qin, Zhaoji Lan, Ling Li, and Ling Xie for their support of sample collection and management.

      Author contribution

      Jian Liu and Yujian Shi conceived of the presented idea and supervised the findings of this work. Ang Li developed the theory and performed the statistical analysis. Ang Li wrote the manuscript with support from Rong Xie, Yujian Shi and Jian Liu. Qihuan Zhi performed the sequencing experiments with the assistant of Jiajia Zhang. Yixiao Deng, Weiwei Li and Lu Yang performed the annotation processes. Yangming Wu carried out the bioinformatic analysis. Zinan Jiao contributed to the date preparations. Jiaqi Luo and Yi Zi participated in major revision process. Gang Sun contributed to sample collection. All authors discussed the results and contributed to the final manuscript.

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