2017, Vol. 38
表 1
(Tab 1)
中国遗传高风险乳腺癌患者BRCA1/2编码区SNP突变频率及其与肿瘤临床病理特征相关研究
引用本文
方敏, 张小曦, 于跃, 李恒宇, 吴凯男, 林健, 盛湲. 中国遗传高风险乳腺癌患者BRCA1/2编码区SNP突变频率及其与肿瘤临床病理特征相关研究[J]. 第二军医大学学报, 2017, 38(3): 276-281 
FANG Min, ZHANG Xiao-xi, YU Yue, LI Heng-yu, WU Kai-nan, LIN Jian, SHENG Yuan. Mutation frequency of SNP in BRCA1/2 coding region and its relationship with clinicopathological features of Chinese patients with hereditarily high-risk breast cancer[J]. Academic Journal of Second Military Medical University, 2017, 38(3): 276-281 (in Chinese with English abstract)
中国遗传高风险乳腺癌患者BRCA1/2编码区SNP突变频率及其与肿瘤临床病理特征相关研究
第二军医大学长海医院甲乳外科, 上海 200433
摘要:
目的
研究中国遗传高风险乳腺癌患者乳腺癌易感基因（BRCA）1/2编码区单核苷酸多态性（SNP）的突变频率，并探讨其相应位点多态性与肿瘤临床病理特征的关系。
方法
收集提取69例遗传高风险乳腺癌患者的外周血单个核细胞DNA，运用二代测序技术对BRCA1/2编码区的49个外显子序列进行检测分析。同时收集所有患者的临床病理资料，包括发病年龄、初潮年龄、首次妊娠年龄、肿瘤的TNM分期、免疫组化特征、是否为双侧乳腺癌及是否具有家族史等，分析BRCA1/2编码区相应位点多态性与肿瘤临床病理特征的相关性。
结果
研究共发现34个SNP位点，其中14个位于BRCA1，20个位于BRCA2；有高频位点18个及低频位点16个。BRCA1编码区rs80356892位点多态性与肿瘤临床病理特征存在相关性，该位点突变的患者倾向于双侧乳腺癌（P=0.005）、存在家族史（P=0.029）以及三阴性乳腺癌（P < 0.001），且rs80356892位点突变与雌激素受体、孕激素受体表达呈负相关。
结论
BRCA1/2编码区SNP与乳腺癌发病风险及乳腺癌临床病理特征相关，因此SNP的检测将有助于发病风险的评估及遗传性乳腺癌的筛查、防治。
关键词:
疾病遗传易感性
乳腺肿瘤
BRCA基因
单核苷酸多态性
突变频率
病理特征
Mutation frequency of SNP in BRCA1/2 coding region and its relationship with clinicopathological features of Chinese patients with hereditarily high-risk breast cancer
Department of Breast and Thyroid Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Supported by National Natural Science Foundation of China (8150111616).
Abstract:
Objective
To explore the mutation frequency of single nucleotide polymorphism (SNP) in the breast cancer susceptibility gene (BRCA)1/2 coding region and its relationship with clinicopathological features of Chinese patients with hereditarily high-risk breast cancer.
Methods
We extracted the DNA of peripheral blood mononuclear cells from 69 Chinese hereditarily high-risk patients with breast cancer, and detected the sequences of 49 exons in BRCA1/2 coding region by second-generation sequencing technology. At the same time, we collected the clinicopathological data of all patients, including the age at diagnosis, the age at menarche, first pregnancy age, TNM stage, immunohistochemical status, whether it was bilateral breast cancer and/or with family history, and analyzed the relationship of site polymorphism of BRCA1/2 coding region with clinicopathological features of breast cancer.
Results
A total of 34 SNPs were identified, among which 14 were located in the BRCA1 and 20 in the BRCA2 coding region, including 18 high frequency sites and 16 low frequency sites. The rs80356892 site polymorphism of BRCA1 coding region was significantly correlated with the clinicopathological features of breast cancer. The patients with rs80356892 site mutation were inclined to have bilateral breast cancer (P=0.005), family history (P=0.029) and triple negative breast cancer (P < 0.001). In addition, SNP in rs80356892 site was negatively correlated with the expressions of estrogen receptor and progesterone receptor.
Conclusion
SNP in BRCA1/2 coding region are associated with the onset risk and the clinicopathological features of breast cancer. The detection of SNP may contribute to risk assessment of breast cancer and screening and prevention of hereditary breast cancer.
Key words:
genetic predisposition to disease
breast neoplasms
BRCA genes
single nucleotide polymorphism
mutation frequency
pathological features
近年来，乳腺癌在中国女性癌症发病率排名中占首位，约占每年新发癌症的15%[1]。其中，遗传性乳腺癌约占5%~10%[2]。对于遗传高危乳腺癌患者，仅在不足30%的患者中能鉴别出明确的有害突变位点，其余可能与中、低度外显基因及基因的单核苷酸多态性 (single nucleotide polymorphism，SNP) 相关[3]。大量研究表明乳腺癌易感基因 (breast cancer susceptibility gene，BRCA)1/2的SNP影响乳腺癌的发病风险[4-6]。随着二代测序 (next-generation sequencing，NGS) 技术的发展，现已可对多基因进行检测分析并评估复发风险[7]。除了有明显家族史的患者，对具有其他临床病理特征的患者是否需要行基因检测，目前尚未见报道。本研究对69例遗传高风险乳腺癌患者BRCA1/2编码区的49个外显子序列进行检测分析，比较不同SNP与肿瘤临床病理特征的关系，旨在更好地筛查与防治遗传性乳腺癌。
1 材料和方法 1.1 入组标准选取2014年5月至2016年5月就诊于第二军医大学长海医院、经病理证实的乳腺癌患者，符合以下任何一项者纳入本研究：(1) 发病年龄≤50岁，且至少有1个一级或二级亲属也为发病年龄≤50岁的乳腺癌患者或至少有1个一级或二级亲属为任何发病年龄的卵巢上皮癌或输卵管癌或原发性腹膜癌患者；(2) 单一个体患2种原发性乳腺癌，并且首次发病年龄≤50岁；(3) 至少有2个一级或二级亲属患有任何发病年龄的乳腺癌或卵巢上皮癌或输卵管癌或原发性腹膜癌；(4) 发病年龄≤35岁的早发性乳腺癌患者；(5) 具有血缘关系的男性近亲患有乳腺癌；(6) 合并有卵巢上皮癌或输卵管癌或原发性腹膜癌的既往史。本研究共纳入符合标准的患者69例，对其外周血BRCA1和BRCA2编码区的49个外显子序列进行检测分析；同时收集患者完整的临床及病理资料，包括发病年龄、肿瘤病理类型、临床分期、免疫组化特征及是否为双侧或遗传性乳腺癌等，其中双侧乳腺癌患者的两侧肿瘤相关数据分别按两个独立个体进行统计。所有患者均签署了BRCA基因检测知情同意书，该研究获得第二军医大学长海医院伦理委员会批准。
1.2 基因检测方法每位患者抽取5 mL外周血，从外周血白细胞中提取基因组DNA作为实验材料，基因组DNA提取方法参照QIAGEN DNeasy Blood & Tissue Kit。按照Illumina标准操作流程制备DNA小片段文库:取1 μg基因组DNA，用Biorupter非接触式全自动超声波破碎仪 (Diagenod) 将其打断成200 bp的小片段，然后依次进行末端修复、末端加A和末端加接头 (adapter)，并使用KAPA HiFi DNA Polymerase对文库进行扩增。使用Qubit3.0 Fluorometer (Invitrogen) 检测制备好的DNA小片段文库的浓度，使用Agilent 2100 Bioanalyzer检测文库插入片段的大小。按照Roche NimbleGen SeqCap EZ Choice标准操作流程制备外显子文库：取12~16个DNA小片段文库行pooling杂交，之后进行目标区域捕获，并使用1×KAPA HiFi Hot Start Ready Mix对杂交捕获到的目的片段进行LM-PCR扩增富集。扩增富集后所得的外显子文库中插入片段的大小使用Agilent 2100 Bioanalyzer进行检测，并使用qPCR进行文库上机前定量检测。最后用Illumina HiSeq 2500行PE100测序。
1.3 统计学处理采用SPSS 22.0软件进行数据处理。计量资料比较采用t检验; 计数资料比较采用χ2检验。检验水准 (α) 为0.05。
2 结果 2.1 一般情况本研究共入组乳腺癌患者69例，男性2例，女性67例；发病年龄24~75岁，平均 (37.3±10.1) 岁，肿瘤发病年龄≤35岁的占61.0%(47/77)。11.6%(8/69) 的患者患有双侧原发性乳腺癌，43.5%(30/69) 的患者有乳腺癌家族史。入组患者的肿瘤总数为77，主要病理类型为浸润性导管癌，占88.3%(68/77)，其余为小叶癌、黏液癌等其他类型肿瘤。
2.2 核苷酸多态性研究共发现34个SNP位点，其中14个位于BRCA1，20个位于BRCA2。在18个高频SNP (频率>5%) 位点中，有17个位点的突变频率大于正常人群携带频率 (正常人群携带频率来源：PubMed ClinVar)；而rs799917位点的突变频率小于正常人群携带频率，推测该位点发生突变可能是乳腺癌发病风险的保护因素。此外，研究还检测到16个低频SNP位点。见表 1。
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表 1 BRCA1/2编码区SNP位点突变频率比较 Tab 1 Comparison of mutation frequency of SNP site in BRCA1/2 coding region |
2.3 BRCA1编码区rs80356892位点多态性
研究共发现5例患者携带rs80356892位点突变，其中3例为双侧原发性乳腺癌患者, 具体临床特征见表 2。将患者是否携带rs80356892位点多态性与肿瘤的临床病理特征进行比较，结果发现两者具有相关性，携带rs80356892位点突变的患者倾向于双侧乳腺癌 (60.0% vs 7.8%，P=0.005)、存在家族史 (100.0% vs 39.1%，P=0.029) 以及三阴性乳腺癌[雌激素受体 (estrogen receptor，ER)、孕激素受体 (progesterone receptor，PR) 和人类表皮生长因子受体2(human epidermal growth factor receptor-2，Her2) 均阴性；87.5% vs 18.8%，P < 0.001]。此外，在携带rs80356892位点突变的患者中，肿瘤ER、PR的表达均为阴性，提示该位点多态性与激素受体 (hormone receptor，HR) 表达呈负相关。见表 3。
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表 2 rs80356892位点多态性携带者的临床特征 Tab 2 Clinical characteristics of rs80356892 site polymorphism carriers |
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表 3 BRCA1编码区rs80356892位点多态性与肿瘤临床病理特征的关系 Tab 3 Association of rs80356892 site polymorphism in BRCA1 coding region with clinicopathological features |
3 讨论
遗传性乳腺癌主要与3类基因突变相关，首先是BRCA1/2、PTEN、TP53、CDH1及STK11等基因的高外显性突变，其次是CHEK2、BRIP1、ATM及PALB2等基因的中度外显性突变，此外还有一些低度外显性突变的SNP[3]。BRCA1/2发生特定突变会增加乳腺癌的患病风险。Gabai-Kapara等[8]报道BRCA1突变的女性80岁时患乳腺癌或卵巢癌的风险达83%，BRCA2突变的女性70岁时患乳腺癌或卵巢癌的风险为76%。研究发现超过90%的遗传性乳腺癌与BRCA1/2突变相关[9]。但是目前仅在不到25%的家族性乳腺癌患者中能通过基因检测鉴别出明确的高外显性有害突变位点[3]。BRCA1/2的突变频率在不同人群中差别较大 (1/300~1/800)，其突变位点和类型也各不相同[10]。目前有关BRCA1/2突变的数据库大多以国外人群为主，由于缺乏大规模观测数据，中国人群的突变情况尚未形成统一结论。
随着NGS的发展，越来越多的基因突变位点及SNP被发现，不仅能更全面地筛查单基因相关遗传性乳腺癌，更为分析多基因相关遗传性乳腺癌建立了良好的技术平台。虽然NGS检测费用昂贵，不能作为常规检查项目，但是对于高度怀疑的基因突变携带者，应推荐其行基因检测以助于临床治疗[3]。因此建立适合中国人群的基因检测标准至关重要，对突变携带者临床病理特征的探讨将有助于基因检测目标人群的筛选。既往研究报道BRCA1可以影响ER在人乳腺癌和前列腺癌细胞中的转录活性，因而BRCA1有害突变携带者发生的乳腺癌往往倾向于基底细胞样乳腺癌[11-16]。BRCA2有害突变携带者发生的乳腺癌的病理特征与散发性乳腺癌相似[14-17]。但是关于中国人群BRCA1/2编码区SNP与肿瘤临床病理特征的关系目前尚未见报道。本研究对69例中国遗传高风险乳腺癌患者BRCA1/2编码区的49个外显子序列进行检测分析，结果共检出34个SNP位点，在其中的18个高频SNP中有17个位点的突变频率高于正常人群携带频率，提示这些位点的多态性可能是乳腺癌发病的危险因素。但该结果尚需通过对正常中国人群相应位点的检测及分析来进行验证。在被检出的SNP位点中，位于BRCA1编码区10号外显子，第2566位的T被C所替代，造成酪氨酸转变为组氨酸，但目前尚不清楚该位置氨基酸的转变与BRCA1蛋白功能的关系。该突变在本研究中的检出频率为7.2%，明显高于正常人群携带频率，且该位点的核苷酸多态性与乳腺癌的临床病理特征相关。该突变携带者的临床病理特征更倾向于双侧乳腺癌、有家族史及分子分型为三阴性乳腺癌。结合BRCA1蛋白的功能，推测该位点突变可能与抑制肿瘤组织中HR的表达相关。因此，我们提出几点设想：(1) 对于具有上述临床病理特征的患者，可进行该位点突变的筛查；(2) 对于该位点突变阳性的患者，可对其家属进行基因检测，以评估乳腺癌发病风险并实施相应的预防措施；(3) 对该位点突变的筛查可能成为评估对侧乳腺癌风险的一项新指标。但是目前尚没有证据支持单个SNP位点在乳腺癌发病中的作用，它可能与同一基因的其他突变位点发生协同作用或参与到多基因相关遗传性乳腺癌的发病过程中[3, 18]。本研究是一项单中心、小样本的高危人群检测，得到的仅是初步结果，尚不能得出相关SNP与遗传性乳腺癌有直接关系的结论，需要后续对多中心、大样本、高选择性人群进一步进行检测分析来证实。
近年来，人们越来越重视遗传性乳腺癌的筛查、预防及治疗。欧洲临床肿瘤协会 (European Society for Medical Oncology，ESMO) 及美国国立综合癌症网络 (National Comprehensive Cancer Network，NCCN) 指出，携带遗传性乳腺癌相关基因的突变者应该提高自检及乳房体检意识[19-20]。对于有明确家族史的患者，绝经前行预防性卵巢输卵管切除术可明显降低乳腺癌、卵巢癌的发病风险[21-22]。预防性乳房切除可降低乳腺癌高危患者的发病风险及对侧乳腺癌的发病风险[23]。此外，有研究显示，口服他莫昔芬可降低BRCA1/2突变携带者乳腺癌的发病风险[24-25]。目前对于遗传性乳腺癌的治疗，大多还是依据肿瘤本身的组织学及免疫组化特点进行的，但一项正在进行的Ⅱ期临床试验发现，BRCA相关乳腺癌可能对Poly聚合酶 (PARP) 抑制剂更加敏感[26]。Arun等[27]研究发现BRCA1突变携带者可从蒽环类、紫杉烷类方案中获益。因此，如何准确地筛选出遗传性乳腺癌患者及乳腺癌相关基因突变携带者尤为重要。目前仅小部分有遗传倾向的乳腺癌患者能明确致病性突变位点，这提示我们：(1) 尚存在大量的有害突变位点未被发现；(2) 某些突变是否与特定的临床病理特征相关，尚需进一步研究验证；(3) 对具有相应临床病理特征的患者应进行基因检测，以更好的筛查、预防及治疗遗传性乳腺癌。
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