Genetic diagnosis of familial breast cancer using clonal sequencing

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at‐risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA‐deficient breast cancers. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Clinical,pathological and genetic features of women at high familial risk of breast cancer undergoing prophylactic mastectomy

Prophylactic mastectomy (PM) is a risk-management option for women at high familial risk of breast cancer (BC). This study describes the PM experience of women enrolled in a large observational cohort study involving families with a history of hereditary breast cancer. Within 357 multiple-case BC families [119 (33%) BRCA1 or BRCA2 mutation positive], identified via family cancer clinics, 49 cases of PM [21 (43%) BRCA1 or BRCA2 mutation positive] were identified and their clinical, pathological and genetic features reviewed. Families with at least one incidence of PM displayed stronger breast/ovarian cancer histories than did families without PM. Median age at time of PM was 45 years (range 28-58). Ten cases (21%) were bilateral PMs in unaffected women and 39 cases were contralateral PMs in women with prior invasive BC (71%) or ductal carcinoma in situ (DCIS) (8%). Most (88%) underwent total mastectomy. Unnecessary axillary surgery occurred in eight subjects (16%). Malignant histology was found in three PM specimens (6%). Prior to genetic testing, PM was performed in two women who were subsequently shown not to carry the mutation specific to their family. Optimal utilization of genetic testing to guide surgical decision making, appropriate surgical technique and careful pathology examination of PM specimens, are important issues to consider prior to PM in women at high familial risk of BC.     

中国上海家族性乳腺癌BRCA1和BRCA2基因的突变

目的研究上海地区家族性乳腺癌中BRCA1／BRCA2基因的突变位点及携带情况。方法研究对象来自35个汉族家族性乳腺癌家系，家系中至少有一个一级亲属乳腺癌患病史。共35例患者，其中13例发病年龄≤加岁。由静脉血提取基因组DNA，对BRCA1／BRCA2基因的全部编码序列进行扩增。扩增产物突变分析先由变性高效液相色谱分析进行筛查，之后进行DNA直接测序证实。结果在BRCA1基因中发现有4个突变位点，其中2个为新发现位点——拼接点突变（IVS17-1G〉T；IVS21＋1G〉C）；另两个为已报道的致病突变位点——移码突变（1100delAT；5640delA）。BRCA2基因的1个致病突变位点位于11号外显子上，为移码突变（5802delAATT）。另外，共发现有12个新的单核苷重复多态位点，都未引起氨基酸编码改变；其中，8个在BRCA1基因上，4个在BRCA2基因上。在家族性乳腺癌中，BRCA1突变频率（11．4％）高于BRCA2基因（2．9％）。结论新发现的2个BRCA1基因的拼接点突变可能是中国上海人群家族性乳腺癌的特有突变位点；在我国上海地区人群中，BRCA1基因突变起着比BRCA2基因更大的作用；该研究丰富了中国人群中BRCA基因的突变谱，并为未来的临床基因检测提供了筛查模式。     

BRCA1 and BRCA2 mutations in Russian familial breast cancer

We have screened index cases from 25 Russian breast/ovarian cancer families for germ‐line mutations in all coding exons of the BRCA1 and BRCA2 genes, using multiplex heteroduplex analysis. In addition we tested 22 patients with breast cancer diagnosed before age 40 without family history and 6 patients with bilateral breast cancer. The frequency of families with germline mutations in BRCA was 16% (4/25). One BRCA1 mutation, 5382insC, was found in three families. The results of present study, and those of a separate study of 19 breast‐ovarian cancer families, suggest that BRCA1 5382insC is a founder mutation in the Russian population. Three BRCA2 mutations were found in patients with breast cancer without family history: two in young patients and one in patients with bilateral breast cancer. Four novel BRCA2 mutations were identified: three frameshift (695insT, 1528del4, 9318del4) and one nonsense (S1099X). © 2002 Wiley‐Liss, Inc.     

BRCA1 and BRCA2 mutation analysis of early-onset and familial breast cancer cases in Mexico

The entire coding regions of BRCA1 and BRCA2 were screened for mutations by heteroduplex analysis in 51 Mexican breast cancer patients. One BRCA1 and one BRCA2 truncating mutation each was identified in the group of 32 (6%) early-onset breast cancer patients (< or =35 years). Besides these two likely deleterious mutations, eight rare variants of unknown significance, mostly in the BRCA2 gene, were detected in six of 32 (19%) early-onset breast cancer cases and in three of 17 (18%) site-specific breast cancer families, one containing a male breast cancer case. No mutations or rare sequence variants have been identified in two additional families including each an early-onset breast cancer case and an ovarian cancer patient. The two truncating mutations (BRCA1 3857delT; BRCA2 2663-2664insA) and six of the rare variants have never been reported before and may be of country-specific origin. The majority of the alterations appeared to be distinct, with only one of them being observed in more than one family.     

BRCA1 and BRCA2 mutations among familial breast cancer patients from Costa Rica

Gutiérrez Espeleta GA, Llacuachaqui M, García-Jiménez L, Aguilar Herrera M, Loáiciga Vega K, Ortiz A, Royer R, Li S, Narod SA. BRCA1 and BRCA2 mutations among familial breast cancer patients from Costa Rica. The contribution of mutations in BRCA1 and BRCA2 genes to the burden of breast cancer in Costa Rica has not been studied. We estimated the frequency of BRCA mutations among 111 Costa Rican women with breast cancer and a family history of breast cancer. These women were mainly from the metropolitan area of San José. A detailed family history was obtained from each patient and a blood sample was processed for DNA extraction. Mutations in BRCA1 and BRCA2 were sought using a combination of techniques and all mutations were confirmed by direct sequencing. Four different mutations were identified in five patients (four in BRCA2 and one in BRCA1) representing 4.5% of the total. Two unrelated patients were found to have a BRCA2 5531delTT mutation. Other BRCA2 mutations included C5507G and 6174delT. Only one BRCA1 mutation was found (C3522T). The family with the BRCA1 mutation had five cases of gastric cancer. Families with BRCA2 mutations were also reported to have cases of gastric and prostate cancers; however, the full range of cancers associated with BRCA1 and BRCA2 mutations in Costa Rica has not yet been established.     

BRCA1 germline mutations in Indian familial breast cancer

Germline mutation analysis of BRCA1 gene has demonstrated significant allelic heterogeneity. These differences represent historical influences of migration, population structure and geographic or cultural isolation. To date, there have been no reports of Indian families with mutations in BRCA1. We have screened for mutations in selected coding exons of BRCA1 and their flanking intron regions in three breast or breast and ovarian cancer families with family history of three or more cases of breast cancer under age 45 and/or ovarian cancer at any age. We have also analyzed 10 female patients with sporadic breast cancer regardless of age and family history, as well as 50 unrelated normal individuals as controls. Thus a total of 90 samples were analyzed for BRCA1 mutations using polymerase chain reaction-mediated site directed mutagenesis (PSM) and single stranded conformation polymorphism (SSCP) analysis for various selected exons followed by sequencing of variant bands. Eight point mutations were identified. Two deleterious pathogenic, protein truncating non-sense mutations were detected in exon 11 (E1250X) and exon 20 (E1754X) and six novel and unique amino acid substitutions (F1734S, D1739Y, V1741G, Q1747H, P1749A, R1753K). One complex missense mutation of exon 20 [V1741G; P1749A] was seen in two out of three families and another complex combination of missense and non-sense mutations of the same exon [V1741G; E1754X] was observed in only one family. These complex mutations exist only in breast cancer families but not in control populations of women. Three splice site variants (IVS20+3A>C, IVS20+4A>T, IVS20+5A>T) and two intronic variants (IVS20+21_22insG, IVS20+21T>G) were also detected. In the group of 10 sporadic female patients no mutations were found.     

Mutation spectrum and prevalence of BRCA1 and BRCA2 genes in patients with familial and early‐onset breast/ovarian cancer from Tunisia

The contribution of BRCA1/BRCA2 mutations to hereditary breast cancer in the Tunisian population has not been accurately estimated. The purpose of our study was to estimate the incidence and spectrum of pathogenic mutations in BRCA1/2 genes in early onset and familial breast/ovarian cancer among Tunisian women. To identify predictive factors for BRCA1/2 mutations, we screened the entire coding sequences and intron/exon boundaries of BRCA1/BRCA2 genes in 48 patients by direct sequencing. Twelve pathogenic mutations were detected (25%); three in BRCA1 (c.211dupA in four families, c.5266dupC in three families and c.1504_1508delTTAAA in one family) and two novel mutations in BRCA2 (c.1313dupT in two families and c.7654dupT in two families). We also identified 23 different polymorphisms and unclassified variants. These results indicate that our population has a spectrum of recurrent BRCA mutations.     

三阴性乳腺癌患者BRCA1和BRCA2基因突变的临床特征及预后因素

目的:探究三阴性乳腺癌患者BRCA1和BRCA2基因突变检测的临床意义及预后因素分析.方法:研究对象选取为2003年1月至2015年12月之间的三阴性乳腺癌156例,所有患者均通过PCR法和DNA序列测定检验BRCA1和BRCA2基因突变情况,分析乳腺癌患者BRCA1和BRCA2基因突变特点.应用Log-Rank检验对BRCA1和/或BRCA2基因突变的三阴性乳腺癌患者的各项指标如年龄、ECOG状态、临床分期、淋巴结阳性数、月经状态和给药方式进行单因素分析.应用Cox风险比例回归模型分析患者年龄、ECOG状态、临床分期、淋巴结阳性数、肿瘤大小、月经状态和给药方式等多因素分析.结果:三阴乳腺癌患者发生基因突变21例,总体发生率13.46％,BRCA1突变15例,BRCA2突变6例.Log-Rank检验结果显示,发病年龄越大,ECOG评分越高,临床分期越晚,淋巴结阳性数越多,预后越差(P＜0.05),而发病时月经状态和给药方式与预后无关.COX风险比例回归模型显示,肿瘤大小(相对危险度,3.163;95％CI:1.455～9.287;P＜0.05)和淋巴结转移数(相对危险度,1.859;95％CI:1.254～6.875;P＜0.05)是BRCA基因突变三阴性乳腺癌独立的预后因素.结论:BRCA1和BRCA2基因突变可能与乳腺癌尤其是三阴乳腺癌可能有着密切的相关性,发病年龄、ECOG评分,临床分期和淋巴结阳性数及肿瘤大小与BRCA基因突变的三阴乳腺癌预后有关.     

Family history, BRCA mutations and breast cancer in Vietnamese women

The purpose of this report is to estimate the proportions of familial and hereditary breast cancers among unselected cases of breast cancer in Vietnam. Two hundred and ninety-two unselected cases of incident breast cancer were recruited from the National Cancer Hospital, Hanoi, the largest cancer centre in Vietnam. Family histories were collected for 292 cases and a DNA sample was obtained for 259 cases. DNA samples were screened for mutations in the large exons of BRCA1 and BRCA2 using the protein truncation test and by allele-specific testing for 17 founder mutations which have been reported in other Asian populations. Complete gene sequencing was performed on two cases of familial breast cancer. Seven of 292 cases reported a relative with breast cancer and one patient reported a relative with ovarian cancer. A pathogenic BRCA mutation was detected in 2 of 259 cases; one BRCA1 carrier was diagnosed at age 51 and one BRCA2 carrier was diagnosed at age 42. Neither case reported a relative with breast or ovarian cancer. A family history of breast cancer is very uncommon among Vietnamese breast cancer patients. The frequency of pathogenic BRCA mutations in Vietnamese breast cancer patients is among the lowest reported worldwide.     

The risk of breast cancer in BRCA1 and BRCA2 mutation carriers without a first‐degree relative with breast cancer

The objective of this study was to estimate the lifetime risk of breast cancer in women with a BRCA1 or BRCA2 mutation with and without at least 1 first‐degree relative with breast cancer. A total of 2835 women with a BRCA1 or BRCA2 mutation were followed. Age‐ and gene‐specific breast cancer rates were calculated. The relative risks of breast cancer for subjects with a family history of breast cancer, compared to no family history were calculated. The mean age at baseline was 41.1 years, and they were followed for a mean of 6.0 years. The estimated penetrance of breast cancer to age 80 years was 60.8% for BRCA1 and 63.1% for BRCA2. For all BRCA carriers, the penetrance of breast cancer to age 80 for those with no first‐degree relative with breast cancer was 60.4% and 63.3% for those with at least 1 first‐degree relative with breast cancer. The risk of breast cancer for BRCA carriers with no first‐degree relative with breast cancer is substantial, and as a result, clinical management for these women should be the same as those for women with an affected relative.     

山东半岛地区家族性和早发性乳腺癌BRCA2基因突变研究

目的 研究山东半岛地区家族性和早发性乳腺癌中乳腺癌易感基因BRCA2的突变位点及携带情况.方法 应用PureGene DNA纯化系统提取52例家族性和早发性乳腺癌患者的外周血单核细胞DNA,对BRCA2基因的全部编码序列及内含子和外显子拼接区进行扩增,扩增产物用变性高效液相色谱进行初筛,对发现异常片段经重新扩增后进行DNA测序证实.结果 在52例乳腺癌患者中发现3个(5.8%)BRCA2的致病性突变(2001delTTAT,4099＞T,5873C＞A).其中,家族性乳腺癌突变率达到12%(3/25),在单纯早发性乳腺癌病例中未发现致病性突变.结论 在家族性乳腺癌患者中,BRCA2基因突变可能具有重要作用,在此人群中有必要进行相关的基因检测.     

Contribution of BRCA1 and BRCA2 germline mutations to the incidence of early-onset breast cancer in Cyprus

In Cyprus, the prevalence of breast cancer associated with BRCA1 and BRCA2 mutations in young women is unknown. In this study, we present the results of mutational analysis of the BRCA1 and BRCA2 genes in 26 Cypriot women diagnosed with breast cancer by the age of 40. The entire coding regions, including splice sites, of the BRCA1 and BRCA2 genes were sequenced using cycle sequencing. We identified four pathogenic mutations: two in BRCA1 [c.1840A>T (K614X), c.5310delG (5429delG)] and two in BRCA2 [c.3531-3534delCAGC (3758del4), c.8755delG (8984delG)] in six of 26 unrelated patients. The BRCA2 mutation c.3531-3534delCAGC (3758del4) is novel and the BRCA1 mutation c.1840A>T (K614X) is reported for the first time in Cypriot patients. The BRCA2 Cypriot founder mutation c.8755delG (8984delG) was detected in three unrelated patients. Additionally, we identified one novel BRCA1 missense mutation, two novel polymorphisms and three novel intronic variants of which BRCA1 c.4185+3A>G (IVS12+3A>G) may be pathogenic. Of the six BRCA1/2 mutation carriers, only four had a family history. These results show that the prevalence of BRCA1 and BRCA2 mutations in Cypriot women diagnosed with early-onset breast cancer is high. We conclude that Cypriot women with early-onset breast cancer should be offered BRCA1/2 testing irrespective of their family history.     

The contribution of founder mutations to early-onset breast cancer in French-Canadian women

In an ethnically‐homogeneous population, it is valuable to identify founder mutations in cancer‐predisposing genes. Founder mutations have been found in four breast‐cancer‐predisposing genes in French‐Canadian breast cancer families. The frequencies of the mutant alleles have been measured neither in a large series of unselected breast cancer patients from Quebec, nor in healthy controls. These estimates are necessary to measure their contribution to the hereditary burden of breast cancer in Quebec and to help develop genetic screening policies which are appropriate for the province. We studied 564 French‐Canadian women with early‐onset invasive breast cancer who were treated at a single Montreal hospital. Patients had been diagnosed at age 50 or less, and were ascertained between 2004 and 2008. We screened all 564 patients for nine founder mutations: four in BRCA1, three in BRCA2 and one each in PALB2 and CHEK2. We also studied 6433 DNA samples from newborn infants from the Quebec City area to estimate the frequency of the nine variant alleles in the French‐Canadian population. We identified a mutation in 36 of the 564 breast cancer cases (6.4%) and in 35 of 6443 controls (0.5%). In the breast cancer patients, the majority of mutations were in BRCA2 (54%). However, in the general population (newborn infants), the majority of mutations were in CHEK2 (54%). The odds ratio for breast cancer to age 50, given a BRCA1 mutation, was 10.1 (95% CI: 3.7–28) and given a BRCA2 mutation was 29.5 (95% CI: 12.9–67). The odds ratio for breast cancer to age 50, given a CHEK2 mutation, was 3.6 (95% CI: 1.4–9.1). One‐half of the women with a mutation had a first‐ or second‐degree relative diagnosed with breast or ovarian cancer. Thus, it can be concluded that a predisposing mutation in BRCA1, BRCA2, CHEK2 or PALB2 is present in approximately 6% of French‐Canadian women with early‐onset breast cancer. It is reasonable to offer screening for founder mutations to all French‐Canadian women with breast cancer before age 50. The frequency of these mutations in the general population (0.5%) is too low to advocate population‐based screening.     

BRCA1 and BRCA2 in Indian breast cancer patients

Incidence of breast cancer in Indian women is not as high as in Western countries, nonetheless age-adjusted incidence rates (AAR) have risen from 17.9 to 24.9 per 100,000 from 1965 to 1985. Although these rates are still approximately one quarter to one third of incidence rates in North America and Europe, respectively, due to the large population of women at risk, nearly 80,000 new cases were diagnosed in India in 2000. Although identification of BRCA1 and BRCA2 has greatly increased our understanding of breast cancer genetics in populations of Western European descent, the role of these genes in Indian populations remains unexplored. Analysis of a series of 20 breast cancer patients from North India with either family history of breast and/or ovarian cancer (2 or more affected first degree relatives) or early age of onset (<35 years) led to identification of two novel splice variants (331+1G>T; 4476+2T>C) in BRCA1 (10%). In addition, two BRCA2 missense variants were each identified in more than one patient (two unrelated individuals each) and likely represent population-specific polymorphisms.     

Familial breast cancer

Since the localization and discovery of the first high-risk breast cancer (BC) genes in 1990, there has been a substantial progress in unravelling its familial component. Increasing numbers of women at risk of BC are coming forward requesting advice on their risk and what they can do about it. Three groups of genetic predisposition alleles have so far been identified with high-risk genes conferring 40-85% lifetime risk including BRCA1, BRCA2 and TP53. Moderate risk genes (20-40% risk) including PALB1, BRIP, ATM and CHEK2, and a host of low-risk common alleles identified largely through genome-wide association studies. Currently, only BRCA1, BRCA2 and TP53 are used in clinical practice on a wide scale, although testing of up to 50-100 gene loci may be possible in the future utilizing next-generation technology.     

散发性乳腺癌及乳腺不典型导管增生组织BRCA1基因启动子区甲基化分析

目的 了解散发性乳腺癌及癌旁增生组织、乳腺不典型导管增生组织BRCA1基因启动子区甲基化状态,探讨其与乳腺癌发生的关系.方法 采用甲基化特异性PCR(MSP)结合巢式PCR技术,研究23例散发性乳腺癌及其癌旁增生组织、6例乳腺不典型导管增生组织及5例健康成人女性外周血淋巴细胞中BRCA1基因启动子区甲基化状态.结果 5例健康成人女性外周血淋巴细胞均表现BRCA1基因启动子区甲基化阴性;23例原发性乳腺癌组织中,BRCA1基因启动子区CpG岛甲基化率为65.22%(15/23);癌旁增生组织检出CpG岛甲基化者11例,甲基化率为47.83%(11/23),且均为癌组织阳性患者;6例乳腺不典型导管增生组织中,BRCA1基因启动子区CpG岛甲基化阳性者2例,甲基化率为33.33%(2/6);统计学检验结果表明,乳腺癌、癌旁增生组织之间,BRCA1基因启动子区甲基化阳性率无显著差异.结论 BRCA1基因启动子区CpG 岛甲基化是散发性乳腺癌发生过程中的早期事件,可能在乳腺癌发生中和乳腺增生病癌变过程中起重要生物学作用.     

Prevalence of BRCA1 and BRCA2 mutations in unselected breast cancer patients from Peru

The prevalence of BRCA1 and BRCA2 mutations among breast cancer patients in Peru has not yet been explored. We enrolled 266 women with breast cancer from a National cancer hospital in Lima, Peru, unselected for age or family history. DNA was screened with a panel of 114 recurrent Hispanic BRCA mutations (HISPANEL). Among the 266 cases, 13 deleterious mutations were identified (11 in BRCA1 and 2 in BRCA2), representing 5% of the total. The average age of breast cancer in the mutation‐positive cases was 44 years. BRCA1 185delAG represented 7 of 11 mutations in BRCA1. Other mutations detected in BRCA1 included: two 2080delA, one 943ins10, and one 3878delTA. The BRCA2 3036del4 mutation was seen in two patients. Given the relatively low cost of the HISPANEL test, one should consider offering this test to all Peruvian women with breast or ovarian cancer.