Analysis of point mutations in BRCA1 gene using hybridization on hydrogel microchips

Germ-line mutations in BRCA1 gene account for a substantial proportion of inherited breast and ovarian cancers. Identification of these mutations allows molecular diagnosis for breast cancer susceptibility. We have developed method for identification of 185delAG, 300T>G, 4153delA, 4158A>G and 5382insC mutations in BRCA1 gene using hybridization with microarray of geL-immobilized oligonucleotides (microchip). The microchip was tested with 36 control samples, carrying the above-mentioned mutations and 65 clinical cases with breast cancer. Our data demonstrated that developed microchip can be very effective and realible tool, easily introduced in ordinary medical and genetic laboratories.     

Analysis of Loss of heterozygosity and immunohistochemistry in BRCA1 gene in sporadic breast cancers

BRCA1 is a tumour suppressor gene (TSG), which predisposes cancer to both breast and ovary. The primary objective of the present study is to ascertain the involvement of BRCA1 gene in the pathogenesis of sporadic breast cancer women in Chennai (South India) by analysing its protein expression by immunohistochemistry (IHC) and loss of heterozygosity (LOH) for confirmation of the involvement of TSG in the study population. We found down regulation of BRCA1 protein (54%) in IHC and it was correlated with the clinicopathological parameters of the patients. We found near significant correlation (P < 0.063) between BRCA1 protein expression and clinicopathological parameters. We found 30% LOH in our study and it was also correlated with the clinicopathological parameters. No correlation was found between LOH and clinicopathological parameters. Though we found no correlation, the results revealed in this study support the involvement of BRCA1 TSG in the pathogenesis of sporadic breast cancer women in Chennai (South India).     

Analysis of NAT2 point mutations with biological microchips

The NAT2 product, N-acetyltransferase 2, is involved in biotransformation and detoxification of several aromatic amines (in particular, 2-aminofluorene, 4-aminobiphenyl, and 4-naphthylamine), which are strongly mutagenic and carcinogenic, and acetylates some drugs, affecting their metabolism. A biological microchip was developed to detect 16 point mutations, which determine 36 alleles and 660 genotypes of NAT2. The genotypes can be divided into four groups according to the acetylator phenotype: groups with rapid (R/R), intermediate (R/S), or slow (S/S) acetylation and a group combining intermediate and slow alleles (“R/S or S/S”). The last group includes the alleles determined by combinations of seven mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, and 857G/A), whose cis or trans position is detectable by restriction enzyme analysis. The NAT2 genotype was unequivocally established for 37 out of 71 DNA specimens, while the other 34 specimens were characterized by more than two genotypes. By the acetylator phenotype, 16 out of the 34 genotypes were assigned to the group “R/S or S/S,” combining mutations 282C/T, 341T/C, 481C/T, 590G/A, and 803A/G. Thus, the biochip allows primary analysis of most NAT2 polymorphic substitutions, the acetylator genotype being important to know in predictive medicine and individualized therapy.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

Role of BRCA1 and BRCA2 gene mutations in epithelial ovarian cancer in Indian population: a pilot study

Ovarian cancer is a silent killer as most patients have non-specific symptoms and usually present in advanced stage of the disease. It occurs due to certain genetic alterations and mutations namely founder mutations, 187delAG and 5385insC in BRCA1 and 6174delT in BRCA2 which are associated with specific family histories. These highly penetrant susceptibility genes responsible for approximately half of families containing 2 or more ovarian cancer cases account for less than 40% of the familial excess malignancy risk. The remaining risk may be due to single nucleotide polymorphisms (SNPs) which are single base change in a DNA sequence with usual alternatives of two possible nucleotides at a given position. Preliminary study involving 30 women with histologically proven epithelial ovarian cancer was conducted and their detailed genetic analysis was carried out. Regions of founder mutations on BRCA1 and BRCA2 were amplified and sequenced using primers designed based on 200 bp upstream and downstream regions of the mutation sites. Five sequence variants in BRCA1 were identified of which three novel sequence variants were found in 23 patients while in BRCA2, one novel sequence variant was found. The three founder mutations 187delAG, 5385insC in BRCA1 and 6174delT in BRCA2 were not seen in any of the subjects.     

Hereditary breast cancer; Genetic penetrance and current status with BRCA

The most important cause of developing hereditary breast cancer is germline mutations occurring in breast cancer (BCs) susceptibility genes, for example, BRCA1, BRCA2, TP53, CHEK2, PTEN, ATM, and PPM1D. Many BC susceptibility genes can be grouped into two classes, high- and low-penetrance genes, each of which interact with multiple genes and environmental factors. However, the penetrance of genes can also be represented by a spectrum, which ranges between high and low. Two of the most common susceptibility genes are BRCA1 and BRCA2, which perform vital cellular functions for repair of homologous DNA. Loss of heterozygosity accompanied by hereditary mutations in BRCA1 or BRCA2 increases chromosomal instability and the likelihood of cancer, as well as playing a key role in stimulating malignant transformation. With regard to pathological features, familial breast cancers caused by BRCA1 mutations usually differ from those caused by BRCA2 mutations and nonfamilial BCs. It is essential to acquire an understanding of these pathological features along with the genetic history of the patient to offer an individualized treatment. Germline mutations in BRCA1 and BRCA2 genes are the main genetic and inherited factors for breast and ovarian cancer. In fact, these mutations are very important in developing early onset and increasing the risk of familial breast and ovarian cancer and responsible for 90% of hereditary BC cases. Therefore, according to the conducted studies, screening of BRCA1 and BRCA2 genes is recommended as an important marker for early detection of all patients with breast or ovarian cancer risk with family history of the disease. In this review, we summarize the role of hereditary genes, mainly BRCA1 and BRCA2, in BC.     

乳腺癌易感蛋白BRCA1的BRCT1结构域染色质伸展活性的定位

乳腺癌易感基因BRCA1（Breast cancer susceptibility gene 1）在乳腺癌的发生、发展中起重要作用。BRCA1 C末端含有2个BRCT结构域（BRCT1和BRCT2）,许多乳腺癌易感突变发生在BRCA1的BRCT结构域中。利用染色质结构检测技术表明,BRCT结构域具有染色质伸展活性。本文利用缺失突变技术构建了6种BRCT1结构域（1642-1736 aa）缺失突变体并将BRCT1结构域中与染色质伸展相关的重要区域定位到1691-1721之间的氨基酸残基;用丙氨酸扫描技术构建了10种BRCT1结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到1707-1711之间的IAGGK。利用定位的重要区域进行Blast分析,结果找到一新型同源蛋白质。BRCT1结构域的定位有助于预测BRCT1结构域突变后发生乳腺癌的风险,也为进一步研究BRCT1结构域的功能机制提供了有用的材料。     

Deciphering the BRCA1 Tumor Suppressor Network

The BRCA1 tumor suppressor protein is a central constituent of several distinct macromolecular protein complexes that execute homology-directed DNA damage repair and cell cycle checkpoints. Recent years have borne witness to an exciting phase of discovery at the basic molecular level for how this network of DNA repair proteins acts to maintain genome stability and suppress cancer. The clinical dividends of this investment are now being realized with the approval of first-in-class BRCA-targeted therapies for ovarian cancer and identification of molecular events that determine responsiveness to these agents. Further delineation of the basic science underlying BRCA network function holds promise to maximally exploit genome instability for hereditary and sporadic cancer therapy.     

乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .     

利用染色质结构检测技术研究乳腺癌易感蛋白BRCA1转录激活区突变

乳腺癌易感基因BRCA1突变引起的遗传性乳腺癌中40％－50％,其突变引起的遗传性乳腺癌和卵巢癌的比例至少为80％,许多乳腺癌易感突变发生在BRCA1 C末端转录激活结构域（1560－1863aa),但该区域大部分突变导致何种表型（良性多态性或乳腺癌易感突变）目前还不清楚,由于染色质结构调节是基因转录调节的早期事件,该文基于lac阻遏物识别和结合lac操纵基因的原理,利用染色质结构检测技术比较BRCAI转录激活结构域不同突变体与野生型的染色质伸展活性,将1种野生型,2种良性多态型（S1613G和M16521）和4种乳腺癌易感突变型（A1708E,M1775R,W1837R和Y1853term)转录激活区片段以正确相位融合于lac阻遏物的下游,得到野生型重组质粒pwt和pS1613G,pM1652I,pA1708,pM1775R,pW1837R及pY1853tem6种突变型重组质粒,Western blot检测表明,这些重组质粒分别转染A03－1细胞后均表达了相应的融合蛋白。对这些重组质粒的染色质伸展活性检测表明：野生型pwt和两种良性多态性突变体不具有染色质伸展活性或只有极微弱的染色质伸展活性,而其他4种乳腺癌易感突变体均具有过强的染色质伸展活性,提示利用染色质伸展技术可预测BRCA1转录激活区基因型与乳腺癌发生风险的表现型的关系。     

A clinical case of diagnosis of breast cancer in patients with family history of BRCA mutations 1

BackgroundThe incidence of breast cancer is growing rapidly worldwide (1.7 million new cases and 600,000 deaths per year). Moreover, about 10% of breast cancer cases occur in young women under the age of 45. The aim of the study was to report a rare case of BRCA 1-mutated breast cancer in a young patient with multiple affected relatives. Breast cancer is due to a genetic predisposition with BRCA1 and BRCA2 representing a significant proportion of families with a very high risk of developing the disease over a lifetime of up to 50–80%.Case presentationIn this paper we report a case of a 29-year-old woman with a confirmed diagnosis of left breast carcinoma.ConclusionsMutations of the BRCA1 gene were revealed in the patient, in two of her sisters, brother and brother’s daughter.     

Reduced expression of the BRCA1 gene and increased chromosomal instability in MCF-7 cell line.

Using clonal cell cultures, a significant increase in chromosomal aberrations (aneuplolidy, dicentrics and chromatid breaks) were observed in MCF-7 cells compared with HeLa. BRCA1 expression was lower in MCF-7 cells than in HeLa cells. Since BRCA1 is known to play a role in the maintenance of chromosomal integrity, the increase in chromosomal aberrations in MCF-7 clones suggests that downregulation of BRCA1 expression could be one of the possible mechanisms for increased chromosomal instability in this cell line.     

大豆苷元增强多西紫杉醇体外杀伤三阴性乳腺癌作用研究

目的:研究大豆苷元通过调节BRCA1表达逆转多西紫杉醇耐药的具体机制。方法:应用三阴性人乳腺癌细胞系MDA-MB-231、SUM-1315作为研究对象,通过MTT法评估分析不同治疗组的抑瘤率,应用流式细胞仪检测各治疗组细胞的凋亡情况;应用western-blot分析各治疗组BRCA1及凋亡相关蛋白的表达情况。结果:研究发现大豆苷元、多西紫杉醇及联合组对乳腺癌MDA-MB-231、SUM1315细胞的抑制呈浓度依赖的原则。并且BRCA1表达的细胞系MDA-MB-231比BRCA1突变的细胞系SUM1315抑瘤率更高,两个细胞系不同治疗组分别与对照组相比较,凋亡率明显升高,均具有统计学意义;且随着大豆苷元的加入,可使多西紫杉醇的凋亡率显著增加,并且可减少多西紫杉醇的用量。在SUM-1315细胞系,大豆苷元逆转多西紫杉醇耐药,增加凋亡比率更为显著。大豆苷元可使BRCA1突变型的三阴性乳腺癌细胞系SUM-1315表达BRCA1,并具有浓度依赖性。对各组细胞的凋亡蛋白的变化进行免疫印迹分析表明大豆苷元联合多西紫杉醇可通过调节caspase依赖的凋亡通路活性来增强多西紫杉醇的凋亡作用,从而增强乳腺癌细胞杀伤。结论:大豆苷元可通过调节BRCA1表达量,协同增强多西紫杉类药物杀伤三阴性乳腺癌细胞,促进凋亡进而逆转化疗耐药。     

Array-based mutation detection of BRCA1 using direct probe/target hybridization

We describe here an efficient microarray-based multiplex assay to detect Korean-specific mutations in breast cancer susceptibility gene BRCA1 using direct probe/target hybridization. Allele-specific oligonucleotides were covalently immobilized on an aldehyde-activated glass slide to prepare an oligonucleotide chip. From a wild-type sample, a two-step method was used to generate labeled multiplex polymerase chain reaction (PCR) amplification products of genomic regions containing the mutation sites. Amino allyl-dUTP, an amine-modified nucleotide, was incorporated during multiplex PCR amplifications and a monofunctional form of cyanine 3 dye was subsequently attached to the reactive amine group of the PCR products. Hybridization of the labeled PCR products to the oligonucleotide chip successfully identified all of the genotypes for the selected mutation sites. This work demonstrates that oligonucleotides chip-based analysis is a good candidate for efficient clinical testing for BRCA1 mutations when combined with the indirect strategy to prepare labeled target samples.     

Characterization of cancer‐linked BRCA1‐BRCT missense variants and their interaction with phosphoprotein targets

The breast cancer tumor suppressor protein BRCA1 is involved in DNA repair and cell cycle control. Mutations at the two C‐terminal tandem (BRCT) repeats of BRCA1 detected in breast tumor patients were identified either to lower the stability of the BRCT domain and/or to disrupt the interaction of BRCT with phoshpopeptides. The aim of this study was to analyze five BRCT pathogenic mutations for their effect on structural integrity and protein stability. For this purpose, the five cancer‐associated BRCT mutants: V1696L, M1775K, M1783T, V1809F, and P1812A were cloned in suitable prokaryotic protein production vectors, and the recombinant proteins were purified in soluble and stable form for further biophysical studies. The biophysical analysis of the secondary structure and the thermodynamic stability of the wild‐type, wt, and the five mutants of the BRCT domain were performed by Circular Dichroism Spectroscopy (CD) and Differential Scanning Microcalorimetry (DSC), respectively. The binding capacity of the wt and mutant BRCT with (pBACH1/BRIP1) and pCtIP were measured by Isothermal Titration Calorimetry (ITC). The experimental results demonstrated that the five mutations of the BRCT domain: (i) affected the thermal unfolding temperature as well as the unfolding enthalpy of the domain, to a varying degree depending upon the induced destabilization and (ii) altered and/or abolished their affinity to synthetic pBACH1/BRIP1 and pCtIP phosphopeptides by affecting the structural integrity of the BRCT active sites. The presented experimental results are one step towards the elucidation of the effect of various missense mutations on the structure and function of BRCA1‐BRCT. Proteins 2009. © 2009 Wiley‐Liss, Inc.