| Objective:Breast cancer is the most common malignancy among women. It is estimated that about 5% 10% of all breast cancer cases may be due to inherited predisposition. Early genetic linkage analysis suggested that breast cancer susceptibility gene-1 (BRCA1) would be responsible for 45% the site-specific breast cancer families and the majority of breast-ovarian cancer families. The data to the BRCA1 mostly came from foreign countries. Although there are some BRCA1 mutation studies in the Chinese population,they focused on women with sporadic or early breast cancer,or limited to the part coding regions of BRCA1 in breast cancer families. The aim of our study is to screen all exons mutations of BRCA1 gene in Chinese breast cancer families and some sporadic breast cancer patients.Methods:Breast cancer families and sporadic breast cancer patients,who visited or revisited our hospital between 1998 2001,were collected according to selecting standard. Ten millilitres peripheral blood sample was drawn from each of participants. Then,mononuclear cells were obtained by using Ficoll hypaqul lymphocyte separation,and genome DNA of mononuclear cells was extracted by using method of hydroxybenzene -chloroform. Twenty-two coding exons of BRCA1 gene were amplified by polymerase chain reaction (PCR) using 40 pairs of primers. PCR products were analysed by single strand conformation polymorism (SSCP). Shifted bands were detected in the polyacrylamide gel electrophoresis (PAGE). Amplified segments related to the shifted and standard bands weresequenced by using an automated fluorescence based cycle sequencer (ABI PRISM 377) according to the manufacturer's instructions.Results:(1) In this study,41 participants,which including 23 breast cancer patients and 18 volunteers,were obtained from 15 breast cancer families. Average age of patients is 49.7 years. Twenty-seven cases of sporadic breast cancer were collected,average age of which is 54.6 years. (2) PCR-SSCP:PCR products were analyzed by electrophoresis in 1.5% agaragar gels. They all showed singleness bands,unanimous with corresponding products. It illuminates that PCR products have no obvious segments absent or inserted. Abnormal bands were determined by appearing shifted bands,unexpected or broadened bands in polyacrylamide gel electrophoresis (PAGE). As a result,6 shifted bands were found in 6 patients respectively,five of which belonged to 15 breast cancer families and the other one belonged to sporadic breast cancer. Those 6 samples and with 6 control samples were then sequenced. (3) DNA sequence:Mutations were not found in 6 contrast samples and one sample from breast cancer family. Five mutations were found in the 5 samples. Four mutations belonged to 4 breast cancer families,and also belonged to 4 of 23 familiar breast cancer patients. No mutations were found in eighteen of non-patients from breast cancer families. The 4 mutations took place on the exon 11. One of mutations is inserting C at nt2228,and resulting in chain termination at codon 711. The others have one nucleotide variation and result in single amino acid changing respectively. Among of them,one variation is at nt!884A-T,resulting in cysteine substitute for serine at codon 589;another two are changes at nt3232A-G,resulting in glycine substitute for glutamic acid at codon 1038. One mutation,4804C-G,happened on exon 16,belonged to sporadic breast cancer patient (SI 7),which resulted in arginine substitute for proline at codon 1562. (4) In our study,theproportion of BRCA1 mutations was 26.7% (4/15)in breast cancer families. Mutations in family breast cancer patients were 17.4% (4/23). In sporadic breast cancer patients,BRCA1 mutations were 3.7% (1/27),and also was 8.3% under 50 years (1/12).Conclusions:Our results demonstrated that mutations of BRCA1 were lower in Chinese breast cancer families than that of Europe and American breast cancer families. There must have been existed other breast cancer susceptibility genes. The missense mutation of 3232 AG might be the hot spot mutation in Chinese...
|
PDF Full Text Request