title: Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples.
abstract: Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.
authors: Milford Hospital and Milford Molecular Diagnostics Laboratory, 2044 Bridgeport Avenue, Milford, CT 06460, USA. shlee01@snet.net.
authors: Sin Hang
authors: SH
authors: Lee
authors: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China. sxzhou@njau.edu.cn.|College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. sxzhou@njau.edu.cn.
authors: Shaoxia
authors: S
authors: Zhou
authors: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China. tjzhou@sibs.ac.cn.
authors: Tianjun
authors: T
authors: Zhou
authors: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China. gfhong@sibcb.ac.cn.
authors: Guofan
authors: G
authors: Hong
mesh_terms: D000483:Alleles; D052685:Alphapapillomavirus / Q000145:classification / Q000235:genetics; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D055088:Early Detection of Cancer; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D005820:Genetic Testing / Q000379:methods; D060005:Genotyping Techniques; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000235:genetics; D065006:Papanicolaou Test; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D057170:Translational Research, Biomedical
chemical_list: D036022:Capsid Proteins; C114580:HPV L1 protein, Human papillomavirus; D009856:Oncogene Proteins, Viral
keywords: BRCA1 c.5266dup; BRCA1 c.68_69del; BRCA2 c.5946del; HPV; Pap smear cytology; Sanger sequencing; population screen