Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples

Int J Mol Sci. 2016 Feb 8;17(2):229. doi: 10.3390/ijms17020229.

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.

Keywords: BRCA1 c.5266dup; BRCA1 c.68_69del; BRCA2 c.5946del; HPV; Pap smear cytology; Sanger sequencing; population screen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Alphapapillomavirus / classification
  • Alphapapillomavirus / genetics
  • Breast Neoplasms / diagnosis
  • Breast Neoplasms / genetics
  • Capsid Proteins / genetics
  • Cell Line, Tumor
  • Early Detection of Cancer
  • Female
  • Genes, BRCA1*
  • Genes, BRCA2*
  • Genetic Testing* / methods
  • Genotyping Techniques
  • Humans
  • Oncogene Proteins, Viral / genetics
  • Ovarian Neoplasms / diagnosis
  • Ovarian Neoplasms / genetics
  • Papanicolaou Test*
  • Reproducibility of Results
  • Sequence Analysis, DNA
  • Sequence Deletion*
  • Translational Research, Biomedical

Substances

  • Capsid Proteins
  • HPV L1 protein, Human papillomavirus
  • Oncogene Proteins, Viral