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

Sin Lee,Shaoxia Zhou,Guofan Hong,Tianjun Zhou

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.

Highlights

In the United States an estimated 231,840 new cases of invasive breast cancer are expected to be diagnosed in women and about 40,290 are expected to die from breast cancer in 2015 death rates have been decreasing slightly since 1989 as the result of treatment advances, earlier detection through screening, and increased disease awareness [1]
All nucleic acid-based molecular diagnostic tests are designed to determine the order of the four nucleotides within a gene of a pathogen or in the genome of a person, directly by DNA sequencing or indirectly by restriction fragment length polymorphism (RFLP), or by probe hybridization, including the different versions of polymerase chain reaction (PCR)-based assays
When indirect methods are used for BRCA mutations testing, such as the multiplex-PCR test for 185delAG mutation [18], ambiguous results may be generated [19]

Summary

Introduction

In the United States an estimated 231,840 new cases of invasive breast cancer are expected to be diagnosed in women and about 40,290 are expected to die from breast cancer in 2015 death rates have been decreasing slightly since 1989 as the result of treatment advances, earlier detection through screening, and increased disease awareness [1]. The newly developed generation sequencing platforms can detect all these mutations To analyze their relevance as to the risk of developing breast cancer and ovarian cancer on every woman found to be a carrier of these mutations is challenging. Several panels each consisting of a limited number of mutations have been proposed for selective screening [7,8,9,10] These BRCA1 and BRCA2 mutation testing panels still cost up to US $4,000 per test in addition to the cost for genetic counseling, and population screening is not considered cost-effective in the U.S unless the price can be lowered to US $250 per test, a threshold almost impossible to reach [11]

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Conclusion