Allelic Variation in BRCA Genes Could Be the Key to...

Allelic Variation in BRCA Genes Could Be the Key to Understanding Familial Breast Cancer Introduction Breast cancer is an uncontrollable division of cells within the breast tissue that affects about 12% of women in their lifetime. Cancer can be caused by sporadic mutations influenced by environment or by genetic disposition. Several genes play a role in cell division: Oncogenes are responsible for directing mitosis, and tumor suppression genes prevent the expression of genes involved in cell division. Mutations that occur in either gene can lead to carcinogenic tumor growth and immortal cell lines. BRCA1 and BRCA2 are tumor suppression genes known as caretakers that are responsible for maintaining the cell genome during cell†¦show more content†¦A quantitative allelic imbalance assay was developed to determine differences in gene expression from individual BRCA1/2 alleles. Allele-specific assays quantify gene expression specific to the allele being tested. For the BRCA1 gene, two individuals homozygous for the BRCA1-c.4308T/T or BRCA1-c.4308C/C polymorphism were tested. Complementary DNA (cDNA) was created from reverse-transcription polymerase chain reaction (RT-PCR) using RNAs extracted from blood lymphocytes. RT-PCR uses reverse transcriptase to form an RNA/cDNA heteroduplex that is then amplified by normal polymerase chain reaction techniques to produce a large quantity of cDNA. Ratios of the cDNA from the two alleles were mixed for use in real-time PCR (qPCR). qPCR uses fluorescent probes that anneal to the cDNA during PCR. These probes contain a reporter and a quencher; the reporter fluoresces when separated from the quencher, allowing a computer to measure the number of cycles needed for the fluorescence to exceed background levels (cycle threshold or CT). Using the ratios of cDNAs and ∆CT, a linear regression was computed to form an allelic expression standard curve that can be used to evaluate allelic imbalance. These same methods were repeated with BRCA2 with two individuals homozygous for the BRCA2-c.3396A/A or BRCA2-c.3396G/ G allele. The allelic imbalance assay developed was now used with specific single-nucleotide polymorphisms in BRCA1