Digital PCR assays for detecting breast cancer gene variants

BRCA1, or Breast Cancer Gene 1, is a tumor suppressor whose mutations are associated with a significant increase in the risk of breast and ovarian cancers. These mutations hinder the gene's role in DNA repair, leading to genomic instability and cancer. BRCA1 mutations are particularly impactful in hereditary breast cancer syndromes, significantly raising the risk of early-onset breast cancer. Targeting these genetic vulnerabilities is crucial in the development of therapeutic strategies and management of cancer risk.

• 185delAG: The deletion of two nucleotides, adenine (A) and guanine (G), at position 185 in the BRCA1 gene occurs early in the gene's coding sequence and results in a frameshift and the production of a significantly truncated BRCA1 protein, lacking critical regions essential for its role in DNA repair and tumor suppression. The absence of these functional domains prevents BRCA1 from participating in the repair of double-strand breaks via homologous recombination, leading to genomic instability and significantly increasing the risk of breast cancer.
• 5382insC: The insertion of a cytosine (C) at position 5382 causes a frameshift mutation that alters the reading frame of the BRCA1 gene, resulting in a premature stop codon. The truncated protein produced is unable to effectively engage in DNA repair processes, diminishing the cell's ability to maintain genomic stability and promoting tumorigenesis.
  
   

BRCA2, or Breast Cancer Gene 1, functions similarly to BRCA1, aiding in DNA repair and maintaining genomic stability. Mutations in BRCA2 disrupt these critical processes, significantly increasing the risk of developing breast, ovarian, and other cancers. BRCA2 mutations are inherited and have a profound impact on cancer risk, underlining the importance of genetic testing for individuals at risk. These mutations offer insights into personalized prevention and treatment options.

• 6174delT: This mutation involves the deletion of thymine (T) at position 6174, producing a truncated BRCA2 protein that lacks a portion of its DNA binding domain, essential for repairing DNA double-strand breaks. The mutant protein's impaired DNA repair capability leads to genetic mutation accumulation and elevated breast cancer risk.
• 5946delT: Similar to 6174delT, this mutation's deletion of thymine (T) at position 5946 results in a truncated protein. This loss compromises the critical BRCA2 functionality in DNA repair, undermining genomic integrity and contributing to cancer development.
  
   

PALB2 (Partner and Localizer of BRCA2) interacts with BRCA2 in DNA repair and, when mutated, can elevate breast cancer risk. While PALB2 mutations confer a lower risk compared to BRCA1/2 mutations, their discovery is crucial for understanding individual risk profiles and guiding cancer prevention strategies. PALB2 is an emerging marker for targeted therapy research, emphasizing the need for comprehensive genetic screening in affected families.

• • 509_510delGA: This frameshift mutation involves the deletion of two nucleotides, guanine (G) and adenine (A), which alters the reading frame of the PALB2 gene. This change results in the production of a truncated protein that is unable to fulfill its role in DNA repair alongside BRCA2. The loss of function in this crucial repair pathway increases the susceptibility to breast cancer by allowing DNA damage to accumulate, potentially leading to cancerous changes within cells.
• 1592delT: This mutation represents a deletion of a single thymine (T) nucleotide at position 1592. It is another frameshift mutation that disrupts the normal sequence of the PALB2 gene, resulting in an abnormal protein product. The consequent deficiency in the gene's repair function compromises genomic integrity and significantly raises the risk of developing breast cancer, highlighting the critical role of PALB2 in maintaining cellular health and preventing tumorigenesis.
• 3113+5G>A: This splice site mutation occurs in the intronic region near exon 13 of the PALB2 gene, specifically changing a guanine (G) to adenine (A) at the +5 position of the intron following exon 12. By altering the gene's natural splicing process, this mutation can lead to an incorrectly spliced PALB2 mRNA, potentially resulting in a dysfunctional protein that is incapable of effectively participating in the homologous recombination repair of DNA double-strand breaks. The compromised DNA repair mechanism significantly contributes to the elevated risk of breast cancer associated with this mutation, underscoring the importance of precise genomic maintenance for cancer prevention.

   

PIK3CA, or Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, is a critical component of the PI3K/AKT signaling pathway, and mutations in this gene are prevalent in breast cancer. These mutations activate the PI3K/AKT signaling pathway, promoting tumor growth and survival. The occurrence of PIK3CA mutations underscores the potential for targeted therapy, with inhibitors of PIK3CA showing considerable promise in treating cancers that harbor such genetic alterations. The identification of PIK3CA mutations is essential for customizing treatment to the individual profiles of patients, thereby improving outcomes in hormone receptor-positive, HER2-negative breast cancers. 

• E545K in Exon 9: This point mutation results in the substitution of glutamic acid with lysine, enhancing the lipid kinase activity of the PI3K enzyme abnormally. The increased activity promotes cell growth and survival pathways, such as mTOR signaling, that are typically tightly regulated, contributing to breast cancer progression.
• E542K in Exon 9: Similar to E545K, this mutation alters the enzyme's structure, increasing its activity and contributing to the deregulated growth signaling pathways, such as mTOR signaling, that are characteristic of cancer cells.
• H1047R in Exon 20: The substitution of histidine with arginine in the kinase domain leads to a gain of function, with increased PI3K enzymatic activity. This mutation drives the constitutive activation of downstream signaling pathways, such as AKT, promoting cell proliferation and survival in the absence of growth signals and facilitating tumorigenesis.

   

   

BRIP1, or BRCA1 Interacting Protein C-terminal Helicase 1, works closely with BRCA1 in DNA repair processes, particularly in the repair of double-strand breaks. Mutations in BRIP1 can compromise the effectiveness of DNA repair, leading to increased genomic instability and a higher risk of developing breast and ovarian cancer. Like mutations in BRCA1, BRIP1 mutations can elevate the risk of early-onset breast cancer, although the overall risk increase is less pronounced than that associated with BRCA1 or BRCA2 mutations. Identifying mutations in BRIP1 is important for understanding individual risk profiles and can influence decisions regarding cancer surveillance and preventive strategies.

ATM, or Ataxia-Telangiectasia Mutated, is a key player in the cellular response to DNA damage. It phosphorylates several downstream proteins, including p53, CHK2, and BRCA1, activating the DNA repair machinery. Mutations in the ATM gene diminish the cell's ability to repair DNA damage, leading to an accumulation of mutations and an increased risk of breast cancer. The role of ATM mutations in breast cancer is complex, with some mutations having a more profound impact on cancer risk than others. Individuals with ATM mutations may benefit from increased surveillance and, in some cases, preventive measures to mitigate their heightened risk of breast cancer.

CHEK2, or Checkpoint Kinase 2, acts as a tumor suppressor that interacts with several other proteins, including BRCA1 and p53, to halt cell division in response to DNA damage. Mutations in CHEK2 can impair this checkpoint function, allowing cells with DNA damage to continue dividing, thereby increasing the risk of cancer development. CHEK2 mutations are associated with a moderately increased risk of breast cancer. The detection of CHEK2 mutations can offer valuable insights into an individual’s cancer risk, guiding recommendations for cancer screening and preventive strategies.

PTEN, or Phosphatase and Tensin Homolog, is a tumor suppressor gene that negatively regulates the PI3K/AKT signaling pathway, crucial for controlling cell growth and survival. Mutations or deletions in PTEN lead to unregulated cell proliferation and survival, contributing to the development of breast cancer and other types of cancer. PTEN mutations are often seen in patients with Cowden syndrome, a disorder that significantly increases the risk of breast, thyroid, and endometrial cancers. Understanding PTEN status in patients can be critical for assessing cancer risk and determining appropriate surveillance and treatment options.

TP53 is a pivotal tumor suppressor gene involved in DNA repair, apoptosis, and cell cycle control. Mutations in TP53 are one of the most common genetic alterations in human cancers, including breast cancer. These mutations can lead to the production of a dysfunctional p53 protein, incapable of performing its normal protective roles against cancer development. The loss of functional p53 protein allows cells with damaged DNA to proliferate, significantly increasing the risk of breast cancer and other types of cancer. TP53 mutations are particularly associated with more aggressive and treatment-resistant forms of breast cancer, making the identification of these mutations important for prognosis and treatment planning.